**A** *LEA* **Gene from a Vietnamese Maize Landrace Can Enhance the Drought Tolerance of Transgenic Maize**

### **Bui Manh Minh 1, Nguyen Thuy Linh 1, Ha Hong Hanh 1, Le Thi Thu Hien 1,2, Nguyen Xuan Thang 3, Nong Van Hai 1,2 and Huynh Thi Thu Hue 1,2,\***


Received: 20 December 2018; Accepted: 29 January 2019; Published: 31 January 2019

**Abstract:** Maize (*Zea mays*) is a major cereal crop worldwide, and there is increasing demand for maize cultivars with enhanced tolerance to desiccation. Late embryogenesis abundant (LEA) proteins group 5C is involved in plants' responses to various osmotic stresses such as drought and salt. A putative group 5C LEA gene from *Z. mays* cv. Tevang 1 was isolated, named *ZmLEA14tv*, and cloned into a T-DNA for expression in plants. The deduced amino acid of ZmLEA14tv showed a conserved Pfam LEA\_2 domain and a high proportion of hydrophobic residues, characteristic of group 5C LEA proteins. Transgenic tobacco and maize plants expressing *ZmLEA14tv* were generated. During drought simulation conditions, the *ZmLEA14tv*-expressing plants of tobacco showed improved recovery ability, while those of maize enhanced the seed germination in comparison with the non-transgenic control plants. In addition, the survival rate of *ZmLEA14tv* transgenic maize seedlings was twice as high as the control. These results indicated that *ZmLEA14tv* might be involved in the drought tolerance of plants and could be a candidate gene for developing enhanced drought-tolerant crops.

**Keywords:** drought tolerance; *LEA*; Tevang 1 maize; tobacco

#### **1. Introduction**

Late embryogenesis abundant (LEA) proteins are mostly hydrophilic proteins, which can reduce the damage caused by severe environmental conditions. LEA proteins were reported to contribute to various developmental processes and to accumulate in response to drought, low temperature, salt stress, or treatment with the phytohormone ABA [1–4]. The first LEA was reported in cotton seeds [5,6]. LEA proteins accumulated during the late stages of embryogenesis and associated with the desiccation of seeds' embryos [7,8]. The members of the LEA protein family are also expressed during water deficit in bacteria (*Escherichia coli*) and yeast (*Saccharomyces cerevisiae*), suggesting a ubiquitous protective role of these proteins against osmotic stresses [9,10].

Following the Battaglia's classification, LEA proteins are categorized into seven different groups [11]. The LEA proteins of groups 1, 2, 3, 4, 6, and 7 are hydrophilic or typical LEA proteins, which have a low proportion of cysteine and tryptophan residues, and a high proportion of glycine, glutamic acid, lysine, and threonine residues. In contrast, the group 5 LEA protein has high content of hydrophobic residues. Based on amino acid sequences and conserved motifs, the group 5 LEA protein

was classified into three subgroups, namely 5A, 5B, and 5C [11]. Subgroup 5C LEA proteins were characterized by a low instability index, low proportion of polar (hydrophilic) and small residues, a higher proportion of non-polar residues, and heat-unstable conformation [11–13]. Moreover, the 5C LEA proteins are folded intrinsically and have more β-sheets than α-helices, which is also different from group 5A and 5B [8,13]. These differences in residue proportion and physical characteristics of group 5C from other LEA protein groups may refer to alternative functions involving stress tolerance.

Recently, due to the development of new sequencing technologies, the whole genome sequences of valuable plants such as rice (*Oryza sativa* L.), maize (*Zea mays* L.), and cotton were published and made available to researchers [14–16]. Based on the conserved domains of LEA proteins, LEA protein families could be identified and characterized through whole-genome prediction approaches. In rice, 34 rice candidate *LEA* (*OsLEA*) genes were identified through a HMMER search (http://hmmer.janelia. org/) [17]. By using a similar method, 242, 136, and 142 candidate DNA regions that encode for LEA proteins were identified in three upland cotton namely *Gossypium hirsutum*, *G. arboreum*, and *G. raimondii*, respectively [18]. The LEA protein profile of maize was also reported with 32 LEA genes distributed non-randomly across chromosomes [19]. The accumulation of LEA profiles in various plants provided fundamental knowledge for functional analysis and *LEA* gene engineering in the future.

A small number of group 5C LEA proteins have been characterized, but their physical characteristics and biological functions are largely unknown. Some members of group 5C were identified in other plants such as cotton LEA14A, soybean D95-4, tomato ER5, hot pepper CaLEA6, *Arabidopsis* AtLEA14A, sweet potato IbLEA14A, rice OSLEA5, foxtail millet SiLEA14A, and wild peanut LEA [8,13,20–26]. The expression of LEA 5C proteins is upregulated by ABA and multiple abiotic stresses including salt and drought [13,23]. Functional studies of group 5C proteins showed that an overexpression of CaLEA6 protein, which originates from hot pepper (*Capsicum annuum*), could improve drought and salt tolerance significantly in tobacco [23]. In addition, the overexpression of other *LEA14A* genes such as *IbLEA14A* and *SiLEA14A* remarkably raised the level of lignification, free proline, and soluble sugar in transgenic sweet potato (*Ipomoea batatas*) calli, *Arabidopsis*, and foxtail millet (*Setaria italica*) [13,23,24]. Recombined OsLEA5 in *E. coli* could protect lactate dehydrogenase from misfolding under different abiotic stresses, resulting in stress tolerance [25].

Maize (*Zea mays* L.) is an important monocot crop worldwide; its production was more than 1.06 billion tons in 2016 [27]. Drought is the major factor that accounts for significant losses in maize productivity. A water reduction of 40% could decrease maize production by 39.3% [28]. Recently, a predicted profile of LEA family in maize has been reported through both bioinformatic and practical approaches [19]. A putative maize *LEA* gene located on chromosome 8 that contained a Pfam LEA\_2 domain was described; however, any function of this gene in protecting plants against osmotic stress remains unknown [19]. In the current study, a putative *LEA* gene was isolated from the Tevang 1 landrace, designated as *ZmLEA14tv* and cloned into a T-DNA for expression in plants. The expression patterns of transgenic *ZmLEA14tv* in both tobacco and maize models were investigated to determine the importance of this gene in enhancing drought tolerance in selected plants.

#### **2. Materials and Methods**

#### *2.1. Plant Materials and Drought-Stimulating Growth Conditions*

Maize seeds (*Z. mays* cv. Tevang 1) were provided by the Maize Research Institute in Vietnam. Tevang 1 cultivar is a well-known landrace maize from the rocky mountain region in Northern Vietnam. The cultivar is well adapted to low annual rainfall and water-deficit cultivation. Maize seeds were germinated and grown under greenhouse conditions at 22/26 ◦C (night/day) and a photoperiod of 14/10 h (day/night) for two weeks. The genetic background line for transformation was K7, a selected maize that has a higher rate of regeneration and successful transformation through *A. tumefaciens*-mediated methods. The plant material was evaluated for several morphological and physiological traits. For analysis of drought tolerance at the germination stage, 30 seeds of both WT

and homozygous *ZmLEA14tv* maize (T2 generation) were germinated on filter paper in a Petri dish wetted with water (as control) or 10% PEG, 20% PEG (*w*/*v*) solution for one day at 30 ◦C. Shoot and root lengths were measured after eight days of the treatment, followed by taking photographs. For the drought tolerance assay, the five-leaf-stage maize seedlings were assigned to a withholding water period for 14 days followed by a three-day re-watering. At least five seedlings were grown in each plot. The survival rate, fresh stem weight, and fresh root weight of drought-treated and control seedlings were measured. The experiment was replicated three times.

Seeds of WT tobacco (*Nicotiana tabacum*) cultivar K326 and transgenic tobacco were sterilized with 70% ethanol and 5% bleach, which follows a method described previously [29]. The tobacco explant was germinated in Murashige and Skoog (MS) medium containing 200 mg/μL kanamycin under light/dark cycle conditions of 16/8 h at 25 ◦C. The 20-day-old T3 tobacco plants were put under drought conditions for 15 days and then re-watered to observe the morphological modification.

#### *2.2. Isolation of the Putative LEA Coding Region in Tevang 1 Landrace Maize*

Based on the putative *ZmLEA14A* sequence published in GenBank (accession number EU976614.1) and the flanking sequence of this gene located on maize chromosome 8 in the maizeGBD database (http://www.maizegdb.org/), PCR-specific primers for amplification of this region were designed. The DNA regions containing the putative *ZmLEA14A* open reading frame (ORF) and 5 UTR of this gene were amplified from genomic DNA of Tevang 1 cultivar using the *ZmLEA14tv\_F* forward primer sequence (5 TCACTTCTCTTCCAGCGAGTAC3 ) and *ZmLEA14tv\_R* reverse primer sequence (5 TCTCGTACTACTCAAGCAGCAC3 ). The PCR product was denoted as *ZmLEA14tv* and purified by Thermo Scientific PCR product purification kit (Cat. number #K0702, Waltham, MA, USA) then cloned into a pJET1.2 cloning vector following the manual of the producer. The cloning vector pJET1.2, which contains the PCR fragment, was transformed into *E. coli* DH10β competent cell by heat shock at 42 ◦C for 1 min. The colonies harboring the pJET1.2–ZmLEA14tv plasmid were checked by colony PCR with isolation primers and the restriction enzyme *Bgl*II.

#### *2.3. ZmLEA14tv Expression Vector Construction and Transgenic Plant Generation*

The coding region of *ZmLEA14tv* was amplified with the *ZmLEA14tv\_CloneF* forward primer sequence (5 ATTACCATGGCGCAGTTGGTG3 ) and *ZmLEA14tv\_CloneR* reverse primer sequence (5 ATTAGCGGCCGCGAAGATGCTGG3 ) that generates the recognition sites of *Nco*I and *Not*I at the 5 and 3 ends of the PCR products, respectively. The thermocycler program (Eppendorf, Hamburg, Germany) starts at 95 ◦C for 3 min, followed by 30 cycles of amplification (95 ◦C for 1 min; 56 ◦C for 30 s; 72 ◦C for 1 min); the final extension step was 72 ◦C for 10 min. The 463-bp PCR product was then treated with *Nco*I and *Not*I, purified, and ligated into the pRTRA7/3 vector to generate a 35S promoter-ZmLEA14tv-35S terminator construct. The cassette was cut and combined into the T-DNA region of the pCAMBIA1300 (8958 bp) binary vector. The recombined binary vector pCAMBIA1300/ZmLEA14tv was transformed into *A. tumefaciens* strain EHA 105 after validation by sequencing. The transgenic tobacco and maize plants were regenerated by modified *Agrobacterium*-mediated transformation methods described in the studies of Topping (1998) and Frame et al. (2011), respectively [30,31].

#### *2.4. Sequence Alignment and Gene Evolution Analysis*

The sequence of PCR products was verified by 3500 Series Genetic Analyzers (Applied Biosystems, Foster City, CA, USA) followed the Sanger method. A deduced amino acid sequence of ZmLEA14tv protein was generated using the ExPaSy web tool (https://web.expasy.org). The isoelectric point molecular mass, the proportion of amino acid, and grand average of hydropathy (GRAVY) index of the putative ZmLEA14tv peptide were estimated using the ProtParam web tool (https://web.expasy. org/protparam/). Motif analysis was performed using the Pfam program (http://www.ebi.ac.uk/ Tools/InterProScan/). The completed amino acid and deduced amino acid sequences of subgroup 5C

LEA proteins were used to construct a phylogenetic tree. Sequence alignment was carried out with ClustalW and adjusted manually. The phylogenetic tree was constructed with the neighbor-joining bootstrap method using the MEGA v6.0 program [32].

#### **3. Results**

#### *3.1. Isolation and Vector Construction of ZmLEA14tv Gene*

A putative LEA coding sequence from a native maize landrace Tevang 1 (*Z. mays* cv. Tevang 1) was isolated and named *ZmLEA14tv*. The isolated *ZmLEA14tv* had a size of 693 base pairs (bp) with an open reading frame of 459 bp in length encoding for a deduced 152 amino acid (aa) protein. Sequence analysis exhibited the highest similarity at 99% with a *Z. mays LEA14A* coding sequence (accession number NM\_001159174), followed by *LEA14A* of *Shorgum bicolor* (XM\_002454858.2) and *LEA14A*-like gene of *S. italica* (93% and 87%, respectively). The comparison with the reference sequence number NM\_001159174 in GenBank showed two variations, G381A and C456T; however, the deduced amino acid sequence was not changed. The putative protein was predicted at 15.96 kDa in molecular weight with a pI of 6.08. The protein was rich in Val (12.6%), Leu (11.3%), and Gly (9.2%), but contained low quantities of Trp (0.7%), Asn (1.3%), Cys (0.7%), and Gln (1.3%). The GRAVY and instability index of the putative *ZmLEA14tv* were 0.047 and 16.01, respectively, suggesting stability and hydrophobic nature. A conserved "LEA\_2" motif (PF03186), which was classified into subgroup 5C according to Battaglia's classification of LEA proteins, was found on the ZmLEA14tv through an InterProScan search [11]. Further analysis showed that ZmLEA14tv contained a low percentage of polar amino acids (23.02%) and a high level of hydrophobic residues (47.02%). The ZmLEA14tv protein displayed diverse homology with other group 5C LEA proteins and broadly matches similar segments in related LEA proteins, indicating a close evolutionary relationship among these proteins (Figure 1A,B). The phylogenetic analysis also showed that the ZmLEA14tv protein has the closest relationship with maize LEA14A protein (accession number NM\_001159174), followed by an LEA-like protein from rice, namely Os01g0225600 (accession number NM\_001048996), supported by high bootstrap values (99% and 95%, respectively).

**Figure 1.** *Cont.*

**Figure 1.** Sequence alignment and phylogenetic relationship for the putative ZmLEA14tv protein and its homologs. (**A**) Multiple sequence alignment of ZmLEA14tv with its homologs (LEA group 5 protein) from various plant species. The conserved positions were marked as stars. (**B**) Neighbor-Joining phylogenetic trees of ZmLEA14tv and its homologs. The clades of monocots and dicots are marked. ZmLEA14tv in *Z. mays* cv. Tevang 1 branch is highlighted by a solid dark circle. The GenBank accession numbers are as follows: SiLEA14 (*S. italic*, KJ767551), AtLEA14 (*Arabidopsis thaliana*, NM100029), Lea14-A (*Z. mays*, NM001159174), D95-4 (*Glycine max*, U08108), IbLEA14 (*Ipomoea batatas*, GU369820), ER5 (*Solanum lycopersicum*, U77719), Lemmi9 (*S. lycopersicum*, Z46654), CaLEA6 (*Capsicum annuum*, AF168168), OsLEA5 (*Oryza sativa*, JF776156), pcC27-45 (*Craterostigma plantagineum*, M62990), pcLEA14 (*Pyrus communis*, AF386513), At1g01470 (*A. thaliana*, BT015111), D95-4 (*G. max*, U08108), At2g46140 (*A. thaliana*, NM130176), Os01g0225600 (*O. sativa*, NM001048996), LEA14-A-like (*Brachypodium distachyon*, XM003567779), BdLEA14-like (*B. distachyon,* XM003567779), LOC100274480 (*Z. mays*, NM001148839), SORBIDRAFT (*Sorghum bicolour*, XM002441543), LEA-like protein (*Cenchrus americanus*, AY823547), OsI21161 (*O. sativa*, CM000130), Os05g0526700 (*O. sativa*, NM001062639), Os05g0584300 (*O. sativa*, NM001062985), At2g44060 (*A. thaliana*, BT024723), LOC100285131 (*Z. mays*, EU970969) and umc2111 (*Z. mays*, NM001155750).

#### *3.2. ZmLEA14tv Gene Expression in Drought Resistance for Transgenic Tobacco*

To evaluate the function of the *ZmLEA14tv* transgenic structure in plant osmotic tolerance, the transgenic tobacco plants that expressed ZmLEA14tv under the control of the CaMV 35S promoter were selected for further analysis (Figure 3A). Thirty transgenic plants were obtained, and three homozygous T3 transgenic lines (LEAtv-L1, LEAtv-L3, LEAtv-L7) with high expression levels of ZmLEA14tv (Figure 2B) were chosen for further investigation. To investigate the drought tolerance of the transgenic tobacco, the seedlings were treated with a shortage of water for 15 days. Subsequently, the plants were re-watered and grown for three days. Under normal and drought conditions, there were no significant differences in morphological features such as height, weight, and leaf surface between the transgenic and non-transgenic wild-type (WT) plants. Leaves of the transgenic and control plants became curled and wilted after 15 days of drought. However, 100% of the transgenic tobacco was

restored after a three-day re-watering, unlike the control plant (Figure 2C). The fastest recovery was observed in LEAtv-L1, which also expressed the highest level of the *ZmLEA14tv* transgene. This result indicated a correlation between the *ZmLEA14tv* expression and the recovery ability of the plant after the drought conditions.

**Figure 2.** The expression of the *ZmLEA14tv* in tobacco (*Nicotiana tabacum*). (**A**) Schematic description of T-DNA involving in the pCAMBIA1300/ZmLEA14tv plasmid for the expression of *ZmLEA14tv* in plants. LB: left T-DNA border; RB: right T-DNA border; HygR: Hygromycin resistant gene; CaMV35S pro: Cauliflower mosaic virus 35S promoter; 35S ter: 35S terminator; *ZmLEA14A*: coding region of *ZmLEA14tv* gene; *Nco*I, *Not*I, and *Hin*dIII: restriction site of *Nco*I, *Not*I và *Hin*dIII, respectively. (**B**) RT-PCR analysis of ZmLEA14tv in transgenic tobacco lines (LEAtv-L1, LEAtv-L3, LEAtv-L7). WT: the wild type was used as a control. (**C**) The phenotype of transgenic and WT tobacco explants under normal and drought stress conditions.

#### *3.3. Transgenic Maize with ZmLEA14tv Gene in Drought Tolerance*

The ZmLEA14tv integration was confirmed by genomic PCR using pairs of primer sets specific to the Hpt and 35S promoter regions, respectively. RT-PCR and qRT-PCR of ZmLEA14tv were performed to validate the expression of the transgenic structure of T2 transgenic maize lines. Three T2 lines, namely L1453, L1482, and L1510, showed the highest expression level (2.7-, 9.2-, and 5.8-fold higher than the control, respectively) and the T2 seeds of these re-watered lines were used for further analysis (Figure 3A,B).

The drought tolerance of transgenic maize seeds during germination was examined. When germinated in water for eight days, the transgenic lines showed better growth than the WT line used as the regeneration material. Under normal conditions (H2O), no significant differences in shoot height were observed between the transgenic lines and the WT ones. However, the root length of the L1510 and L1482 lines, which have higher ZmLEA14tv transcripts accumulation, was significantly higher than that of the wild type. In comparison with the control group in water, the germination of both the WT and transgenic lines was severely suppressed under 10% and 20% PEG stress (Figure 3C). None of the experimental seeds could germinate in 20% PEG. However, better germination and subsequent development were observed with the seeds of L1510 and L1482, which had higher expression of ZmLEA14tv transcripts in 10% PEG than the wild type (Figure 3D).

**Figure 3.** The expression of *ZmLEA14tv* in maize (*Z. mays*). (**A**,**B**) *ZmLEA14tv* expression in three lines of T2 transgenic maize (L1453, L1482, and L1510) determined by RT-PCR (**A**) and qRT-PCR (**B**). Data in (**B**) represent means and standard errors for three biological replicates. (**C**) The phenotype of transgenic and WT maize under various abiotic stress treatment during the germination stage. The T2 of transgenic seeds were soaked in water (as control) or in 10% PEG, 20% PEG solution for drought simulation for one day at 30 ◦C and then placed on filter paper in plastic boxes wetted with the same solutions mentioned above for eight days. Each experiment was replicated three times. (**D**,**E**) The root and shoot length of transgenic and wild-type maize germinated under control (H2O) and drought-simulating conditions (10% PEG and 20% PEG). Statistical significance was determined by Student's *t*-test. \* *p* < 0.05; \*\* *p* < 0.01.

Furthermore, the drought tolerance of transgenic maize seedlings in soil was examined (Figure 4B–E). No significant differences in survival rate and fresh weight were observed between the transgenic and WT plants under well-wateredconditions (H2O). However, after drought stress for 14 days and re-watering, only 40% of WT seedlings could be restored, while this ratio in transgenic lines (LEAtv-L1 and LEAtv-L2) was almost doubled (87% and 80%, respectively) (Figure 4C). In addition, the fresh stem weight and fresh root weight of transgenic lines were significantly higher than the K7 wild type, suggesting a better growth rate of these lines in drought condition (Figure 4D,E). Taken together, these results indicate that the *ZmLEA14tv* gene showed improved drought resistance in transgenic maize.

**Figure 4.** Drought tolerance of maize seedlings overexpressing *ZmLEA14tv***.** (**A**) The RT-PCR analysis of *ZmLEA14tv* expression in transgenic maize lines (LEAtv-L1, LEAtv-L2). (**B**) The phenotype of transgenic and WT maize seedlings under drought stress treatment and re-watering conditions. The five-leaf-stage maize seedlings had watering withheld for 14 days, followed by a three-day re-watering. At least five seedlings were grown in each plot and the experiment was replicated three times. WT: wild type. (**C**–**E**) The survival rate, fresh stem weight, and fresh root weight of WT and transgenic maize seedling after the drought and re-watering treatments. Each experiment was replicated three times. Statistical significance was determined by Student's *t*-test. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

#### **4. Discussion**

The identification and characterization of ZmLEA14tv, a putative atypical LEA group 5C member of the Tevang 1 maize cultivar, were reported in the present study. The deduced amino acid sequence of ZmLEA14tv possessed characteristics of a 5C LEA protein that contains a "LEA\_2" domain (Pfam cluster PF03168). The LEA proteins are normally known as hydrophilins with a hydrophilicity index of more than 1 and a glycine (Gly) content more than 6% [11]. Typical LEA proteins can retain water and protect other soluble protein from the aggregation due to their highly hydrophilic properties [33]. Group 5C LEA proteins had a higher proportion of hydrophobic residues than typical LEA proteins; however, they were also involved in various kind of stress tolerance. The estimated GRAVY index of ZmLEA14tv was 0.047, much lower than typical LEA proteins. The ZmLEA14tv protein sequence deduced from the isolated DNA showed a "LEA\_2" domain that was characteristic of 5C LEA proteins and a high level of homology with other 5C members.

Functional analysis of 5C LEA proteins showed that the molecular mechanisms of the protective ability against desiccation stress were diverse. The overexpression of AdLEA, a 5C LEA protein from wild peanut, could help maintain the photosynthetic efficiency, reduce the ROS level, and induce the expression of some drought-responsive genes in transgenic tobacco [26]. Meanwhile, overexpressing *SiLEA14* from foxtail millet enhanced a higher level of proline and sugar accumulation in transgenic *Arabidopsis* [13]. The present study showed that the expression of *ZmLEA14tv* in tobacco significantly improved the recoverability of transgenic plants suffering from a short desiccation (Figure 2). This result suggested that *ZmLEA14tv* could function properly in tobacco and enhance its drought tolerance.

One of the methods to generate drought-tolerant maize is enhancing the expression of superior drought-tolerant genes in commercial lines. The overexpression of the *OsSta2* gene, encoding for a AP2/ERF protein, under a maize ubiquitin promoter improved the salt tolerance and grain yield of transgenic rice [34]. Furthermore, enhanced expression of rice dehydrin, namely *OsDhn1*, could increase the tolerance to oxidative stress under salt and drought conditions [35]. Group 5C LEA is well known as an atypical group of proteins that are involved in various abiotic stress response in plants [8,13,24]. In this study, a putative LEA gene namely *ZmLEA14tv* was isolated from the genomic DNA of *Z. mays* cv. Tevang 1, which is well adapted to drought stress in the northern mountains of Vietnam. Our data revealed the potential application of *ZmLEA14tv* in genetic engineering for improving crop performance in the context of climate change. Interestingly, transgenic maize seeds expressing *ZmLEA14tv* showed an improved germination ability in drought-simulated condition in comparison with the WT and did not cause any delay in shoot and root development in transgenic plants.

#### **5. Conclusions**

In summary, the coding region of *ZmLEA14tv* was isolated from *Z. mays* cv. Tevang 1 and then cloned into an overexpression cassette. This gene encoded a hydrophobic deduced protein that has a high similarity in structure and a close relationship with other 5C LEA proteins. In addition, the expression of *ZmLEA14tv* in model dicot plants such as tobacco significantly improved the recovery ability, while the enhanced *ZmLEA14tv* transgenic maize showed better germination and growth in drought simulation conditions. These results suggested that *ZmLEA14tv* could act as a potential candidate for genetic engineering to improve drought and other osmotic stress tolerance.

**Author Contributions:** N.V.H. and H.T.T.H. conceived and designed the experiments; B.M.M., N.T.L., H.H.H., and N.X.T. performed the experiments; B.M.M., L.T.T.H., and H.T.T.H. analyzed the data; B.M.M., N.V.H., and H.T.T.H. prepared the manuscript.

**Funding:** This research was funded by the Vietnam Ministry of Agriculture and Rural Development (MARD) grant number 16HĐ/KHCN-VP for the period 2014–2018.

**Acknowledgments:** The authors thank Doan Thi Bich Thao and Nguyen Thu Hoai for participating in some greenhouse experiments.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

1. Chandler, P.M.; Robertson, M. Gene expression regulated by abscisic acid and its relation to stress tolerance. *Annu. Rev. Plant Physiol. Plant Mol. Biol.* **1994**, *45*, 113–141. [CrossRef]


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### *Article* **Silicon and the Association with an Arbuscular-Mycorrhizal Fungus (***Rhizophagus clarus***) Mitigate the Adverse Effects of Drought Stress on Strawberry**

### **Narges Moradtalab 1,\*, Roghieh Hajiboland 1, Nasser Aliasgharzad 2, Tobias E. Hartmann <sup>3</sup> and Günter Neumann <sup>3</sup>**


Received: 26 November 2018; Accepted: 18 January 2019; Published: 21 January 2019

**Abstract:** Silicon (Si) is a beneficial element that alleviates the effects of stress factors including drought (D). Strawberry is a Si-accumulator species sensitive to D; however, the function of Si in this species is obscure. This study was conducted to examine the effect of Si and inoculation with an arbuscular mycorrhizal fungus (AMF) on physiological and biochemical responses of strawberry plants under D. Plants were grown for six weeks in perlite and irrigated with a nutrient solution. The effect of Si (3 mmol L−1), AMF (*Rhizophagus clarus*) and D (mild and severe D) was studied on growth, water relations, mycorrhization, antioxidative defense, osmolytes concentration, and micronutrients status. Si and AMF significantly enhanced plant biomass production by increasing photosynthesis rate, water content and use efficiency, antioxidant enzyme defense, and the nutritional status of particularly Zn. In contrast to the roots, osmotic adjustment did not contribute to the increase of leaf water content suggesting a different strategy of both Si and AMF for improving water status in the leaves and roots. Our results demonstrated a synergistic effect of AMF and Si on improving the growth of strawberry not only under D but also under control conditions.

**Keywords:** silicon; strawberry; total antioxidants; drought; stress responses; arbuscular mycorrhizal fungus (AMF); *Rhizophagus clarus*

#### **1. Introduction**

Although silicon (Si) is not considered an essential element for higher plants, numerous studies have demonstrated that Si is a beneficial element that alleviates abiotic and biotic stresses in plants [1–3]. Si is a quasi-essential element for the growth of rice, wheat, sorghum, potato, cucumber, zucchini, and soybean, under various biotic and abiotic stress conditions [4]. According to the Si tissue concentration, plants are classified into Si-accumulators and non-accumulators. The differences in Si accumulation among species can be attributed to the differential ability of roots to take up Si [2].

Drought (D) adversely influences several features of plant growth and development, and a prolonged D severely diminishes plant productivity [5]. Water loss through transpiration is reduced by stomatal closure as an immediate response of plants upon being exposed to D; however, it reduces also nutrient uptake and limits plant ability for dry matter production. In addition, reduced intercellular CO2 concentration leads to an excess excitation energy that causes enhanced leakage of electrons to molecular oxygen and increases the production of reactive oxygen species (ROS) [6,7]. These cytotoxic ROS destroy normal metabolism through oxidative damage to lipids, proteins, and nucleic acids [8]. Plants have developed complex physiological and biochemical adjustments to tolerate D, including the activation of antioxidative enzymes, maintenance of cell turgor, and water status through the accumulation of organic osmolytes such as soluble carbohydrates and free amino acids, particularly proline [9,10].

Si supplementation of plants alleviates D stress. Several mechanisms including the activation of photosynthetic enzymes [11], the activation of enzymatic antioxidant defense systems, increased water use efficiency [12,13], nutrient uptake [14], root growth and hydraulic conductance [15], and the accumulation of organic osmolytes [16] are involved in Si-mediated growth improvement under D [11,17].

The association of roots with arbuscular mycorrhizal fungi (AMF) is the most abundant symbiosis in the plant kingdom [18]. The colonization of roots by AMF enhances the plant growth by increasing nutrient uptake and plant tolerance to stress [19,20]. Several studies evaluated the effects of AMF-inoculation in horticultural plants such as citrus, apple, and strawberry [21–23]. AMF symbiosis increased the rate of photosynthesis, stomatal conductance, and leaf water potential in colonized plants under D [24]. Moreover, AMF had a significant direct contribution to the uptake of phosphorus (P), zinc (Zn), and copper (Cu) under water stress [25].

Strawberry (*Fragaria x ananasa* Duch.) plants are extremely sensitive to drought because of a shallow root system, large leaf area, and high-water content of fruits. When the strawberry plants are not sufficiently irrigated, both yield and fruit size are reduced [22]. As a Si-accumulating species [26,27], strawberry has both functional influx (Lsi1) and efflux (Lsi2) transporters for Si uptake, and under a constant soluble Si application can absorb 3% Si per dry weight [26]. However, to the best of our knowledge, there is no study on the effect of Si on strawberry under abiotic stresses including D. Another obscure aspect in this regard is Si effect on the association of roots with AMF in this species. Therefore, given the potential of both Si and AMF for mitigation of drought stress effects, the objectives of the present study are (1) to elucidate the influence of Si on photosynthesis, water status, and activity of antioxidative defense system in strawberry plants under D conditions and (2) to investigate the Si effect on the response of mycorrhizal plants when exposed to D stress. We hypothesized the existence of a synergistic effect of Si and AMF on the protection against D in strawberry plants.

#### **2. Materials and Methods**

#### *2.1. Preparation of Plant and Fungus Materials*

The first-generation strawberry (*Fragaria* × *ananassa* var. Paros) plantlets of genetically different individuals originating from a strawberry field were prepared as donor mother plants. Second-generation strawberry plantlets from 10 cm stolons of these genetically different mother plants were propagated in a growth chamber. Four independent biological replicates were used per treatment. The offset plants were grown in a standard peat–perlite (1:1) mixture for one week to allow root development.

Inoculum of *Rhizophagus clarus* (Walker & Schüßler; isolated in symbiosis with *Poa annua* L. in a grassland in Cuba) (MUCL 46238–GINCO–BEL; Synonymy: *Glomus clarum* Nicolson & Schenck; [28]) was provided by the Department of Soil Science, University of Tabriz, Iran. Originally, fungi were obtained from Pal Axel Lab, Lund University, Sweden. *R. clarus* was propagated with *Trifolium repens* L. plants in 3.5 L pots containing sterile sandy loam soil. Rorison's nutrient solution, prepared with deionized water [29] with 50% strength of phosphorus, was added to the pots twice a week to bring the soil moisture to water holding capacity (WHC). The pots were incubated in a greenhouse with 28/20 ◦C day/night and 16/8 h light/dark periods. After four months, the tops of the plants were excised and the pot materials containing soil and mycorrhizal roots were thoroughly mixed and used as fungal inoculum.

#### *2.2. Plant Treatments*

The experiment was conducted using a completely randomized design with three factors including irrigation regimes (three levels), Si treatments (two levels), and AMF inoculation (two levels). Each treatment combination was represented by four independent pots as four replicates.

One-week-old strawberry seedlings were transferred to 3 L pots (one plant per pot) filled with washed perlite and containing 60 g autoclaved and non-autoclaved AMF inoculum in −AMF and +AMF treatments, respectively. The pots were irrigated daily with water or Hoagland nutrient solution at WHC of the perlite after weighing. The total volume of nutrient solution applied to the plants was 200 mL pot−<sup>1</sup> week−1. To avoid the accumulation of salts in the substrate, electric conductivity in the perlite was measured in samples taken weekly from the bottom of the pots. Si as sodium silicate (Na2SiO3, Sigma–Aldrich, Munich, Germany) prepared as the solution (0.6 mM, pH = 6.1) was added to the pots weekly by irrigation leading to a concentration of 3 mmol L−<sup>1</sup> perlite (~84 mg L−<sup>1</sup> perlite) at the end of the experiment after 6 weeks. One week after starting the Si application, the different irrigation regimes (IR) included well-watered (WW, 90% WHC), mild drought (MD, 75% WHC), and severe drought (SD, 35% WHC) and were assigned randomly to the pots, and watering was omitted from D treatments until they reached the respective WHC. This was achieved 4 and 6 days after starting a different IR for the MD and SD treatments, respectively. Well-watered and D plants received the same amount of nutrient solution, and the respective WHC was achieved by adjusting the volume of water used for irrigation.

In order to determine the possible effect of Na as the accompanying ion in the Si salt applied to the plants, an experiment was conducted parallel to the main experiment with an additional control (without the addition of salt or Si) and 6 mmol L−<sup>1</sup> NaCl containing an equivalent Na with 3 mmol <sup>L</sup>−<sup>1</sup> Na2SiO3. The dry weight (g plant−1) of plants under control (0.48 ± 0.05) and 6 mmol L−<sup>1</sup> salt (0.51 ± 0.04) was not significantly different (Tukey test, *p* < 0.001).

Plants were grown under controlled environmental conditions with a temperature regime of 25 ◦C/18 ◦C day/night, 14/10 h light/dark periods, a relative humidity of 30%, and at a photon flux density of about 400 μmol m−<sup>2</sup> s<sup>−</sup>1.

#### *2.3. Plant Harvest*

Six-week-old plants (five weeks after starting Si treatments and four weeks after reaching the respective WHC) were harvested. Shoots and roots were separated, washed with distilled water, and blotted dry on filter paper. After determination of the fresh weight (FW), the dry weight (DW) was determined after drying at 60 ◦C for 48 h. Subsamples were taken for biochemical analyses before drying. Before harvest, the gas exchange parameters were determined in attached leaves.

For evaluation of the AMF colonization, the fine roots (1 g FW) were cleared in 10% (*v*/*v*) KOH and stained with 0.05% (*v*/*v*) trypan blue in lacto–glycerin. The colonization rate of the roots (%) was estimated by counting the proportion of root length containing fungal structures (arbuscules, vesicles and hyphae) using the gridline intersect method [30,31]. In brief, stained root segments were spread out evenly in a 10 cm diameter Petri dish. A grid of lines was marked on the bottom of the dish to form 0.5 cm2. Vertical and horizontal gridlines were observed with a binocular device, and the presence or absence of fungal structures was recorded at each point where the roots intersected a line. Three sets of observations were made recording all the root-gridline intersects. Each of the three replicate records was made on a fresh rearrangement of the same root segments [30,31].

#### *2.4. Leaf Osmotic Potential and Relative Water Content*

The leaf osmotic potential (ψs) was determined in the second leaves harvested 1 h after the light was turned on in the growth chamber. The leaves were homogenized in a prechilled mortar and pestle and centrifuged at 4000 g for 20 min at 4 ◦C. The osmotic pressure of the samples was measured by an osmometer (Micro–Osmometer, Herman Roebling Messtechnik, Germany), and the milliosmol

data were recalculated to MPa. For the determination of the relative water content (RWC%), the leaf disks (5 mm diameter) were prepared, and after the determination of the fresh weight (FW), they were submerged for 20 h in distilled water; thereafter, they were blotted dry gently on a paper towel, and the turgid weight (TW) was determined. The dry weight (DW) of the samples was determined after drying in an oven at 70 ◦C for 24 h, and the RWC% was calculated according to the formula (FW − DW)/(TW − DW) × 100.

#### *2.5. Measurements of Photosynthetic Gas Exchange*

Before the harvest gas exchange parameters were determined with the attached leaves. The net CO2 fixation rate (μmol m−<sup>2</sup> s−1), transpiration rate (mmol m−<sup>2</sup> s−1), and stomatal conductance (mol m−<sup>2</sup> s−1) were determined with a calibrated portable gas exchange system (LCA–4, ADC Bioscientific Ltd., Hoddesdon, UK). Water use efficiency (WUE) was calculated as the ratio of photosynthesis/transpiration (μmol mmol<sup>−</sup>1).

#### *2.6. Biochemical Determinations*

For the determination of carbohydrates, leaf and root samples (100 mg) were homogenized in a 100 mM potassium phosphate buffer (pH 7.5) at 4 ◦C. After centrifugation at 12,000 g for 15 min, the supernatant was used for the determination of total soluble sugars. An aliquot of the supernatant was mixed with an anthrone–sulfuric acid reagent and incubated for 10 min at 100 ◦C. After cooling, the absorbance was determined at 625 nm. The standard curve was created using glucose (Sigma–Aldrich, Munich, Germany) [32]. The total soluble protein was determined by the Bradford (1976) method using a commercial reagent (Roti®Quant, Roth GmbH, Karlsruhe, Germany) and bovine serum albumin (BSA) as standard. Total free α-amino acids were assayed using a ninhydrin colorimetric method. Glycine (Sigma–Aldrich, Munich, Germany) was used to produce a standard curve [33]. For the determination of proline, samples were homogenized with 3% (*v*/*v*) sulfosalicylic acid and the homogenate was centrifuged at 3000 g for 20 min. The supernatant was treated with acetic acid and acid ninhydrin and boiled for 1 h, and then the absorbance was determined at 520 nm. Proline (Sigma–Aldrich, Munich, Germany) was used to produce a standard curve [34].

#### *2.7. Determination of Enzyme Activities and Concentration Of Oxidants*

Fresh leaf samples (100 mg) were ground in liquid nitrogen using a mortar and pestle. Each enzyme assay was tested for linearity between the volume of crude extract and the measured activity. All measurements were undertaken through spectrophotometry (Specord 200, Analytical Jena AG, Jena, Germany) according to optimized protocols described elsewhere [35]. The activity of ascorbate peroxidase (APX, EC 1.11.1.11) was measured by determining the ascorbic acid oxidation; one unit of APX oxidizes ascorbic acid at a rate of 1 μmol min−<sup>1</sup> at 25 ◦C. The catalase (CAT, EC 1.11.1.6) activity was assayed by monitoring the decrease in absorbance of H2O2 at 240 nm; unit activity was taken as the amount of enzyme which decomposes 1 μmol of H2O2 in one min. Peroxidase (POD, EC 1.11.1.7) activity was assayed using the guaiacol test. The enzyme unit was calculated as the enzyme protein required for the formation of 1 μmol tetra–guaiacol for 1 min. The total superoxide dismutase (SOD, EC 1.15.1.1) activity was determined using the mono–formazan formation test. One unit of SOD was defined as the amount of enzyme required to induce a 50% inhibition of nitro blue tetrazolium (NBT) reduction as measured at 560 nm compared with control samples without enzyme aliquot. The concentration of H2O2 was determined using KI at 508 nm. Lipid peroxidation was estimated from the amount of malondialdehyde (MDA) formed in a reaction mixture containing thio-barbituric acid (Sigma–Aldrich, Munich, Germany) at 532 nm. The MDA levels were calculated from a 1,1,3,3–tetraethoxypropane (Sigma–Aldrich, Munich, Germany) standard curve [35].

#### *2.8. Mineral Nutrient Analysis*

For the determination of the plant nutritional status, 250 mg of dried leaf material was ashed in a muffle furnace at 500 ◦C for 5 h. After cooling, the samples were extracted twice with 2.5 mL of 3.4 M HNO3 until dryness to precipitate SiO2. The ash was dissolved in 2.5 mL of 4 M HCl, subsequently diluted ten times with hot deionized water, and boiled for 2 min. After the addition of a 0.1 mL cesium chloride/lanthanum chloride buffer to the 4.9 mL ash solution, Fe, Mn, and Zn concentrations were measured by atomic absorption spectrometry (AAS, UNICAM 939, Offenbach/Main, Germany) [36].

#### *2.9. Silicon Determination*

Dry leaf material (0.2 g) was microwave digested with 3 mL concentrated HNO3 + 2 mL H2O2 for 1 h. Samples were diluted with circa 15 mL deionized H2O and transferred into 25 mL plastic flasks; 1 mL concentrated Hydrofluoric acid was added and left overnight. After the addition of 2.5 mL 2% (*w*/*v*) H3BO3, the flask volume was adjusted to 25 mL with deionized H2O, and Si was determined by ICP–OES (Vista−PRO, Varian Inc., Palo Alto, USA) [36].

#### *2.10. Statistical Analyses*

A primary statistical analysis was carried out using the Sigma Plot 11.0 software Systat Software Inc. San Jose, USA. Experimental data were checked for normality using the Shapiro–Wilk test. Where necessary, data were transformed through standard methods to meet the requirements of statistical analysis. In a second analytical step, a so-called insert-and-absorb algorithm was used to truthfully present all relevant significant differences for the main factors and interactions between the main factors. The algorithm was implemented using the SAS 9.4 macro% (Multi factors)based on the work of Piepho, 2012 [37]. The %MULT macro uses output generated from the MIXED, GLIMMIX, or GENMOD procedures. It allows up to three by-variables for factorial experiments but can process the least squares means for one effect only. If Least Squares Means (LSMEANS) are needed for several effects, the linear model procedure must be run several times, each time using only one LSMEANS statement with only one effect. It means each level of one main factor (e.g. IR) was compared separately for each level of the remaining two factors (e.g. AMF and Si) as pairwise comparisons. In our three-factorial analyses (IR, Si and AMF factors), the main effects of the experiment (IR, AMF, Si, IR×AMF, IR×Si, Si×AMF, IR×Si×AMF) were compared using a proc mixed model (MIXED procedures) in the SAS environment at a significance level of α = 0.05. LSMEANS of the main and interaction effects were determined.

#### **3. Results**

#### *3.1. Effect of Si and Inoculation with AMF on Plants Biomass And Root Colonization*

Mycorrhization or Si as single treatments did not significantly affect shoot biomass under well-watered (WW) conditions while the combination of both treatments resulted in a higher shoot biomass suggesting a synergistic effect between AMF and Si. In the plants exposed to mild drought (MD) and severe drought (SD) stresses, in contrast, Si and AMF as single treatments increased the shoot biomass; however, the effect of AMF was not significant in SD plants (Figure 1A). Root biomass was increased by the AMF treatment under WW conditions. Under MD and SD, in comparison, the effect of both Si and AMF as single treatments was significant on root biomass; the effect of AMF was much higher than Si particularly under MD (Figure 1B).

The relative water content decreased with the severity of D. Under WW conditions, there was no significant effect of Si or AMF as single treatments on RWC while the combination of both treatments resulted in higher RWC. In MD and SD plants, in contrast, the effect of single treatments was mainly significant (Figure 1C). The osmotic potential of the leaves and roots was affected by an inverse trend of RWC (Figure 1D). There was a significant interaction among the three main factors including IR, Si, and AMF on the shoot and root biomass, RWC, and osmotic potential where all decreased with the severity of D (IR factor) but were modified by Si and AMF applications (Figure 1).

**Figure 1.** The biomass of shoot (**A**) and root (**B**), the leaf relative water content (RWC) (**C**), and the osmotic potential (**D**) in strawberry plants at harvest after six experimental weeks under three irrigation regimes (IR): well-watered (WW), mild drought (MD), and severe drought (SD) without (−AMF) or with inoculation with arbuscular mycorrhizal fungus (+AMF) *Rhizophagus clarus* (Walker & Schüßler), in the absence (−Si) or presence of silicon (+Si, 3 mmol L−<sup>1</sup> Na2SiO3). The bars show the treatment means (4 replicates) ±SE of the mean. The interactions among the main factors are in the table (**F**); \*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.05, and ns is not significant (Tukey test, alpha = 0.05).

There was a low colonization percentage detectable even in –AMF plants, which might be caused by carryover of some fungal populations from the field-grown mother plants or from the peat culture substrate used for the preculture (Table 1). Interestingly, D decreased the hyphal and arbuscular colonization rates (%) in the –AMF plants while not influencing them in the +AMF ones (Table 1). The pairwise comparison indicated that the hyphal colonization percentage of +AMF plants was

increased by Si under all IR treatments. The same was true for the frequency of arbuscules that was significant even for the –AMF plants under MD and SD conditions. The frequency of vesicles increased in the –AMF plants under SD conditions. In the +AMF plants, a significant effect was observed under both MD and SD conditions. Si did not affect this parameter. Interestingly, inoculation with AMF decreased the frequency of vesicles in the WW while it increased in the MD and SD plants (Table 1).

**Table 1.** The root colonization rate (%) in strawberry plants at harvest after six experimental weeks under three irrigation regimes (IR): well-watered (WW), mild drought (MD), and severe drought (SD) without (−AMF) or with inoculation with arbuscular mycorrhizal fungus (+AMF) *Rhizophagus clarus* (Walker & Schüßler), in the absence (−Si) or presence of silicon (+Si, 3 mmol L−<sup>1</sup> Na2SiO3). The numbers show the treatment means (4 replicates) ±SE of the mean. Means with the same letters are not significantly different. The interactions among the main factors include \*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.05, and ns as not significant (Tukey test, alpha = 0.05).


#### *3.2. Effect of Si and Inoculation with AMF on the Leaf Gas Exchange Parameters*

The single application of Si or AMF did not influence the rate of photosynthesis under WW conditions. A significant effect of Si as the single treatment, however, was observed under the MD and SD conditions, and a significant AMF effect was observed under MD conditions. The combined application of Si and AMF, in contrast, increased the rate of photosynthesis under WW, MD, and SD conditions, and the highest photosynthesis rate was obtained under the combination of both treatments with a significant difference with each single treatment (Figure 2A). In the absence of AMF and Si treatments, SD decreased the transpiration rate. This parameter increased by AMF only under SD

conditions and by Si under MD and SD conditions. Under WW conditions, in contrast, the rate of transpiration was only increased by the combined application of Si and AMF (Figure 2B). The stomatal conductance showed a similar pattern to the rate of photosynthesis (Figure 2C). The sater use efficiency (WUE) decreased by D irrespective of the AMF or Si treatments. Significant effects of the single treatments were observed in the SD plants for Si and in both MD and SD plants for AMF, and the highest value of WUE was obtained in the combination of both treatments (Figure 2D). There was a significant interaction among the three main factors on photosynthetic activity, transpiration rate, and stomatal conductance. There was not any three-way interaction evident for water use efficiency. Significant differences were observed in IR, AMF, Si, and IR×Si (Figure 2).

**Figure 2.** The net photosynthesis rate (**A**), transpiration rate (**B**), stomatal conductance (**C**), and water use efficiency (**D**) of strawberry plants at harvest after six experimental weeks under three irrigation regimes (IR): well-watered (WW), mild drought (MD), and severe drought (SD) without (−AMF) or with inoculation with arbuscular mycorrhizal fungus (+AMF) *Rhizophagus clarus* (Walker & Schüßler), in the absence (−Si) or presence of silicon (+Si, 3 mmol L−<sup>1</sup> Na2SiO3). The bars show the treatment means (4 replicates) ±SE of the mean. The interactions among the main factors are in the table (**F**); \*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.05, and ns is not significant (Tukey test, alpha = 0.05).

#### *3.3. Effect of Si and Inoculation with AMF on the Concentrations of Osmolytes*

Under WW conditions, there was no effect of either AMF or Si on the proline concentrations (Table 2). Under MD and SD, in comparison, both Si and AMF treatments decreased leaf proline concentrations; a synergistic effect, however, was observed only under SD conditions (Table 2). The opposite trend of the proline concentration was observed in the root under SD, which was increased by Si and AMF applications where the combined application was not significantly different from the single application. There was a significant interaction among the three main factors including IR, Si, and AMF on the leaf proline concentration (Table 2). D conditions decreased the concentration

of proteins while increased the concentration of free amino acids (AA) in leaf and root tissues. The application of Si in the −AMF plants increased leaf protein concentrations under D (but not under WW) conditions while decreasing that of free AA. In the roots, in contrast, both protein and free AA concentrations increased by Si in the −AMF plants under SD conditions. Similar to Si as a single treatment, AMF application as a single treatment decreased the concentration of free AA in the leaves while increased that in the roots under SD conditions (Table 2). The total free AA concentration of the leaf was significantly affected by all two-way and three-way interactions, while there was not IR×AMF interaction regarding leaf protein concentration. For the roots, there was only an interaction of AMF and Si factors on protein concentrations and of IR and Si on free AA concentration (Table 2).

The concentration of soluble sugars increased under D conditions in both leaves and roots irrespective the AMF and Si treatments. Upon the application of Si and AMF, the soluble sugars concentration decreased in the leaves under MD and SD conditions while increased in the roots of SD plants. The lowest and the highest concentrations of soluble sugars was observed in the leaves and roots in the +AMF+Si plants, respectively. Under WW conditions, the effects of Si and AMF as single treatments were not statistically significant in the leaves and of Si in the roots. There was a three-way interaction among the main factors on shoot sugar concentrations (Table 2).

#### *3.4. Effect of Si and Inoculation with AMF on the Function of Enzymatic Antioxidant Defense*

The activity of CAT, SOD, and POD in the leaves and the activity of CAT and SOD in the roots were increased under D conditions irrespective the Si and AMF treatments (Table 3). The highest activity of antioxidative enzymes was observed in the combination of Si and AMF treatments (+AMF+Si). A significant effect of Si and AMF as single treatments was found in SD plants for all analyzed antioxidative enzymes while this effect in the leaves was not significant for POD in MD and for SOD and POD in WW plants. Among all analyzed leaf antioxidative enzymes, only SOD was significantly affected by a three-way interaction. In the roots, the effect of AMF on the CAT and SOD activity was higher than Si as single treatments. There was a significant interaction of the three main factors in CAT but not SOD activities of the root. The activity of root SOD was affected only by IR×AMF (Table 3).

In the absence of Si and AMF, MDA concentration as an indicator of damage to the membrane increased with increasing severity of D. Both Si and AMF treatments decreased the concentration of the leaf MDA that was observed only in D plants. AMF was more effective than Si as single treatment on the reduction of MDA concentrations; the lowest value was observed in +AMF+Si plants. A significant three-way interaction affected the leaf MDA (Table 3).

D treatment led to the accumulation of H2O2 in the roots that increased with increasing severity of stress. Si treatment decreased H2O2 concentration that was significant only in the D treatments. AMF inoculation caused a significant reduction of the H2O2 concentration only under D treatment that was significant in SD plants. The H2O2 concentration of the root was decreased by a significant interaction among IR, Si, and AMF factors (Table 3).

#### *3.5. Effect of Si and Inoculation with AMF on the Leaf Concentrations of Nutrients and Si*

The Si concentration significantly decreased in SD plants and increased by Si application in the presence or absence of AMF under WW and D conditions (Table 4). The effect of AMF on Si concentration was significant only in +Si plants under WW and in −Si ones under SD conditions (Table 4). The interaction effects between two (IR×AMF, IR×Si, and AMF×Si) and among three main factors (IR×AMF×Si) on Si concentration were significant (Table 4).

A significant effect of D on the leaf concentrations of Mn, Fe, and Cu was observed only in the SD treatment while leaf Zn concentration decreased under both MD and SD conditions (Table 4). Si and AMF treatments alone or in combination did not influence the concentrations of Mn, Fe, and Cu. However, Si and AMF significantly increased the leaf Zn concentration under MD conditions (Table 4). Furthermore, significant two-way (IR×Si) and three-way (IR×AMF×Si) interactions were observed for the leaf Zn concentration (Table 4).


*Agronomy* **2019** , *9*, 41

**Table 2.** The in the leaf and roots of strawberry plants at harvest after six

severe drought (SD) without (−AMF) or with inoculation

concentrations

 of proline (μg g −1 FW), total free amino acids (AA, μg g −1 FW), total soluble proteins (mg g −1 FW), and soluble sugars (mg g −1 FW)

experimental

 with arbuscular mycorrhizal

 weeks under three irrigation regimes (IR):

 fungus (+AMF)

*Rhizophagus*

 *clarus* (Walker & Schüßler), in the absence (−Si)

well-watered

 (WW), mild drought (MD), and


**Table 3.** The activity of catalase (CAT, μmol mg −1 protein min −1), superoxide

guaicol mg −1 protein min −1) and the

hydrogen peroxide (H2O2, μmol g −1 FW) in the roots of strawberry plants at harvest after six

concentration

 of

malondialdehyde

 (MDA, nmol g−1 FW) in the leaf and the activity of CAT and SOD and the

experimental

 weeks under three irrigation regimes (IR):

 dismutase (SOD, Unit mg −1 protein min −1), and peroxidase

 (POD, (μmol tetra

concentration

 of well-watered

**Table 4.** The concentrations of Si (%), Zn (μg g <sup>−</sup><sup>1</sup> DW), Mn (μg g <sup>−</sup><sup>1</sup> DW), Fe (μg g <sup>−</sup><sup>1</sup> DW), and Cu (μg g <sup>−</sup><sup>1</sup> DW) in the leaf of strawberry plants at harvest after six experimental weeks under three irrigation regimes (IR): well-watered (WW), mild drought (MD), and severe drought (SD) without (−AMF) or with inoculation with arbuscular mycorrhizal fungus (+AMF) *Rhizophagus clarus* (Walker & Schüßler), in the absence (−Si) or presence of silicon (+Si, 3 mmol L−<sup>1</sup> Na2SiO3). The numbers show the treatment means (4 replicates) ±SE of the mean. Means with the same letters are not significantly different. Interactions among the main factors are indicated as \*\*\* *p* < 0.001, \*\* *p* < 0.01, and \* *p* < 0.05 and ns as not significant (Tukey test, alpha = 0.05).


#### **4. Discussion**

Our results showed that the application of Si and AMF in strawberry might alleviate the adverse effects of D stress in a synergistic manner. Different mechanisms could be involved in this synergistic effect, including Si-mediated improvement of the carbon supply for fungi and likely an increase in the formation of arbuscules. Our results also provide evidence for the effect of Si and AMF on the improvement of strawberry growth under optimum growth conditions through an elevated photosynthesis and water use efficiency.

#### *4.1. Effect of Si and AMF on Growth and Photosynthesis of Plants under Water Stress*

Biomass production, water content, and photosynthetic activity of leaves decreased under D conditions in the strawberry plants of this work. Both the Si and AMF treatments alleviated the effects of D and increased leaf water content and photosynthesis rate, leading to a higher biomass production. The observations of gas exchange parameters indicated a D-induced decrease in CO2 assimilation caused by the closure of stomata. The application of Si and AMF increased net photosynthesis rate through an elevation of stomatal conductance. Our results on the effect of Si are in agreement with those of Ma, 2004 [38] for cucumber, Chen et al. 2011 [14] for rice, and Pilon et al. 2013 [39] for potato. Further research has shown that AMF significantly increased leaf area, carboxylation efficiency, chlorophyll content, net photosynthetic rate, and the photochemical efficiency of PS II under water

stress [40,41]. Although an improved stomatal conductance upon Si and AMF treatments resulted also in a higher transpiration rate, a greater stimulation of photosynthetic capacity than water loss led to higher water use efficiency in +AMF and +Si plants.

Despite lower photosynthesis activity, soluble sugars accumulated in the leaves of D plants following an impaired growth. It has been stated that water stress triggers sugar accumulation and leads to an adjustment of the rate of photosynthesis [42]. This accumulation of soluble sugars under water stress, in turn, causes an impaired plant metabolism by changing either the composition or the translocation of sugars in the leaves [43]. In the leaves, the concentration of soluble sugars decreased by AMF and Si treatments most likely because of the growth resumption and consumption of carbohydrates for biomass production. Thus, Si and AMF may modulate the accumulation of soluble sugars in water-stressed leaves in a negative feedback mechanism of biochemical limitations.

The same effect of D on the soluble sugars concentration was observed in the roots. However, in contrast to the leaves, the soluble sugars concentration in the roots increased by AMF and Si treatments. This increase may be resulted from an improved net CO2 assimilation and/or allocation of photosynthates to the roots and may, in turn, contribute to the stimulation of root growth under these conditions. Considering the osmotic effect of soluble carbohydrates, elevated soluble sugars pool may also improve root water uptake capacity from a dry substrate (see below).

#### *4.2. Effect of Si and AMF on the Water Status and Concentration of Organic Osmolytes*

The accumulation of organic osmolytes leading to an osmotic gradient with the environment, as a common response in plants under water stress [44], was observed in the strawberry plants in this work for proline, free AA, and soluble sugars, concomitant with the reduction of osmotic potential. The alleviating effect of AMF and Si, however, was not mediated by an osmo-adjustment, and the concentration of organic osmolytes rather decreased in the leaves of +AMF and +Si plants. These results suggest that the Si-mediated increase in leaf water uptake was not due to an increase in the osmotic driving force in strawberry plants under water stress. An increase in the leaf RWC was achieved apparently by an increased capacity for water uptake that in turn hindered triggering the stomatal closure and allowed the maintenance of a high photosynthetic capacity for supporting growth and dry matter production. Increasing levels of organic compounds under osmotic stress are usually thought to adversely affect growth because of the cost associated with their synthesis [45]. Thus, the method of stress alleviation of AMF and Si for an increase in water uptake capacity may be less expensive than the strategy of osmo-adjustment. This result is in contrast with our previous observation on tobacco plants showing a Si-mediated improvement of plant water status through the leaf accumulation of organic osmolytes including soluble sugars, free amino acids, and proline [13].

In contrast to the leaves, the root concentration of organic osmolytes increased by AMF and Si treatments, suggesting a different strategy for the adjustment of the water economy triggered by AMF and Si in the roots than in the leaves of strawberry. In tomatos, water stress did not change the root osmotic potential in Si-treated plants [46], and in cucumbers, the role of the osmotic driving force in the Si-mediated enhancement of water uptake was genotype-dependent [47]. Collectively, these results suggest different strategies for the improvement of water content and uptake capacity under osmotic stress in Si-treated plants depending on plant organ, species, and genotypes. There are reports on the increased root hydraulic conductance by Si, and the increase was attributed to the Si-mediated upregulation of transcription of some aquaporin genes [48].

Under D conditions, proline accumulated in the leaves while the application of AMF and Si reduced leaf proline concentrations. The accumulation of proline in the leaves under water stress is a well-documented phenomenon, but the role of proline in osmotolerance remains controversial. In some studies, the accumulation of proline has been correlated with stress tolerance [49], but other researchers suggest that proline accumulation is a symptom of stress impairment rather than stress tolerance [50]. Our results support the view that proline accumulation under stress is a symptom of stress and, thus, the Si-mediated reduction of proline concentrations is a sign of stress alleviation. Similarly, the AMF-mediated reduction of the proline concentration suggests that the AMF colonization of plants, to an extent, mitigated the effects of drought stress and reduced proline concentrations in leaves. These results are in agreement with a previous report [51].

An inhibited formation of proteins from amino acids, which could be judged by the accumulation of free AA concomitant with a reduced protein concentration, was observed under water stress of leaves. Both AMF and Si treatments caused the reduction of the free AA pool associated with an increase in soluble proteins. The accumulation of proteins helps the plant to maintain the water-status of leaves, reduce negative effects from active and reactive oxygen species [52] under severe and long-term drought, and maintain the water-status of leaves [10].

#### *4.3. Effect of Si and AMF on the Antioxidative Defense System*

Water stress caused the activation of antioxidative defense enzymes in the leaves and roots. However, this activation was not obviously sufficient for the protection of the plants against ROS that was reflected well in the increasing MDA concentrations in the D plants. The application of AMF and Si to the D plants similarly increased the activity of antioxidative defense enzymes (particularly of SOD). However, compared to the stress-induced activation of enzymes, it led to a decline of stress metabolites (MDA, H2O2). It may be suggested that AMF and Si contributed to the alleviation of oxidative damage not only by an elevated capacity of defense system but also through less production of the stress metabolites. It has been frequently shown that plants with higher root colonization with AMF exhibit greater enzymatic and non-enzymatic antioxidative defense systems activity [21,35] than non-inoculated plants. A clear biochemical link between Si and antioxidative capacity in stressed plants, however, has not yet been found. It has been argued that the biochemical enhancement of antioxidant defense mechanisms is a beneficial, physical result of Si-deposition in the cell membrane [4]. Several investigators argue that the Si-induced increases in the activity of antioxidant enzymes and the levels of non-enzymatic antioxidative substances in plants exposed to abiotic stress lead to an implication of Si in the plant metabolism [46,47,53]. According to Ma and Yamaji, 2006 [54], the Si-mediated increase in antioxidant defense abilities is a beneficial result of Si rather than a direct effect.

#### *4.4. Effect of Si and AMF on Plants Nutrients Uptake*

Water stress reduced the nutritional status of plants, causing deficiencies in Zn, Mn, Cu, and Fe, particularly in more severely stressed plants, but was already partially detectable under MD conditions. The application of AMF and Si led to an improved micronutrient status, equaling or even exceeding the critical deficiency thresholds of Fe, Zn, and Mn. Maksimovi´c et al., 2012 [55] and Pavlovic et al., 2013 [56] found that Si application increased the uptake of Zn and Fe at low concentrations on the rhizoplane. In this work, the effect of Si on nutrient acquisition under D stress was more pronouncedly observed for Zn than other micronutrients. This effect is likely mediated by stimulation of root growth [57] that increases the spatial availability of Zn for plants [58] or by an enhanced concentrations of low molecular weight organic compounds by Si (e.g., citrate) that might contribute to metal uptake and transport from root to shoot, thereby diminishing deficiency symptoms [59]. The higher Zn uptake after the application of Si under D conditions is also likely to result from the effect of the Si on Zn transporters. It has been observed that Si increases the expression levels of the Fe transporters (IRT1 and IRT2) [56] belonging to the ZIP (Zrt/IRT-like protein) family that include also Zn transporters. A limited Zn/Mn availability in the D plants of this work disbalanced Zn/Mn-dependent ROS detoxification systems produced excessive ROS accumulation and caused oxidative damage. The excessive production of ROS can promote oxidative degradation of indoleacetic acid, as was demonstrated in Zn-deficient maize plants under cold stress, which is restored by the Si application [60]. Auxin deficiency is an important factor for growth limitation in Zn-deficient plants [60]. Regarding the role of AMF, plants with a higher root colonization by AMF are more efficient in the uptake and translocation of macro- and micronutrients to the shoot than non-inoculated plants [61,62].

#### *4.5. A Synergistic Effect of Si and AMF*

The synergistic effects of Si and AMF as a combined treatment (+AMF+Si) on the low-Si medium used as growth substrate in this work may partly be related to the contribution of AMF to Si uptake observed in this work and in other works [63–66], the Si-induced stimulation in root growth that in turn promotes AMF colonization in the combination treatment, and the effect of Si on an increase in the root soluble sugars pool, which is important for supporting AMF entry, and further establishment in the roots are other probable mechanisms. The mycorrhizal association is completely dependent on the organic carbon supply from their photosynthetic partner since 4 to 20% of the C fixed through photosynthesis is transferred to the AM fungi [67]. Similarly, the Si-induced increase in the percentage of arbuscules formation observed in this work may result from the improved root growth, the enhancement of nutrients uptake and transfer within the plant, and the induced photosynthesis rate that provides more carbon sources for the fungi partner. A significant increase in the percentage of arbuscule formation in response to Si added to a sand substrate has been reported for Banana [65]. In contrast to our results, in a report on the effect of Si on mycorrhizal chickpea [66], an increase was observed in the salinity tolerance by both Si and AMF, but a synergistic effect was not detected.

Another possible explanation for the synergistic effect of AMF and Si is a Si-induced alteration of the AMF-hosts metabolism. In another report, the authors reported an enhanced metabolism of phenolic compounds (flavonoid-type phenolics) influenced by Si [68]. Phenolic compounds such as flavonoids may play a role in facilitating the interactions between fungus and host [69] and have some positive effects on fungal growth parameters, e.g., hyphal growth and branching, germination of spores [70], and formation of secondary spores. Moreover, they play a role during the fungal invasion and arbuscule formation inside the root [71]. The recent identification of strigolactones as host-recognition signals for AM fungi, however, raises the question about the role of flavonoids as general signaling molecules in AMF-plant interactions [72].

#### *4.6. Effect of Si and AMF on Plants Growth in the Absence of Stress*

In the well-watered (WW) strawberry plants grown as unstressed controls, Si treatment caused a significant increase in the shoot growth, where the highest biomass production of the shoot was observed in the +AMF+Si treatment. This Si effect under WW conditions disagrees with some of the previous reports [4] describing the beneficial effects of Si on plant growth only under stress conditions. The Si application has been frequently related to the stimulation of enzymatic defense strategies involved in the detoxification of ROS [12]. However, the lower growth of –Si plants under WW conditions in our experiment was not associated with significant changes in the physiological stress indicators, such as MDA and proline. Furthermore, the positive effects of Si on plant growth under WW conditions could also not be attributed to the increased concentrations of the micronutrients. Even in −Si control plants, the nutritional status exceeded the critical levels reported for the respective micronutrient deficiencies. The unexpected positive effects of Si supplementation on the growth of WW plants may be attributed to a significant improvement of the leaf photosynthesis and water content. Considering a higher leaf area in the +Si plants, it is expected that the photosynthesis of the whole strawberry plants is considerably higher than the –Si ones. Improved Si supply may increase the physical stability of the leaves, leading to a more horizontal orientation of the leaves and thereby improving photosynthetic efficiency as previously reported for cucumber [73]. A recent unified model, so-called apoplastic obstruction hypothesis (74), argued for a fundamental role of Si as an extracellular prophylactic agent as opposed to an active cellular agent. In this model, Si, rather than being involved directly in the regulation of gene expression and metabolism, regulates plant metabolism through a cascading effect [74]. Here in our work, the highest growth improvement was observed in the WW plants under the combination of Si with AMF treatments because a Si-induced shoot growth was associated with an AMF-mediated increase in the root growth. The soil-free culture systems that are based on perlite or vermiculite and are being widely used in horticultural practices and are characterized by low plant availability of Si [75]. Thus, the significant effect of Si supplementation in

plants cultivated on these potting substrates, in contrast to the soil-grown plants, could be related to supply of plants with Si and meeting their requirement at least in the accumulator species.

#### **5. Conclusions**

The findings of the present study suggest that the major factors determining the sensitivity of strawberry plants to D stress are a reduction of micronutrients uptake, particularly Zn, a reduced photosynthesis rate and protein level, a ROS overproduction, and the consequent membrane damage. In this context, the protective effects of Si and AMF treatments seems to be related to an improved micronutrients status, an increased expression of the enzymatic antioxidative defense system, and an elevated water uptake capacity and use efficiency. Our results indicate that Si and AMF alleviated water stress in a synergistic manner. The AMF colonization and formation of fungal structures were increased by Si, and, in turn, Si uptake was increased upon mycorrhization. Other probable interactions at the metabolic levels need to be elucidated. A conceptual model of these proposed roles of Si and AMF, mediating D tolerance in strawberry plants is presented in Figure 3. Our results provide a theoretical basis for the application of Si fertilizers and AMF in water-conserving irrigation systems for strawberry cultivation under field conditions and for greenhouse production, particularly in the soil-free culture systems.

**Figure 3.** A conceptual model representing the effect of Si and AMF in drought-stressed strawberry plants reverting plant performance to well-watered conditions. Si and AMF 1) enhanced growth and photosynthesis of plants, 2) regulated the water status and concentration of organic osmolytes, 3) promoted the antioxidative defense system, 4) increased plants nutrients uptake, 5) had synergistic effects, and 6) enhanced plant growth even in the absence of stress. Abbreviations: AQP: Aquaporin, AMF: Arbuscular Mycorrhizae Fungi, IAA: Indole 3-Acetic Acid, ROS: Reactive Oxygen Species, SOD: Superoxide Dismutase, POD: Peroxidase, APX: Ascorbate Peroxidase, CAT: Catalase, MAD: Malondialdehyde, NADPH: nicotinamide adenine dinucleotide phosphate hydrogen, NADP+: Nicotinamide adenine dinucleotide phosphate.

**Author Contributions:** R.H. and N.M. conceived and designed the experiments. N.M. conducted the experiments, performed the analyses, and collected the data. N.A., R.H., and G.N. provided the facilities and advised on the preparation of materials. N.M. wrote the manuscript. N.M. and T.E.H. did the statistics evaluations. G.N. and R.H. read and edited the manuscript. All authors approved the final manuscript.

**Funding:** This research was funded by the University of Tabriz, Iran [Ph.D project].

**Acknowledgments:** The University of Tabriz, Iran, is greatly appreciated for its financial support. Thanks to Zarrin Eshaghi (Payame Noor University, Mashhad) for giving support to the analytical facilities and to Filippo Capezzone (Hohenheim University, Biostatistics department) for the support with SAS and statistical analyses. Very special thanks to Hans Lambers (University of Western Australia) for reviewing the manuscript.

**Conflicts of Interest:** The authors declare no competing financial interests.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **What Has Been Thought and Taught on the Lunar Influence on Plants in Agriculture? Perspective from Physics and Biology**

#### **Olga Mayoral 1,2,\*, Jordi Solbes 1, José Cantó <sup>1</sup> and Tatiana Pina 1,\***


Received: 30 April 2020; Accepted: 17 June 2020; Published: 2 July 2020

**Abstract:** This paper reviews the beliefs which drive some agricultural sectors to consider the lunar influence as either a stress or a beneficial factor when it comes to organizing their tasks. To address the link between lunar phases and agriculture from a scientific perspective, we conducted a review of textbooks and monographs used to teach agronomy, botany, horticulture and plant physiology; we also consider the physics that address the effects of the Moon on our planet. Finally, we review the scientific literature on plant development, specifically searching for any direct or indirect reference to the influence of the Moon on plant physiology. We found that there is no reliable, science-based evidence for any relationship between lunar phases and plant physiology in any plant–science related textbooks or peer-reviewed journal articles justifying agricultural practices conditioned by the Moon. Nor does evidence from the field of physics support a causal relationship between lunar forces and plant responses. Therefore, popular agricultural practices that are tied to lunar phases have no scientific backing. We strongly encourage teachers involved in plant sciences education to objectively address pseudo-scientific ideas and promote critical thinking.

**Keywords:** plant growth; agriculture; traditions; pseudo-science; lunar phases; physics; biology; education

#### **1. Introduction**

This paper addresses the existing dichotomy between what science shows regarding agriculture protocols and past and current agricultural practices in much of Europe and Latin America. More specifically, it focuses on some pseudo-scientific questions and beliefs that impregnate a large part of agricultural traditions and agronomic practices according to which certain lunar phases encourage plant growth while others compromise their development. These beliefs share our lives with scientific and technological advances not reached ever before.

After introducing the main features of the Moon and its phases, as well as the factors that determine plant growth, this study continues with a brief historical overview about what has been thought from the agricultural sector concerning the lunar influence on plants and crops. In this overview, we have included references to both earlier ages and the most recent trends within agriculture, such as biodynamic agriculture, which bases part of its operating on the close relationship between the Moon and plant growth.

Then, we analysed monographs on botany, plant biology and physiology—considered as texts of consolidated science—, searching for any mention about the Moon being a factor influencing plant growth. At the same time, we reviewed physics handbooks, focusing on which aspects or natural processes the Moon has influence on, looking for any mention of some type of effect on living beings and, specifically, on plants.

The paper concludes with a reflection on the implications of the different existing visions lasting over time within both the field of agriculture and citizenship in general, being part of the global desirable scientific literacy.

#### *1.1. The Moon*

This section covers the basic key aspects about the Moon required to understand most of the arguments detailed in the subsequent analysis of both scientific literature and explanations provided by some agricultural sectors.

#### 1.1.1. The Gravitational Pull

The Moon is the only natural satellite of our planet describing an elliptical orbit around it with a semi-major axis of 384,000 km, an eccentricity of 0.0549 and an angle of 5◦9 relative to the ecliptic plane. The Moon takes 29.5 days to orbit around the Earth and return to its analogous position with respect to the Sun and the Earth (lunar month or synodic month) [1]. However, it takes 24.8 h for a specific location on the Earth to rotate from one exact point beneath the Moon and back (lunar day) [2,3]. The combined action of these two cycles (lunar month and day) has different effects on the Earth such as changes in tides and in the intensity of illuminance.

So, what is the explanation for the Moon's influence on tides? Tides are due to the difference in gravitational pull (or gravity acceleration) between the part of the oceans which are nearest (A) and farthest (B) to the Moon and the relative acceleration in relation to the Earth's centre of mass (CM) in such points (Figure 1).

**Figure 1.** Representation of how tides are produced. In the drawing, gM represents the acceleration in the Earth's centre of mass (CM) caused by lunar attraction; gA and gB are, respectively, the accelerations of points A and B located at both ends of the Earth's surface over the Earth–Moon line, and grA and grB are the acceleration in relation to the Earth's CM. Modified from Martínez et al. [1].

From the gravitational point of view, the effect of gravity on the Earth's CM produced by the Moon (gM) can be calculated by means of the expression gM = Gm/r2, being G the universal gravitational constant, r the distance Earth–Moon (E–M) and m the mass of the Moon. That is to say, the value of the Moon's gravity on the Earth's surface is approximately 2,951,800 times lower than the Earth's gravity (gM <sup>=</sup> 3.32 <sup>×</sup> <sup>10</sup>−<sup>5</sup> ms<sup>−</sup>2). Therefore, the gravitational pull is negligible. Accordingly, the Sun's gravity (gS) on the Earth is 177 times greater than the Moon's (gS <sup>=</sup> <sup>g</sup>/<sup>1627</sup> <sup>=</sup> 177 gM <sup>=</sup> <sup>6</sup> <sup>×</sup> <sup>10</sup>−<sup>3</sup> ms<sup>−</sup>2).

We can also calculate the Moon's gravity in point A (gA ≈ gM(1 + 2 R/r) being M the mass of the Earth and R its radius) and in point B (gB ≈ gM(1 − 2 R/r) and their relative accelerations in A (grA) and B (grB) regarding Earth's CM (grA = gA <sup>−</sup> gM <sup>≈</sup> 2 GmR/r<sup>3</sup> = 2 RgM/r and grB <sup>=</sup> gB <sup>−</sup> gM ≈ −2 GmR/r<sup>3</sup> <sup>=</sup> <sup>−</sup>2 RgM/r, respectively) (Figure 1). From these calculi, we observe that the relative acceleration in relation to CM depends on the distance between A and B and on the cubed distance between the Earth and the Moon, instead of squared distance, rendering identical values in both A and B points (as r = 60 R, its value is 10−<sup>6</sup> ms−2, 30 times lower than gM) but in the opposite direction: the relative acceleration in point A (grA) is directed towards the Moon and in point B (grB) towards the opposite direction [1,4]. Therefore, there will be high tide in A and B, and low tide in those points located at 90◦ (Figure 2).

**Figure 2.** Diagram to illustrate the Sun (S)–Earth (E)–Moon (M) configuration regarding neap and spring tides. Source: designed by the authors.

For this reason, although the Sun's gravity on the Earth is greater than the one from the Moon, as tides are influenced by the inverse of the distance to cube (1/r3), its effect is lower than the one from the Moon, as its distance is much greater. And as tides depend on the size of the object, in the Mediterranean Sea, for instance, these are negligible due to the fact that it is a semi-enclosed, shallow and small sea (with an average depth of R = 1500 m). In contrast, as all the oceans are communicated, we can consider their size being the size of the Earth and, therefore, tides are apparent. In this sense, the tidal effect of the Moon over a 2 m height living being located on the Earth is about 1000 times lower (tidal acceleration <sup>=</sup> 2 gMh/<sup>r</sup> <sup>=</sup> <sup>3</sup> <sup>×</sup> <sup>10</sup>−<sup>13</sup> ms<sup>−</sup>2) than the effect produced by a mass of 1 kg at 1 m height above it (2.67 <sup>×</sup> <sup>10</sup>−<sup>10</sup> ms<sup>−</sup>2) [5].

Thus, because of the daily rotation of the Earth, the tides rise and fall twice each lunar day in most coastal areas and estuaries at intervals of approximately 12.4 h (tidal cycles) reflecting the lunar 24.8 h day. The amplitude of successive tides is also modulated every 14.77 days, or semi-lunar cycle. So, the highest tides, or spring tides, take place when the Sun and the Moon are aligned with the Earth (i.e., full and new moon), and the lowest, or neap tides, occur when the Sun–Earth axis and the Moon–Earth axis are at right angles (90◦) to each other (i.e., first and third quarter) (Figure 2) [2,6]. These tidal forces due to the Moon and the Sun are also observed in the atmosphere and the Earth's crust [7].

#### 1.1.2. Illuminance

The moonlight we see from the Earth is the sunlight reflected on the greyish-white surface of the Moon. Since the Moon orbits the Earth and the Earth orbits the Sun, the fraction of the Moon we see changes along the lunar month giving rise to the lunar phases, being new moon, first quarter, full moon and third quarter the main ones. The illuminance (defined as the amount of luminous flux striking a surface per unit area) varies depending on the lunar phase [2,8]. In the case of the Moon, as endpoint cases we can find 0.001 lx for a new moon and 0.25 lx for a full moon to 0.01 lx for a crescent or waning moon (Table 1).


**Table 1.** Illuminance according to the Moon phase.

Source: adapted from RCA Corporation [9] and Schlyter [10].

As it can be seen in Table 1, the Moon's maximum illuminance is 128,000 times lower than the minimum of sunlight on an average day or 400,000 times lower than the maximum of sunlight on an average day.

#### *1.2. Factors Influencing Plant Growth and Development*

The revision carried out considers plant growth and development from a holistic point of view. This implies all those changes in structure and function of plants and their parts, the course of genesis, assimilation, growth and development, as well as environmental, physiological and chemical modifications, maturation and decline [11–15].

Plant growth and development is regulated by both endogenous and external factors [12,14]. Regarding endogenous factors, phytohormones are in charge of the coordination of metabolic and developmental processes at the molecular and cellular level. Phytohormones can be divided into two groups depending on their functions: (i) those involved in growth-promoting activities; (ii) those in charge of responding to wounds or to biotic and abiotic stresses [16]. Synthesis or changes in the concentration of these phytohormones transduce the perception of environmental stimulus (i.e., radiation, photoperiod, temperature, gravity or stresses as cold, heat, drought or flooding). However, which plant hormones will be triggered will depend on the plant developmental state, the type of external stimulus, the part of the plant exposed, when this stimulus arrives, etc. [14]. Phytohormones, together with external factors, can activate growth and differentiation processes and allow the synchronization of plant development and seasonal changes. Furthermore, they also regulate plant growth (intensity and direction), the metabolic activity and the storage and transport of nutrients. All these endogenous factors are determined by endogenous genetic components (genome structure and gene expression, i.e., plant genotype) [17].

The growth and development of plants can also be affected by external factors such as quality, intensity, direction and duration of radiation, temperature, position with relation to Earth's gravitational field and stresses conferred by wind, water currents or snow cover, apart from other chemical influences. These external factors can initiate, complete and regulate the timing of developmental processes (inductive mode of action) but can also act quantitatively (by altering the speed and extent of growth) and formatively (by affecting morphogenesis and tropisms) [14]. These external factors are the ones which might be affected by a potential effect of the Moon—specifically, the gravitational and illuminance effects—.

Other authors propose to split the factors that determine quality and quantity into biotic and abiotic factors [18,19]. Within the biotic factors, we find arthropods, nematodes, bacteria, fungus and viruses as well as their relationships with other plants and organisms which can be competitive, mutual or parasitic types, among others [20]. In addition, within the abiotic factors, we find soil composition, salinity, pH, temperatures, pollution, humidity (water), wind and ultraviolet radiation, among others. The interaction of biotic and abiotic factors will determine plant growth, development, and productivity. Understanding their interactions is essential in agriculture when searching for the ideal growth conditions for each particular plant. In this sense, stress physiology research is very valuable, as it focuses on whether the full genetic potential of plants will be fulfilled and if plants will attain maximal growth and reproductive potential depending on different factors [21]. In particular, the study of abiotic stress originated from excess or deficit in the physical, chemical and energetic conditions to which plants are exposed provides farmers with guidelines for optimizing their harvests.

The references to the potential influence of the Moon on plants will have to be searched considering this influence as an abiotic factor. The excess or deficit of this factor should be studied taking into account that the Moon is always present, so the search should focus on those moon-derived sub-factors that can undergo substantial changes. The indirect possible effect of the Moon on the biotic components interacting with plants is a matter which falls outside the scope of the revision carried out in this paper.

#### **2. What Has Been Thought on the Influence of the Moon on Plants**

This section focuses on all those aspects related to agriculture that, according to some traditions, are determined by the Moon. To do so, we have developed a brief overview of what has been thought and written throughout history about the influence of the Moon on living beings and, in particular, plants. This analysis addresses manuals which have been used and are still used in certain agricultural sectors and information present on websites related to agriculture, gardening, agricultural machinery and so on. A special section is dedicated to biodynamic agriculture which links plant growth and lunar phases.

#### *2.1. Brief Historical Overview*

The Moon and the Sun hold a significant place in many mythologies and popular legends throughout the world. In particular, beliefs regarding the relationships between lunar phases and human and other organisms' behaviour are as ancient as human cultural heritage but have hardly ever found any solid scientific support [22].

Assertions concerning the existence of repetitive cycles in the Moon (phases), the Sun (day/night, solstices, and equinoxes/seasons), and Sirius (its heliacal rising) were extremely useful to develop lunar, lunar–solar and solar calendars, and to predict eclipses—just as it happened in the Egyptian, Babylonian, Greek or Chinese world. Such knowledge was continued in different cultures, mainly the Arab or the Mayan, the Aztec, and the Inca in America [23,24]. It is known that the Mayan carried out thorough observations of natural events, finding certain cyclical repetitions which allowed making predictions and organising when to sow or harvest [25].

Botanists and herbalists from the seventeenth century, such as Nicholas Culpeper (1616–1654), believed that plants and ailments were determined by constellations. The Sun ruled our heart, blood circulation and spine, while the Moon had influence on growth, fertility, breasts, stomach, uterus and menstrual flow. In fact, all the body fluids, as the tides, were controlled by the lunar phases. This was the prevalent belief at that time, since astrology was broadly accepted as the key to understanding the universe [26].

Surprisingly, these beliefs are still active, as shown by Phillips [26] in his *Encyclopaedia of Plants in Myth, Legend, Magic and Lore* which includes more than 200 entries linking different plant species or genera with stars or natural elements. This author states, as we have been able to check along with personal interviews made in the agricultural and rural world of the Iberian Peninsula, that garlic (*Allium sativum* L.) has been strongly associated with the Moon, and it was thought to grow stronger as the Moon waned. However, Navazio [27], in his manual dedicated to the organic seed grower, makes no mention of the Moon as an element to consider. Potato growing (*Solanum tuberosum* L.) is also supposed to be influenced by the Moon. According to different beliefs this underground crop should be planted during the black moon, that is to say, when it is waning [26]. But, once again, Navazio [27] does not mention the need to consider this aspect in the organic cultivation. Other examples would be the white clover (*Trifolium repens* L.), which has to be seeded by the darkness of the Moon or "no-Moon"—that is during the 24 h between the waning moon and the crescent moon—if you want it to grow, since if it is seeded under the moonlight, it will not sink into the ground [26]. Or corn (*Zea mays* L.), the seeds of which must be planted by moonlight in order to obtain a good performance [26].

Anglés Farrerons [28], in his work *Influence of the Moon on Agriculture and Other Topics of Main Interest for the Farmer and People from the City*, collects all the existing beliefs among elder farmers regarding the Moon. He dedicates specific chapters to the vine and the wine, the fruit growing, the cereals, the olive tree, several horticultural crops such as chard (*Beta vulgaris* var. *cicla* (L.) K.Koch), artichoke (*Cynara cardunculus* var. *scolymus* (L.) Benth.), garlic, celery (*Apium graveolens* L.), onion (*Allium cepa* L.), etc. Anglés Farrerons [28] also focuses on tree felling, forage harvesting, influence of animal manure or weather forecast. According to these traditions, he states when to sow, prune, harvest, etc., depending on the Moon phase and the crop, being true nonsense in some cases, just as the author suggests in his introduction.

Another work that requires special attention is that of Restrepo [29], a Brazilian agronomist who reflects the beliefs from Latin America and the Caribbean Area. He provides an interesting revision of the calendars of the ancient people and cultures as well as an extensive description of when to carry out all the agricultural practices (e.g., sow, layer, graft, prune, transplant) based on whether they are annual or perennial plants, vegetables, cereals and grains, tubers, bulbs and rhizomes. He also includes a description of how lunar phases and Moon illuminance affect the movement of the sap in plants (Figure 3).

**Figure 3.** Explanation of how lunar phases affect sap dynamics in plants according to Restrepo [29]. Redrawn and translated from Restrepo [29].

Which is the cause–effect explanation proposed to link lunar phases and sap movement? Restrepo [29] links it with the tides:

"Therefore, in certain positions of the Moon, the water from the oceans rises to reach a maximum height, and then goes down to a minimum level, maintaining this oscillation regularly and successively. It has also been checked that this phenomenon makes itself felt in plant sap". (Translated from Restrepo [29]).

It has already been shown that the effect of the tide of the Moon ona2m height living being is absolutely negligible (3 <sup>×</sup> <sup>10</sup>−<sup>13</sup> ms<sup>−</sup>2), compared to the Earth's gravity (9.8 ms−2) [5]. But considering the tides, there are two high tides and two low tides each day, so if the tide caused any effect on a plant, there should be two sap rises and falls per day and none with the lunar phases. If the latter wanted to

be introduced, we have already seen that at both new moon and full moon, the tides are a bit stronger due to the fact that the Sun and the Moon are aligned (as the Sun is much farther away, its effect on the tide is much lower), but the effects of the tide are symmetrical making the water rise (Figures 1 and 2). Yet, to make matters more contradictory, Restrepo [29] assigns them different effects: the full moon takes up to the leaves the waters of the plant, and the new moon takes them to the roots. On the other hand, the illuminance is the only thing that surely varies with the Moon phases (Table 1), but it does not generate any force that can cause the movement of the sap.

Finally, it has to be pointed out that there are beliefs and practices contained in many different manuals which we did not pretend to either analyse or introduce in detail in this paper. Only as an example, we outlined best sellers, such as *The Secret Life of Plants* [30], in which certain physical, emotional and spiritual relationships between plants and our species are explained in such an appealing manner that have clearly helped to strengthen several pseudo-scientific beliefs among society and many farmers. This best seller has been explicitly refuted by texts, such as *The Not-So-Secret Life of Plants*, in which the historical and experimental myths about emotional communication between animals and plants are put to rest by researchers such as Galston and Slayman [31] or Horowitz et al. [32].

#### *2.2. Agricultural Astronomy Manuals and Websites*

Some authors summarise the situation such as follows [26]:

"There has been a certain amount of interest in planting according to the phase of the Moon. The basic premise being that 'above ground crops' should be planted in the light of the Moon, i.e., on the days between the new moon and the full moon. 'Below ground crops' must be planted in the dark of the Moon, that is between the full moon and the next new moon. Refinements on this require that leaf crops are planted at the new moon and fruit crops or flowers planted at the full moon".

Apart from the agricultural traditions that could explain the use of the Moon as a calendar to organise the crops, the time of seeding, harvesting, reaping, etc., and the advice given by some authors on an individual basis, some companies have taken a step forward by developing a range of documents and even manuals appearing in the guise of scientific advice, which raise popular wisdom to the category of regulated recommendations [33–38]. Such manuals are used as reference books by many farmers at the small and large scales, and they offer a scheduled sequence of agricultural activities and, in many cases, advice regarding health care on the basis of the phases of the Moon and its ascending or descending position in the sky. But in this case, its influence is not obvious since neither gravitation nor illuminance vary.

We can also find these recommendations in the area of gardening with titles such as *Gardening by the Moon Calendar* [39]. This book provides guidance based on the following statements:

"The best rate of germination is achieved just before a full moon, when moonlight and the Moon's gravitational pull are both at their maximum, grafting should be done on a waxing moon, because sap rises in plants during this period and this will help a graft to establish, pruning should be done on a waning moon, because the sap is now falling, and this will help cut surfaces to heal quickly and crops for storage should be harvested while the Moon is waning".

Apart from the Moon, it includes elements about astrology to guide the practice: "the planting of fruit trees and bushes should be done when the Moon is passing through a fire constellation". Besides, this author links the effectiveness of the response to the planetary influence on cultivating without chemicals, pointing out that chemical products desensitize agricultural land.

Many entries to websites link the influence of the Moon to the biodynamic agriculture and the zodiac, which has nothing to do with it since the zodiac are the constellations where the ecliptic passes by (the apparent path of the Sun among the fixed stars).

Some companies within the forestry, agriculture and gardening area also dedicate sections to providing advice on tasks related to the handling of vegetables, fruit trees, etc., on their websites (Table 2).

**Table 2.** Summary of the tasks recommended by a multinational company supplying forestry, agriculture, and gardening machinery according to the lunar phases.


Source: modified and translated from [40].

Moreover, this link between lunar phases and different aspects of cultivation rank highly in major search engines on the internet and in database image repositories, which raises many of the same ideas as Restrepo [29]—that lunar gravity changes according to the phases is the only way to explain popular beliefs concerning the influence of the Moon on plant growth. However, illuminance is the only thing that varies according to the phases, as it can be seen in Table 1. The Moon's gravitational pull does not generate any force able to cause sap movement.

#### *2.3. Biodynamic Agriculture*

Part of the traditions regarding the Moon have been incorporated into biodynamic agriculture, an agricultural management system which is mainly based on the fact that the astronomical bodies influence crop production. As in other forms of organic farming, the use of industrial fertilizers, pesticides and herbicides is avoided. However, the difference lies in the use of plant and mineral preparations as additives to compost and soil sprays—"biodynamic preparations"—and in following a planting schedule for cultivation, sowing and harvesting based on cosmic forces and rhythms and, particularly, on Moon rhythm (Table 3) [41,42].


**Table 3.** Practices and products used in organic and biodynamic agriculture.

Source: modified from Chalker-Scott [41].

This variant of organic agriculture, initiated in a series of lectures given by Rudolf Steiner [43] in 1924, is considered by some authors as an alternative approach to modern agriculture (see review in Brock et al. [44]), while others consider it as not being a science-based practice (see review in Chalker-Scott [41]). The latter brands it as a scam of great implantation in countries as advanced as Germany where it has its origin and from where Brussels is pressured to accept its principles. The restoration of soil quality, of the "harmony" of ecosystems, and of biodiversity can be pointed out as the main objective of biodynamic farmers. The US website for biodynamic certification marks an update of the Moon phases with a quotation from the Natural History of Pliny the Elder (23–79 CE) —the first-century Roman naturalist who wrote extensively about tides—, explaining that the Moon

"replenishes the Earth; when she approaches it, she fills all bodies, while, when she recedes, she empties them" [45].

Kirchmann [46] suggests that biodynamic agriculture has a mystical origin (called spiritualistic research by Steiner, and based on mediation and clairvoyance) that drove Steiner's research to reject scientific inquiry because as he explained in the sixth lecture of the course, "We do not need any confirmation by circumstances or by external methods. Spiritualism is an extension of scientific thought broadening the prevailing one-sided scientific view, being true and correct". A good example of this can be found in what Rudolf Steiner [47] wrote in 2004 insisting on the special care that should be taken when teaching questions about "moon forces", since conventional science considers them pure superstition or mystical fantasy, being a truth that is still difficult to talk about openly. However, Kirchmann [46] maintains that "Steiner's predictions that can be scientifically tested have been found to be incorrect".

#### **3. What Has Been Taught on the Influence of the Moon on Plants? Analyses of Handbooks and Scientific Literature**

This section deals with all those aspects that are known and taught in the agronomic and biological background in relation to plant development. It also analyses the physics books that explain the influence of the Moon on our planet with the intention of clarifying whether they mention a possible relationship with plant development. Consequently, specific sections are devoted to those factors that could depend on or have an influence from the Moon, concretely in relation to the effect of gravity and the light reflected by it, both from the point of view of biology and physics. Likewise, the basic books on botany and plant physiology have been revised, paying special attention to those factors that are determining or causing stress to the development of plants.

The revision of the handbooks has been complemented with the information gathered from the analysis of different scientific articles published in data repositories, such as Web of Knowledge, Scopus or Google Scholar, using the keywords "Moon and plants" or "lunar and plants".

#### *3.1. What Handbooks Say from the Perspective of Physics and Biology*

According to traditional beliefs, the influence of the Moon on plant growth is attributed, among other factors, to the attractive forces that the satellite exerts on the Earth and more specifically on its waters. The gravitational theory of the Moon could be attributed for the first time to Kepler (1571–1630), who claimed that the ocean tides were produced by a hidden force from the Moon. Kepler believed it was due to the affinity that the Moon had for water which was one of the four basic elements [48] in [8]. Gravity was also recognised as an agent of lunar influence with the publication of "Principia" by Newton (1643–1727).

The analyses of various physics textbooks (Table 4) commonly used in science and engineering courses reveals that the term Moon appears in most of them linked to different concepts such as the distance from the Earth (as it was calculated in ancient times or as it is calculated today, with laser telemetry), the Moon's gravity, tides, etc. With regard to the origin of tides, there are many possibilities: (i) it is not approached [49]; (ii) it is introduced in a qualitative way [50,51]; (iii) the exact dependence of R/r3 is provided where r is the distance Earth–Moon, and R is the size of the object on which the tides act—in the case of the oceans, the Earth's radius [5]; (iv) locally, high tides are shown as an effect of the resonance [52] or tidal applications to produce energy are explained [53]. In order to find correct demonstrations of the tides, we have to deal with books on Astronomy (e.g., [4]) which, due to their extraordinary specificity, are beyond our general review.

Another factor that should be considered when approaching sap movement in plants is capillary action or capillarity, described as the spontaneous ability of a liquid to flow against gravity in a narrow space such as a thin tube or pipe (in plants, vascular tissues as xylem and phloem). This rising of liquid is the outcome of two opposing forces: cohesion (the attractive forces among similar molecules or atoms) and adhesion (the attractive forces among dissimilar molecules or atoms). In our case, the contact area between the particles of the liquid and the particles forming the tube. Capillarity is high when adhesion is greater than cohesion and vice versa. There is another important factor in capillarity, which is the contact area, dependent on the diameter of the tube (i.e., vascular tissue). Capillarity interacts with other forces, as gravity, which should be included when considering possible gravitational effects of the Moon on plants. In this sense, Jurin's law is usually introduced, giving information on the height (h) reached when balancing the weight of the column of a liquid and the force h = 2 γ cos θ/ρgr, where γ is the surface tension (Nm<sup>−</sup>1), θ is the contact angle, ρ is the density of the liquid (kgm<sup>−</sup>3), g is the gravity acceleration (ms−2) and r is the radius of the pipe (m). Therefore, the Moon's gravity would have to be subtracted from that of the Earth g = gE − gM, and since it is 288,000 times smaller, its effect on capillarity is negligible.


**Table 4.** Revision of some of the reference handbooks on physics in relation to possible mentions of the Moon affecting plants.

Source: authors' review.

Physics books, even those studying applications of physics in biology [53], do not deal with the Moon's influence on plant growth. This may be due to the fact that the Moon's gravity is, as we have seen in the Introduction (Section 1), negligible compared to that of the Earth. Regarding illuminance, since it is a topic addressed in specialized books on optics [54], it is not usually included in physics books (only one of them does, as shown in Table 4), even less lunar illuminance.

The analysis of reference handbooks and monographs dealing with plant growth and development in the background of biology, environmental sciences, forestry, and agronomy is a key issue to understanding the extent to which this is a question that is limited to agricultural practice and/or the scientific and training field. Table 5 shows a summary of six widespread and commonly used books on botany and plant physiology, making a synoptic review of the endogenous and exogenous factors that determine and modulate plant development. In particular, the focus has been placed on those Moon-dependent factors that could be beneficial or stressful for plants, specifically in relation to Moon gravity or to the light reflected by the Moon.

As mentioned in the Introduction (Section 1) and reflected in Table 5, plant growth and development are regulated by endogenous and exogenous factors. The possible effects of the Moon should be considered as abiotic external factors, either if the effect is considered to be due to the light reflected or to gravitation. Regarding light, we searched for possible quotes of the Moon when addressing light effects on seeds, plant development, phototropism, photoperiodism, phototaxis, photonasties and quantity and quality of light, etc. Focusing on gravitational influence, the search was made on different aspects of gravitropism.


**Table 5.** Revision of some of the reference handbooks on botany and plant physiology in relation to possible references to the Moon's influence on plant growth.

Source: authors' review.

Considering endogenous factors, we searched for possible interactions of Moon radiation and photoperiod, as well as gravity, on the transduction of the perception of those environmental stimuli as well as the possible determination by endogenous genetic components. An important internal process in plants, animals, fungi and cyanobacteria is that related to circadian rhythms that refer to any biological process that displays oscillation, driven by circadian clocks, synchronized with solar time. Plant circadian rhythms are related to seasons and determine, for example, when to flower to maximize the success of pollinator attraction. Circadian rhythms also determine leaf movement, growth, germination, gas exchange or photosynthetic activity, among others. All monographs reviewed mentioning circadian rhythms refer exclusively to synchronization with the light cycle of the surrounding environments of plants, considering the Sun as light source that can determine or influence these cycles.

This search in what is considered consolidated science and is incorporated to handbooks has revealed practically no mention of the Moon (Table 5). We have only found an anecdotic reference, in relation to the possible influence of moonlight on flowering in Thomas and Vince-Prue [57]. These authors explain the work of Salisbury [58], who had indicated that the effective red-light threshold for flowering is higher than the amount of red light produced by the Moon. In addition, it is important to consider that the shade provided by the leaves of the plant itself can reduce the radiation received to 5–10% of the direct moonlight [59]. Thomas and Vince-Prue [57] state that it seems unlikely that full moon light can influence flowering, even in the most sensitive plants, highlighting the scarcity of research on this issue. In this book the authors mention the work of Kadman-Zahavi and Peiper [60], who carried out research with *Pharbitis nil* (L.) Roth —a very sensitive short-day species—which they exposed to moonlight or shielded for different periods. They concluded that, although it is possible that moonlight is perceived, it had no effect on the experience developed

with a short-day species that is particularly sensitive to radiation. The difficulty of isolating the "Moon" factor was highlighted, pointing out the possible influence of shade treatment on plants in other environmental factors that could in turn have an effect on flowering [60]. On the other hand, they indicated that the full moon was only present on very few days of the lunar cycle, so its effect should be negligible under natural conditions.

#### *3.2. What Research Papers Say from the Perspective of Physics and Biology*

We consider a reference and starting point for the review of scientific articles, the brief paper published in *Nature* by Cyril Beeson in 1946, entitled "The Moon and Plant Growth" [61]. In this paper, the author writes "Beliefs that phases of the Moon have a differential effect on the rate of development of plants are both ancient and world-wide" and concludes that the research carried out to that date had not been able to demonstrate a correlation between the Moon and vital processes of terrestrial plants pointing out that, if any research does, the relationship was so unclear that it has no implications for agriculture.

In the 1950s, Frank A. Brown [22,62,63] undertook different investigations in which he studied the possible lunar rhythmicity in organisms. Most of this research was carried out on marine organisms closely linked to the tides—such as algae, crustaceans, molluscs—he also studied the physiological aspects of terrestrial plants. Brown et al. [22] studied the persistent rhythms of O2-consumption in potatoes, carrots (*Daucus carota* L.) and brown seaweed (*Fucus*) and searched for a possible influence of barometric pressure rhythms of primary lunar frequency, noting that they are of much lower amplitude than the solar ones. The study was inconclusive in relation to what external rhythmic forces are involved in the rhythms of O2-consumption, as many of them exhibit some degree of correlation with barometric pressure. In barometric pressure p = ρgh, as its expression depends on g, we would have the same case as with capillarity: the effect of gM should be subtracted from gE and, as we have seen, gM is approximately 300,000 times lower than gM, so the effect of the Moon on barometric pressure is negligible. The authors discuss the possibility that some of the responses attributed to external factors are due to endogenous rhythmic components. This connection between internal and external factors is supported by Wolfgang Schad [64], who states that "all chrono-biological rhythms are always exo-endogenous, sharing their autonomous inner clock to some degree with the periodicity of the environment, both sides being connected by the long process of evolution", remaining unanswered, the question of how the balance between endogenous and exogenous factors oscillates.

Some authors mention the influence of the lunar phases in a tangential way, without getting to clarify anything. One example explores the resistance of circadian clocks to transient fluctuations in night light levels in nature (i.e., change in cloud cover or stellar/lunar illumination) [65]. Van Norman et al. [66], when differentiating the circadian and infradian rhythms, indicate that the former are the best characterised with a period of around 24 h, while the infradians have periods of more than 24 h and can be due to the tides, lunar, seasonal, annual or longer. In other publications, the authors actively search, without finding them, for relationships between the Moon and some organisms. A paradigmatic case is the study conducted by Bitzand Sargent [67], who unsuccessfully tries to relate the growth rate of the fungus *Neurospora crassa* Shear & Dodge to the influence of a supposed lunar magnetic field (which, as we explain in detail in this article, is even more negligible than the gravitational field). Recently Mironov et al. [68] mentioned a circalunar growth rhythm in a research carried out with genus *Sphagnum*. They found an acceleration in the growth of the mosses studied near the new moon, and a slowdown in growth near the full moon.

Regarding biodynamic practices in agriculture, Hartmut Spiess carried out chronobiological investigations of crops grown under biodynamic management, developing experiments to test the effects of lunar rhythms on the growth of winter rye (*Secale cereale* L.) and little radish (*Raphanus sativus* L., cv. Parat) [69,70]. Spiess [69,70] tried to clarify some of the varying results that a number of studies conducted in the 1930s and 1940s had left unclear. This author also focused on studies made by M. and M.K. Thun [71] establishing a relationship between the position of the Moon relative to the zodiac

(sidereal rhythms), planting dates and crop growth, which served as a basis for the publication of calendars. Spiess' [69,70] results pointed out that the effects of lunar rhythms were weak, and especially the effects of the sidereal rhythms described by Thun and Thun were not apparent. In contrast to these papers, Kollerstrom and Staudenmaier [72], pointed out that, although Spiess' [69,70] experiments were well designed, there was a lack of care in the data analysis. According to these authors, the results published to date of its publication suggested that lunar factors may have a practical significance for agriculture.

Without a doubt, one of the botanists who dedicated the most effort and publications to the search for relationships between the Moon and plants was Peter Barlow. Barlow [73–85] devoted part of his research to decoding the influence of the Moon on biological phenomena. Specifically those aspects that take place in plants [73], such as the movements of leaves [74–76], stem elongation [77], fluctuations in tree stem diameters [78], the growth of roots [79–81], biophoton emissions from seedlings [82–84], and chlorophyll fluorescence [85]. According to Barlow et al. [76], and other works of the same author, at least in the cases analysed, the rhythm of leaf movements seem to have been developed or entrained in synchrony with the exogenous lunisolar rhythm experienced either on the Earth or in Space. Barlow [76] believed that plant movements were related with water movements within the plant: as ocean tides are produced by lunisolar gravitational force, water movement in the pulvinus could be responsible for leaf movement, explanation that we have previously discussed.

From all external factors, the perception of light plays a significant role as it can modify biosynthesis by photostimulation and act as a trigger initiating the different stages of development (Table 6). Reversive responses of plant to changes in light conditions can allow them to adjust their leaf or flower position (photonastic and heliotropic movements, respectively) to modulate the incoming radiation. Germination is also severely affected in some plants by light exposition. In fact, some seeds only germinate when they are exposed to a particular red to far-red ratios (660/730 nm), and in a particular moment [14].


**Table 6.** Radiation effects on developmental processes in plants.

<sup>1</sup> I = Inductive; Q = Quantitative; F = Formative. <sup>2</sup> B = Blue light; R/FR = Red-to-Far-Red ratio. <sup>3</sup> P = Photoperiodism; C = Circadian rhythm. Source: modified from Larcher [14], Kronenberg et al. [86] and Salisbury [87].

Despite light being crucial for plant life, just a few studies have explored the effect of moonlight on plant physiology and their results are not conclusive. Kolisko [88] observed that the period and percentage of germination and subsequent plant growth was influenced by the phase of the Moon at sowing time. And according to Bünning and Moser [59], light intensities as low as 0.1 lx, which correspond approximately to moonlight intensities (see Table 1), may influence photoperiodism in plants and animals whose threshold values of photoperiodic time-measurement is on the order of 0.1 lx. They suggest that light intensity may reach 0.7 lx or even 1 lx when the altitude of the Moon is at 60◦ or higher altitudes in tropical and subtropical regions (respectively), clearly influencing photoperiodic reactions. However, they observed that in short-day plants such as *Perilla ocymoides* L. and *Chenopodium amaranticolor* H.J.Coste & Reyn., light intensities similar to those of the full moon favoured rather than inhibited flowering [59]. They justified the circadian leaf movements observed

in *Glycine*, *Arachis* and *Trifolium* plants as an adaptive mechanism to reduce the intensity of full moon received in the upper surface of the leaf avoiding plant misinterpretations of confounding full moonlight as it would be long day [59]. However, Kadman-Zahavi and Peiper [60] rejected this hypothesis concluding "that in the natural environment moonlight may have at most only a slight delaying effect on the time of flower induction in short-day plants" (p. 621). Furthermore, Raven and Cockell [89] suggested that photosynthesis on Earth can occur in the photosynthetically active radiation (PAR) range of (10<sup>−</sup>8–8 <sup>×</sup> <sup>10</sup><sup>−</sup>3) mol of photons m−<sup>2</sup> <sup>s</sup><sup>−</sup>1, and PAR values of moonlight at full moon goes from (0.5–5) <sup>×</sup> <sup>10</sup>−<sup>9</sup> mol of photons m−<sup>2</sup> <sup>s</sup><sup>−</sup>1, suggesting that moonlight is not a significant source of energy for photosynthesis on Earth.

Recently, Breitler et al. [90] described that the photoreceptors present in *Co*ff*ea arabica* L. plants are able to perceive full moonlight and this full moonlight PAR is inadequate for photosynthetically supported growth. Plants perceive it as blue light with a very low R/FR ratio, yet this weak light has a great impact on numerous genes. In particular, it affects up to 50 genes related to photosynthesis, chlorophyll biosynthesis and chloroplast machinery at the end of the night. Moreover, full moonlight promotes the modification of the transcription of major rhythmic redox genes, many heat shock proteins and carotenoids genes suggesting that the moonlight seems to be perceived as a stress factor by the plant.

In other cases, full moonlight is correlated with a successful pollination of *Ephedra* species. Rydin and Bolinder [91] observed a correlation between pollination and the phases of the Moon on the gymnosperm *Ephedra foeminea* Forssk., specifically with the full moon of July. During that period, non-mature cones secreted enough pollination drops to apparently attract pollinators that can use the full moon to navigate and also be attracted to the glittering drops in the full moonlight. According to the authors, when insects are not used as pollinators, as it happens in other species of *Ephedra*, the adaptive value of correlating pollinating with the full moon is lost.

In the literature review carried out, some works were found that deal with two different topics that could have relationship with the Moon: polarization and magnetism. According to Semmens [92–94] during certain periods, moonlight is partially polarised, "the maximum effect being with the oblique reflexion of half-moon, or somewhat later for the waxing and earlier for the waning moon" and that polarised light can favour the diastase, which catalyses the hydrolysis, first of starch into dextrin and immediately afterwards into sugar or glucose, to favour germination, as he observed in crushed mustard seeds in the presence of this polarised light. Macht [95] studied the effect of (not lunar) polarized light on seeds of *Lupinus albus* L. and his results were consistent with previous findings of the action of diastase on starch. However, as far as we know, apart from those works no other research papers have been focused on the role of lunar polarized light. Despite, a full body of evidence supports that polarized moonlight has a biological significance in the vision and orientation of nocturnal animals [96,97]. Although we are at the very beginning of understanding the extent to which and why nocturnal animals use the lunar polarization, we do know that the land area over which it is viewable in pristine form is relentlessly shrinking due to human activity. In this sense, Kyba et al. [98] showed that urban skyglow has a great degree of linear polarization and confirmed that its presence diminishes the natural lunar polarization signal. They also observed that the misalignment between the polarization angles of the skyglow and scattered moonlight could explain the reduction of the degree of linear polarization as the Moon rises. Regarding nocturnal animal navigation systems based on perceiving polarized scattered moonlight, these authors highlighted the necessity of considering polarization pollution models in highly light-polluted areas. In any case, there is almost no doubt that the level of polarization of moonlight would be extremely small: so minimal, that its effect would be completely negligible in plants [98].

On the other hand, some studies suggest an influence of the lunar magnetic field. There is evidence that some animals, fungi, some protists and some bacteria seem to be able to react to the variation of the Earth's magnetic field [99–101]. The question that arises is whether plants are also able to respond to these fields and whether the Moon is capable of producing some magnetic field that plants can respond. There is abundant literature discussing magnetoreception in plants [102–106], but no conclusive results have been reported with direct application to agriculture.

Our planet has a magnetic field, called geomagnetic field, with an intensity of approximately (25–65) <sup>×</sup> 10−<sup>6</sup> T, ridiculously small compared to a commercial magnet (about 0.01 T) or a 0.2 T neodymium magnet. Although there are studies that argue that billions of years ago the Moon generated a magnetic field probably even stronger than the current magnetic field of the Earth, the lunar dynamo ended around one billion years ago [107,108]. The intensity of the present-day magnetic field on the lunar surface is <0.2 <sup>×</sup> 10−<sup>9</sup> T, indicating that the Moon currently does not have a global magnetic field [109]. A magnetic field of this numerical value is approximately 225,000 times less than the Earth's, and if divided by the distance Earth–Moon (3.84 <sup>×</sup> <sup>10</sup><sup>10</sup> m), we can easily conclude that the possible effect of a hypothetical lunar magnetic field on the Earth would be much more negligible than that of the gravitational field.

Other theories claim that it is not the lunar magnetic field that affects, but the disturbance in the Earth's electromagnetic field caused by the lunar gravitational changes that take place during the full moon [4]; or also that Moon effects to the Earth's magnetosphere [110]. In both cases, the assumed effects would be (as we have seen in the calculations for the gravity case) completely insignificant.

A general analysis of the above-mentioned literature highlights the heterogeneity in the information sources regarding year of publication and discipline of the journal. On the one hand, there are very recent papers [68,90] but also literature from more than half a century ago [61,92–94]. On the other hand, there are peer-reviewed papers indexed in the Q1 of JCR in specific publications on Plant Science discipline, as *Annals of Botany* [75,79,80], *BMC Plant Biology* [90], *Frontiers in Plant Science* [104], *Journal of Plant Research* [103], *New Phytologist* [81], *Planta* [76], *Physiologia Plantarum* [68], *Plant Cell* [65,66] or *Plant Physiology* [67], with a long and consolidated trajectory in the field and with a pool of reviewers with solid expertise. Other articles are published in the Q2–Q3 of *JCR* in the same category as *Plant Biology* [77] and *Protoplasma* [78,83], or in other categories as Horticulture or Agronomy (e.g., *Biological Agriculture and Horticulture* [69,70,72]). Other papers included belong to other disciplines: *Astrobiology* [89], *Biology Letters* [91], *Icarus* [109], *Philosophical Transactions of the Royal Society B: Biological Sciences* [96], *Nature* [61,92–94], *Naturwissenschaften* [84], indexed in Q1–Q2 *JCR* lists. Nevertheless, there are also some papers not included in *JCR* lists but in other repositories as *Communicative and Integrative Biology* [73], *Earth, Moon and Planets* [64], *Pathophysiology* [110] and *Star and Furrow* [71].

This analysis also raises the question of the extent to which the authors have a good basis in the physics behind all these phenomena, given that to date Moon has not been proved to affect plant biology regarding consolidated physics.

#### **4. Next Steps from the Perspective of Science Teaching**

We are concerned about the insidious spread of pseudo-scientific ideas, not only in the field of plant science (which determines many of the behaviours, habits and techniques of many farmers in rural areas) but into the broader population through both formal and informal education. As science educators, we are especially concerned about the widespread belief in pseudo-science throughout the general populace and especially in science teachers [111–114]. Solbes et al. [114] showed that 64.9% of a sample of 131 future science teachers agree or partially agree with the expression "The phase of the Moon can affect, to some extent, several factors such as health, the birth of children or certain agricultural tasks".

Given this worrying scenario, teachers must promote critical thinking as an essential part of citizenship development. Critical thinking implies being informed about issues or problems, not limiting oneself to the dominant discourses in the media, understanding alternative, well-argued positions and being able to analyse the evidence supporting each of them, studying the problem in its complexity, so that scientific, technical, social, economic, environmental, cultural and ethical dimensions are involved, etc. [115–117]. We believe that it is crucial for teachers to be aware of

these beliefs in order to address them from a scientific perspective, as has been demanded for some time [118,119].

One way to approach pseudoscience is to involve students in the proper process of reasoning and knowledge building in science, and research-based teaching is postulated as a suitable teaching methodology to address the problem [120]. In the same way that Lie and Boker [121] analysed the perceptions of complementary therapies of medical students who claimed to have pseudo-scientific beliefs related to health, it would be of great interest to address these issues with agronomic students. In this line, a teaching–research sequence has been developed [122] with future science teachers, in which the strategy followed was to plant seeds of different plant species in each of the phases of the Moon and to measure their growth once a lunar cycle was completed. The participants specified the research question and the initial hypothesis. In addition, they established the experimental design as a whole, fixed the dependent, independent, and constant variables, the materials, the sequence of the sessions, etc. [123,124]. As a result of the proposal, still under analysis, it is expected that students will develop a critical attitude towards pseudoscience and improve their training in research methodologies. Didactic proposals of this type could help, not only in teacher training, but also in any scientific study, promoting critical thinking.

With this work, we wanted to draw attention to one of the many facets of current pseudo-scientific ideas, especially in agriculture. However, we want to emphasize that dismantling these ideas, which, as we have seen, lack in any scientific basis, should not be incompatible with knowing and preserving agricultural traditions that are an important part of an ethnographic and anthropological heritage, as some institutions such as the Food and Agriculture Organization of the United Nations (FAO) claim [125–128]. Many of these traditions (e.g., organic farming, traditional and seasonal crops) allow a harmonious and sustainable coexistence with their natural environments, compatible with the conservation of biodiversity, varieties of certain species.

Furthermore, this paper encourages new research on this long-lasting topic of the possible influence of the Moon on plants in order to clarify many aspects that still remain unanswered or which have not been approached. Considering that modern ecophysiology requires a good understanding of both the molecular aspects of plant processes and the environment, future studies will necessarily have to move to a higher level: scaling from physiology to the Globe [129], considering relationships between plant ecophysiological processes and those occurring at ecosystems but also including social aspects—as traditions, farmers' behaviours and protocols, etc.—that can determine the environment where plants grow.

This review opens the door to possible research that would help to complete the picture of the extent to which certain pseudo-scientific ideas have permeated different sectors of the population. In this sense, it would be interesting to carry out an in-depth analysis of what farmers, as well as students in careers related to the agriculture sector and plant biology, think about the relationship of the Moon with the growth and development of plants.

#### **5. Conclusions**

Science has widely established different evidences: (i) the Moon's gravity on the Earth cannot have any effect on the life cycle of plants due to the fact that it is 3.3 <sup>×</sup> <sup>10</sup>−<sup>5</sup> ms<sup>−</sup>2, almost 300,000 times lower that the Earth's gravity; (ii) since all the oceans are communicated and we can consider their size being the size of the Earth, the Moon's influence on the tides is 10−<sup>6</sup> ms−2, but for a 2 m height plant such value is 3 <sup>×</sup> 10−<sup>13</sup> ms−<sup>2</sup> and, therefore, completely imperceptible; (iii) the Moon's illuminance cannot have any effect on plant life since it is, at best, 128,000 times lower than the minimum of sunlight on an average day; (iv) the rest of possible effects of the Moon on the Earth (e.g., magnetic field, polarization of light) are non-existent.

The logical consequence of such evidence is that none of these effects appear in physics and biology reference handbooks. However, many of these beliefs are deeply ingrained in both agricultural traditions and collective imagery. This shows that more research should be undertaken on the possible effects observed on plants and assigned to the Moon by the popular belief, addressing their causes, if any. It would also be interesting to address these issues in both compulsory education and formal higher agricultural education in order to address pseudo-scientific ideas and promote critical thinking.

**Author Contributions:** Conceptualization, O.M. and T.P.; methodology, O.M., J.S., J.C. and T.P.; formal analysis, O.M. and T.P.; investigation, O.M., J.S., J.C. and T.P.; data curation, O.M. and T.P.; writing—original draft preparation, O.M., J.S., J.C. and T.P.; writing—review and editing, O.M., J.S., J.C. and T.P.; supervision, O.M., T.P.; project administration, O.M., J.S. and T.P.; funding acquisition, O.M. and J.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is framed within the project "Proposal for the Improvement of Science Teacher Training Based on Inquiry and Modelling in Context" (EDU2015-69701-P), funded by the Ministry of Economy, Industry, and Competitiveness and the European Regional Development.

**Acknowledgments:** A significant part of the research and bibliographic review was carried out by O.M. and T.P. in the different libraries of Harvard University—especially in the excellent library of the Arnold Arboretum of Harvard University. In this sense, we would like to express our gratitude to the Real Colegio Complutense (RCC) —Harvard, who granted two of the authors (O.M. and T.P.) for a stay at Harvard University during 2019.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


#### *Agronomy* **2020**, *10*, 955


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*Review*

### **Adaptation of Plants to Salt Stress: Characterization of Na**<sup>+</sup> **and K**<sup>+</sup> **Transporters and Role of CBL Gene Family in Regulating Salt Stress Response**

#### **Toi Ketehouli, Kue Foka Idrice Carther, Muhammad Noman, Fa-Wei Wang, Xiao-Wei Li \* and Hai-Yan Li \***

College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, China; stanislasketehouli@yahoo.com (T.K.); kuefokaidricecarther@yahoo.com (K.F.I.C.); mohmmdnoman@gmail.com (M.N.); fw-1980@163.com (F.-W.W.)

**\*** Correspondence: xiaoweili1206@163.com (X.-W.L.); hyli99@163.com (H.-Y.L.); Tel.: +86-0431-84533428 (X.-W.L.); +86-0431-84532885 (H.-Y.L.)

Received: 14 September 2019; Accepted: 21 October 2019; Published: 28 October 2019

**Abstract:** Salinity is one of the most serious factors limiting the productivity of agricultural crops, with adverse effects on germination, plant vigor, and crop yield. This salinity may be natural or induced by agricultural activities such as irrigation or the use of certain types of fertilizer. The most detrimental effect of salinity stress is the accumulation of Na<sup>+</sup> and Cl<sup>−</sup> ions in tissues of plants exposed to soils with high NaCl concentrations. The entry of both Na<sup>+</sup> and Cl<sup>−</sup> into the cells causes severe ion imbalance, and excess uptake might cause significant physiological disorder(s). High Na<sup>+</sup> concentration inhibits the uptake of K+, which is an element for plant growth and development that results in lower productivity and may even lead to death. The genetic analyses revealed K<sup>+</sup> and Na<sup>+</sup> transport systems such as SOS1, which belong to the CBL gene family and play a key role in the transport of Na<sup>+</sup> from the roots to the aerial parts in the *Arabidopsis* plant. In this review, we mainly discuss the roles of alkaline cations K<sup>+</sup> and Na+, Ion homeostasis-transport determinants, and their regulation. Moreover, we tried to give a synthetic overview of soil salinity, its effects on plants, and tolerance mechanisms to withstand stress.

**Keywords:** salinity; sodium; potassium; ion homeostasis-transport determinants; CBL gene family

#### **1. Introduction**

The adverse effects of salinity on plant growth are generally associated with the low osmotic potential of the soil solution and the high level of toxicity of sodium (and chlorine for some species) that causes multiple disturbances to metabolism, growth, and plant development at the molecular, biochemical, and physiological levels [1,2]. In vitro experiments have shown that the enzymes extracted from the halophyte plants *Triplex spongeosa* or *Suaeda maritima (L.)* are sensitive to NaCl to the same degree as those extracted from the glycophyte plants [3,4]. These experiments suggest that tolerance to salinity is not limited to a metabolic response in tolerant plants. Generally, sodium begins to have an inhibitory effect on enzymatic activity from a concentration of 100 mmol/L. Thus, the ability of plants to reduce sodium levels in the cytoplasm appears to be one of the decisive factors in salinity tolerance [5,6]. However, although chloride ions are micro-elements necessary as co-factors, for enzymatic activity, photosynthesis, and the regulation of cell turgor, pH, and electrical membrane potential, they remain no less toxic than Na<sup>+</sup> ions if their concentration reaches the critical threshold tolerated by plants [7]. Ionic cellular homeostasis is an essential and vital phenomenon for all organisms. Most cells maintain a high level of potassium and a low level of sodium in the cytoplasm through the coordination and

regulation of different transporters and channels. There are two main strategies that plants use to cope with salinity—The compartmentalization of toxic ions within the vacuole and their exclusion outside the cell [5,6]. On the other hand, plants modify the composition of their sap; they can accumulate Na<sup>+</sup> and Cl<sup>−</sup> ions to adjust the water potential of tissues necessary to maintain growth [6]. This accumulation should be consistent with a metabolic tolerance of the resulting concentration or with compartmentalization between the various components of the cell or plant. It requires relatively little energy expenditure. If this accumulation does not take place, the plant synthesizes organic solutes to adjust its water potential. It will require a large amount of biomass to ensure the energy expenditure necessary for such a synthesis. Therefore, one adaptation strategy consists of synthesizing osmoprotective agents, mainly amino compounds and sugars, and accumulating them in the cytoplasm and organelles [8,9]. These osmolytes, usually of a hydrophilic nature, are slightly charged but polar and highly soluble molecules [10], suggesting that they can adhere to the surface of proteins and membranes to protect them from dehydration. Another function attributed to these osmolytes is protection against the action of oxygen radicals following salt stress [11]. Under high sodium concentration levels, whether the latter is compartmentalized within the vacuole or excluded from the cell, the osmotic potential of the cytoplasm must be balanced with that of the vacuole and the external environment in order to maintain the cell turgor and the water absorption necessary for cell growth. This requires an increase in osmolyte levels in the cytoplasm, either by the synthesis of solutes (compatible with cellular metabolism) or by their uptake of the soil solution [12,13]. Among these synthesized compounds are some polyols, sugars, amino acids, and betaines, which, energetically, are very expensive to be produced by the cell [14]. The main role of these solutes is to maintain a low water potential inside the cells to generate a suction force for water absorption. Furthermore, the involvement of solutes such as glycine betaine, sorbitol, mannitol, trehalose, and proline in improving tolerance to abiotic stress has been demonstrated by genetic engineering and plant transgenesis [6,14,15]. On the other hand, salt stress induces the production of active forms of oxygen following the alteration of metabolism in the mitochondria and chloroplasts. These active forms of oxygen cause oxidative stress whose adverse effects are reflected in various cellular components such as membrane lipids, proteins, and nucleic acids [16]. As a result, the reduction of these oxidative damages through the deployment of a range of antioxidants could contribute to improving plant tolerance to stress [17]. Early events in plant stress adaptation begin with mechanisms of perception and signaling via signal and messenger transduction to activate various physiological and metabolic responses, including the expression of stress response genes. The main pathways activated during the salt stress signaling include calcium, abscisic acid (ABA), mitogen-activated protein kinases (MAPKinases), salt overly sensitive proteins (SOS), and ethylene [12]. In this chapter, we mainly discuss roles of alkaline cations K<sup>+</sup> and Na+, ion homeostasis-transport determinants, and their regulation. Furthermore, we tried to give a hypothetical overview of soil salinity, its effects on plants, and tolerance mechanisms that allow the plants to withstand stress. A fundamental biological understanding and knowledge of the effects of salt stress on plants is needed to provide additional information for the study of the plant response to salinity and try to find other way for improving the impact of salinity in plants and accordingly enhance crop yields to cope with the starvation that persists in some parts of the world

#### **2. Roles of Alkaline Cations K**<sup>+</sup> **and Na**<sup>+</sup> **in Plants**

Potassium (K) is the third of the three primary nutrients required by plants, along with nitrogen (N) and phosphorus (P). Potassium, with about 100 to 200 mM concentration in the cytosol, is the major inorganic cation of the cytoplasm in plant and animal cells. The reasons for its preferential accumulation compared to Na<sup>+</sup> is probably due to the fact that Na<sup>+</sup> is more "chaotropic" (because of its smaller size and stronger electric field on its surface) [18].

Na<sup>+</sup> is not an essential nutrient for higher plants. For a high concentration of Na<sup>+</sup> in the soil, this cation becomes even toxic to the plant. At lower concentrations, the plant can use it beneficially as a vacuolar osmoticum.

#### *2.1. Physiological Roles*

As most inorganic cations are abundant in the cytoplasm, the potassium is involved in critical cell functions. In addition to its role in the neutralization of the net electric charge of biomolecules, the potassium participates, for example, in membrane transport processes, enzyme activation, and osmotic potential. In plants, in conjunction with osmotic potential [19], K<sup>+</sup> is involved in the control of the turgor pressure [20] and related functions, cell elongation and cell movement. Finally, K<sup>+</sup> plays a direct or indirect role, in the regulation of enzyme activities, the protein synthesis, photosynthesis and homeostasis of the cytoplasmic pH.

These different roles at the cellular level involve potassium in essential functions at the level of the whole plant, for example gas exchange control via regulation of the opening and closing of the stoma, the xylem sap ascension by root thrust, installation of potential osmotic gradient carrying phloem sap flow from original organs to hole organs or even port of herbaceous species.

#### *2.2. E*ff*ect of K*<sup>+</sup> *Deficiency on Plants Physiology*

In K<sup>+</sup> deficiency, the sap flow is disturbed, with spontaneous reduction of the phloem sap velocity of circulation. The photoassimilates then accumulate inside of the leaves. Symptoms of chlorosis and necrosis from the photooxidation of the photosynthetic system are frequently observed. It is well settled that K<sup>+</sup> deficiency induces the acidification of the extracellular medium. Minjian et al. [21], showed that root K<sup>+</sup> absorption depends on the activity of the proton pumps (H+-ATPases) and the occurrence of K<sup>+</sup> transporters on the cellular membrane. The level of H<sup>+</sup> expulsion can be used as a criterion of tolerance to K<sup>+</sup> deficiency. Chen and Gabelman [22] observed in tomato strains that K<sup>+</sup> uptake efficiency is associated with a high net K<sup>+</sup> influx coupled with low pH around root surfaces. The proton-electrochemical gradient may contribute to energizing K<sup>+</sup> uptake, and indeed it is used by some KT/ KUP/HAK transporters, which co-transport K<sup>+</sup> and H<sup>+</sup> [23].

#### *2.3. Toxicity of Na*<sup>+</sup> *in the Cytoplasm*

In plants, the concentration of Na<sup>+</sup> in the cytosol is maintained at a lower value than that of K<sup>+</sup> in animals. In animal cells, the concentration of Na<sup>+</sup> is closely regulated to 10−2mol L−<sup>1</sup> value [24]. In plant cells, the concentration of Na<sup>+</sup> does not seem to be subjected to narrow homeostasis. When the plant grows in salinity conditions, the accumulation of Na<sup>+</sup> in the cytoplasm beyond a certain threshold becomes toxic, but this threshold is not clearly determined.

The toxicity of Na<sup>+</sup> in the cytosol would result from its "chaotropic" character by comparison with K<sup>+</sup> [18]. The toxicity of Na<sup>+</sup> would also probably mean its ability to compete with K<sup>+</sup> during the process of fixing important proteins. More than 50 enzymes require K<sup>+</sup> to be active, and Na<sup>+</sup> would not provide the same function [25]. Therefore, a high concentration of Na<sup>+</sup> in the cytoplasm inhibits the activity of many enzymes and proteins, leading to cell dysfunctions. In addition, protein synthesis requires a high concentration of K<sup>+</sup> for tRNA attachment to ribosomes [26], so the translation would also be affected.

#### *2.4. Na*<sup>+</sup> *Acts as Osmoticum*

If the plant cell cannot substitute Na<sup>+</sup> to K<sup>+</sup> in its cytosol, it can do it so in the vacuoles and use Na<sup>+</sup> as osmoticum. Different studies have actually shown that moderate amounts of Na<sup>+</sup> can improve the growth of many plant species [27]. For example, a beneficial "nutritious" effect of Na<sup>+</sup> has been described in tomato [28,29].

It is likely that the beneficial effect of Na<sup>+</sup> can especially be observed in conditions of K<sup>+</sup> deficiency. In these circumstances, a controlled build-up of Na<sup>+</sup> probably helps to ensure the regulation of cell turgor pressure [30,31]. Similarly, a moderate absorption of Na<sup>+</sup> can be beneficial if it helps the plant, for example, to quickly adjust their osmotic potential from the beginning of salt stress.

Despite these physiological observations, the genetic determinants of improving the growth of plants by sodium and genes may be involved in these processes, however, they are still poorly characterized. Research on rice [32] concerning the function in planta of a transporter HKT family provided genetic proof on the fact that an accumulation of Na<sup>+</sup> in K<sup>+</sup> deficiency conditions can promote the growth of the plant.

#### **3. Interaction between K**<sup>+</sup> **and Na**<sup>+</sup> **Transport and Adaptation to Salt Stress**

The adaptation of the plant to the presence of salt in the soil and salt stress involves various processes, occurring at different levels, from the cell to the whole organism, such as a modification of the metabolic activity leading to the accumulation of organic osmolytes [33], or morphological and developmental changes of the leaves [34]. Within this very complex network of responses, the control of membrane transport activities occurring through a variety of mechanisms, a selective accumulation of K<sup>+</sup> and an exclusion of Na<sup>+</sup> [25,35], appear as a central process. Thus, in a large number of models, from isolated cell culture to the whole plant, adaptation to salt stress appears to be correlated with the ability to selectively remove K+, to control the Na<sup>+</sup> entrance, and maintain the K+/Na<sup>+</sup> ratio of the internal contents of these two cations at a high level. In this context, the molecular and functional characterization of membrane transport systems of K<sup>+</sup> and Na<sup>+</sup> is, therefore, a priority objective. It is probable that the capacity of the channels and transporters to discriminate K<sup>+</sup> from Na<sup>+</sup> is essentially based on the difference of intensity of the electric field at the surface of these two ions, which results from their difference in size and hydration energies. The crystallographic resolution of the bacterial potassium channel structure [36,37] provides an example for understanding how carbonyl groups of the polypeptide chain can be spatially distributed along the permeation pathway to substitute, without energy barrier to the hydration shell of the ion.

#### **4. Physiology of K**<sup>+</sup> **and Na**<sup>+</sup> **Transport in Plants**

#### *4.1. Structure–Function Relationship of the Root*

The movement of the mineral elements by the roots and their transfer to the aerial parts involves at least two membrane steps—Ions *sensu stricto* absorption from the soil solution by the epidermal cells, cortical, and contingently endodermic, and the secretion inside the vessels at the level of xylem parenchyma cells. The ions radial movement from the orbital cells of the root to the stela can, in theory, take three paths [38]—The apoplastic pathway (through cell wall), the symplastic pathway (through cytoplasm), or a mixed path passing the ions alternately from apoplastic compartment to the symplastic compartment (Figure 1). Above the cell differentiation zone, the apoplastic path is interrupted by the endoderm of the root. The walls of these cells are impregnated with lignin and suberin. This deposition of hydrophobic compounds forms the framework of Caspary and constitutes a barrier that blocks water and solutes movement. The very close association of the endodermal cell membrane with the Caspary framework forces the ions and water to undergo membrane control to pass the endodermal barrier and migrate into the stele. However, at several levels in the root, the ions can take a direct apoplastic path from the external environment to the xylem: at the apex, where the endodermis is not yet suberized, at the level of endoderm discontinuity, induced by the appearance of the secondary roots [39], and in some species, at the level of some non-suberized endodermal cells, called passage cells which are thought to serve as cellular gatekeepers, controlling access to the root interior [40].

**Figure 1.** Sodium transport at the cellular level. Schematic representation of transport systems involved in Na<sup>+</sup> transport at the plants through the plasma membrane or the tonoplast. Primary transport systems consisting of proton pump ATPases on the plasmalemma and the tonoplast and a pyrophosphatase on the tonoplast create a pH gradient and a potential difference electric on both sides of the membranes (cytosolic side more alkaline and charged more negatively). Proton concentration gradients allow Na<sup>+</sup> excretion of cytoplasm towards the outside environment or the vacuole via the operation of antiports Na+/H<sup>+</sup> (appealed SOS1 (Salt Overly Sensitive protein 1) on the plasmalemma or NHX1 (K+, Na+/H<sup>+</sup> antiporter), on the vacuole). Potential gradients electric created by the pumps cause the entry of Na<sup>+</sup> in the cytoplasm of the cell since the external environment or the vacuole via non-selective cationic channels (NSCC) (CNGC (Cyclic Nucleotide Gated Channels) on the plasma membrane? TPC1 (Two-Pore Channel 1) on the tonoplast) or possibly carriers of the HKT (High-Affinity K<sup>+</sup> Transporters type in some species. At high external concentration, Na<sup>+</sup> could also enter the cell by borrowing K<sup>+</sup> carriers KUP/HAK (K<sup>+</sup> uptake/High-Affinity K+. ) type.

In the mature root areas of the majority of plants, a second concentric barrier to that formed by the endodermis is formed at the root periphery on the exoderm, subepidermal cell layer. The suberization of the exoderm would occur later during root development than that of the endoderm and would be accelerated in case of drought [40] or salt stress.

#### *4.2. Structure–Function Relationship of Root and Salt Stress*

The current data about root structure and function, as discussed above, indicate that sodium ions can take a direct apoplastic path from the outer medium to the xylem at several levels of the root because endodermal suberization is not yet in place in the young roots area, and leaks remain in secondary roots appearance, which induces a brief discontinuity of the endoderm [41]. The relative contributions of the apoplastic and symplastic pathways of Na<sup>+</sup> transport is therefore largely conditioned by root anatomy and are likely to alter according to plant species and soil salinity. The apoplastic pathway (also called apoplastic leak) could be predominant in Na<sup>+</sup> transport under salt stress conditions.

Studies carried out on rice have shown that there is a strong correlation between sodium transport and the apoplastic tracer. In two different lines of rice, one more tolerant to salt than the other, a significant difference between the proportions of sodium amount and accumulated PTS in their aerial parts was observed [42,43]. This phenomenon results from the fact that the Na<sup>+</sup> entrance into the rice is essentially by free migration in the apoplast up to the stele in spots where the endoplasmic barrier is not functional. This apoplastic leak could occur at the lateral root connection points, at root's

apex before complete differentiation of rhizodermis and endodermis, and even in mature areas with differentiated endoderm because of the inherent permeability of the parietal broad outline [44].

It has been shown in rice that the control of apoplastic leakage of Na<sup>+</sup> into the roots is a critical determinant of salinity tolerance. The addition in the culture medium of silicon in sodium silicate partially blocked the apoplastic pathway and considerably improved the growth and photosynthesis of rice plants under salt stress, especially in the GR4 variety [44,45]. This improvement is correlated with the reduction of the Na<sup>+</sup> concentration in plant aerial parts. Furthermore, the authors found that the addition of sodium silicate in the culture medium reduced the accumulation of Na<sup>+</sup> in the aerial parts of sensitive and tolerant varieties at the same level [44,45].

The apoplastic pathway importance in the overall balance of Na<sup>+</sup> inflow varies with species. Garcia et al. [46] estimated that the contribution of the apoplastic pathway is 10 times greater in rice than in wheat. Moreover, it is important to emphasize that halophytes have root anatomy that can limit the entry of Na<sup>+</sup> via the apoplastic pathway. Indeed, the Caspary band in halophytes is 2–3 times thicker than in glycophytes, and the inner layer of cortical cells in halophytes can differentiate to form the second endoderm [2]. In cotton, considered as salinity-tolerant plant among cultivated species, salinity also accelerates the formation of the Caspary band and induces the formation of an additional exodermal layer [47].

All these findings show that there is a correlation between plant tolerance to salinity and the ability to control the apoplastic influx of Na<sup>+</sup> into the roots. It is, therefore, possible to postulate that reducing apoplastic leakage in sensitive species such as rice is a strategy for increasing plant tolerance to salinity. In this perspective, it is important to write down that complete blockage of apoplastic leakage is not likely to significantly affect water inflow and nutrient ion uptake because this leakage contributes little (less than 6%) in rice) to the incoming flows in the roots [46,48]. Some authors have estimated that the apoplastic flow contributes to the xylem flow feeding in a proportion that cannot exceed 1 to 5% [49]. This means that, concerning K+, the symplasmic transport ensures the essential translocation of this ion from soil solution to the xylem vessels of the stele.

#### **5. Potassium Availability in the Soil and Its Absorption by Plants**

K<sup>+</sup> is an important cofactor in many biosynthetic processes, and in the vacuole, it plays key roles in cell volume regulation [50].

The concentration of K+ in the soil solution is generally between a few tens of μmol. L−<sup>1</sup> and a few mmol. L−<sup>1</sup> (i.e., approximately 10 to 10<sup>3</sup> times lower than that of the cell). The roots are thus confronted with a wide concentration range and the plants possess transport systems allowing them to grow over concentration ranges of K+, ranging from 10−<sup>6</sup> to 10−<sup>1</sup> mol. L−<sup>1</sup> [51].

An enhancement of the absorption capacity of K<sup>+</sup> by the root is observed when the availability of this ion in the soil is limited [52]. In wheat, K<sup>+</sup> deprivation increases the high-affinity transport efficiency, without altering the characteristics of low-affinity transport. This type of response has also been observed in barley and ryegrass [53]. This reaction is not general, but there are many proteins involved in high-affinity potassium transport. However, in Arabidopsis, two proteins have been identified as the most important transporters in this process. Interestingly, one of these transporters, AtHAK5, is a carrier protein and is thought to mediate active transport of potassium into plant roots, whereas the other protein, AKT1, is a channel protein and likely mediates a passive transport mechanism with an increased affinity for K<sup>+</sup> under conditions of potassium limitation [54,55].

Several different natural phenomena could be involved in root absorption capacity enhancement observed in response to K<sup>+</sup> deficiency in the soil. An initial model to account for this response proposes an allosteric regulation of the absorption capacity in terms of the cytosolic concentration of K+, resulting in an inhibition by "feedback" of the transporters when the availability of this ion in the area is high, leading to an increase in its concentration in the cytoplasm [56]. Under this model, the K<sup>+</sup> availability diminution in the area leads to a decrease of K<sup>+</sup> concentration in the cytoplasm, which would lift the allosteric inhibition of transport, thus causing absorption capacity augmentation. Another hypothesis, non-exclusive of the previous one, is based on the observation of modifications of the membrane polypeptide equipment when the plants are cultivated in a weakly concentrated potassium area, confirming the installation of new transport systems in barley [57], especially high-affinity transporters in barley [58], wheat [59], and *Arabidopsis thaliana*, [55,60]. In *Arabidopsis*, studies using the patch-clamp technical revealed that K<sup>+</sup> deficiency increases the activity of IRK-type channels (inward rectifying K<sup>+</sup> channel). This augmentation may reflect a corresponding gene(s) expression enhancement or the existence of a post-translational regulation mechanism (e.g., by dephosphorylation). However, the physiological meaning of the channels activity stimulation—And thus of passive transport systems in response to K<sup>+</sup> concentration diminution in the area—Is unclear, even though it is possible that channels may participate in the absorption function from a relatively low external K<sup>+</sup> concentration. Membrane potentials have indeed been found to be negative enough to be able to involve channels in the influx of potassium from an external solution of which K<sup>+</sup> concentration is less than 10 μM [61].

#### **6. Long-range Transport in Xylem and Phloem**

#### *6.1. Transport into the Xylem*

The Na<sup>+</sup> content of the roots appears to be relatively constant during salt stress. This steady-state probably results in part from root cells' ability to discharge Na<sup>+</sup> in the external area. It also results from Na<sup>+</sup> translocation in the stele and xylem vessels to the aerial parts. The sodium levels of the xylem and phloem may alter during the flow of plant sap. An increase of Na<sup>+</sup> concentration in xylem sap has been described in an "includer" type plant (definition below) *Plantago maritima* [48].

In opposition to this, a decrease of Na<sup>+</sup> concentration in xylem sap has been reported in "excluder" plants type—The sodium contained in the xylem is reabsorbed by roots during the ascent of the sap, and re-excreted toward the outside environment [48]. The amount of sodium that reaches the leaves via xylem sap can be controlled during transport in xylem vessels.

Unfortunately, there is a lack of knowledge about the mechanisms of Na<sup>+</sup> transport in the xylem. However, in *Arabidopsis* under moderate salt stress conditions (40 mM NaCl), *Sos1* mutants (having lost an H+/Na<sup>+</sup> antiport system) accumulate fewer Na<sup>+</sup> in the aerial parts than wild-type plants [34,62]. This suggests that SOS1 plays a role in the transport of Na<sup>+</sup> from the roots to the aerial parts. However, the use of a reporter gene reveals that in the roots, SOS1 is expressed preferentially in the parenchymal cells around the xylem vessels [62]. Together, these data suggest that SOS1 has been involved in Na<sup>+</sup> secretion in xylem sap from stele parenchymal cells under moderate salt stress conditions.

In some plants, there is a reduction of Na<sup>+</sup> accumulation in the aerial parts. This reduction could be explained by sodium removal from the xylem before it reaches the foliar system. The existence of this strategy in plants has been clearly demonstrated by the research work of Adem et al. [63]. The authors have shown that in barley, the Na<sup>+</sup> concentration of the xylem sap varies together with the stem height (10 mM at the base of the stem and only 2 mM at the 8th leaf). This difference of concentration is important particularly for maintaining the photosynthetic activity of young leaves, which in return allows the formation and growth of new leaves. Molecular mechanisms of Na<sup>+</sup> removal from xylem sap ("desalting" of xylem sap) are beginning to be documented. In particular, the genetic analyses revealed that two transporters of the HKT family, *AtHKT1* in *Arabidopsis* and *OsHKT8* in rice, are involved in this desalting process.

The majority of plants maintain a high K+/Na<sup>+</sup> ratio in their aerial parts, so it appears that the selectivity to the benefit of K<sup>+</sup> is ensured during the secretion. The ions are excreted in the xylem bundles via xylem transfer cells that can promote, or delay, the efflux of Na<sup>+</sup> in this vessel. The control of the Na<sup>+</sup> concentration in the xylem can also be carried out all along the stem by reabsorption of the sodium in exchange of potassium in the raw sap by the parenchyma cells [3]. H+-ATPases of the plasma membrane would ensure the energization of the various transports resulting in the exchange of Na<sup>+</sup> against K+. The H<sup>+</sup> gradient created by these pumps would allow the secretion of K<sup>+</sup> via an antiport H+/K+, and a uniporter of Na<sup>+</sup> would ensure sodium reabsorption.

Concerning potassium, the ions absorbed at the level of the plasma membrane of the root superficial cells (epidermal and cortical) are transported towards the tissues of the stele by diffusion from one cell to another through plasmodesmata (symplastic pathway). After migration beyond the endodermal barrier, the ions leave the symplasm crossing a second plasma membrane at the level of the last living cells that border the vessels (xylem parenchyma). Once in the apoplast stellar, the ions are driven by the centripetal flow of water to the vessels, and the convection flow of the raw sap (water and mineral units) carried by transpiration and/or root thrust then exports them to the aerial parts [64].

The inner position of xylem parenchyma cells in the root makes the electrophysiology analyses using microelectrodes difficult. As a result, the mechanisms of secretion of ions in the xylem have been less studied than the mechanisms of absorption. It has been acknowledged that the stela's tissues are not able to accumulate ions and that these ions, inflated at the entrance of the symplasm, passively diffuse to the vessels. This passive diffusion was thought to be the consequence of an oxygen deficiency in the central tissues of the root that results in cell depolarization [65]. The stele cells in hypoxic conditions were then unable to retain the ions. However, Zhu et al. have shown that aeration of root pivotal tissues allows cells sufficient oxygenation. CCCP instantly blocks efflux in the xylem of the 36Cl<sup>−</sup> accumulated in advance but not efflux to the area through the epidermis [66]. These results show that the CCCP affects the existing system at the level of the stele and not the one located in the cells of the epidermis. Since the 1970s, it has been clearly established that the ions efflux in the stellar apoplast depends on specific transporters located on the plasma membrane of xylem parenchyma cells. Several experimental data indicate that absorption and secretion are controlled separately.

In general, the secretion of nutrient ions in the stellar apoplast could in many cases be a passive phenomenon, catalyzed by channels. For example, in *Arabidopsis*, the SKOR potassium channel of the Shaker family plays an important role in K<sup>+</sup> secretion in xylem sap [67]. The knowledge at the molecular level on the mechanisms of secretion of nutrient ions in the xylem sap is, however, still rather small.

#### *6.2. Transport into the Phloem*

The growth and development of the plant require distribution of photosynthesis products. These molecules synthesized in the so-called "source" organs (mature leaves) must then be relocated to the growing organs and non-photosynthetic plant tissues (organs called "wells," young leaves, flowers, seeds, fruits, roots). This relocation requires selective long-distance transport, which is provided by the phloem system.

Data obtained from barley show that the sodium contents of xylem and phloem sap are altered throughout transport in the vessels of the aerial parts [68]. The sodium contained in xylem would be absorbed and stored into leaf cells during its movement, and there would also be a translocation of a part of the sodium from xylem to phloem in the leaf, so that the sodium concentration in phloem sap has increased, as it moves from the top of the leaf to its base. Foliar anatomy, particularly in the area of young veins, suggests that such a transfer could occur either directly from apoplast to symplasm of phloem cells, or by symplasmic transport from parenchymal cells [68]. This recirculation of ions from xylem to phloem thus makes it possible to significantly reduce the salt content of the leaves. This has also been observed in some species such as Lupin [69], pepper [70], corn [71], and barley [13].

Perez-Alfocea et al. [72] have found that Na<sup>+</sup> translocation in the phloem of *Lycopersicon pennellii*, a wild type tomato that is tolerant to salinity, is more important than that observed in domesticated tomatoes. This suggests that the translocation of Na<sup>+</sup> into the phloem would be an adaptation strategy in plants. However, the Na<sup>+</sup> translocation direction and the conditions under which it occurs are probably critical. Indeed, it seems crucial that translocation by phloem does not transport Na<sup>+</sup> to the young tissues—Otherwise, it would completely inhibit their growth. In other words, translocation by the phloem should essentially redirect Na<sup>+</sup> to the roots. In the pepper plant, it has been shown that the translocation of Na<sup>+</sup> from the aerial parts to the roots only occurs when Na<sup>+</sup> is removed from the

nutrient solution, i.e. when there is a favorable gradient between phloem and roots [70]. In *Arabidopsis*, it has been shown that the sodium transporter *AtHKT1*, expressed in phloem tissues, assure Na<sup>+</sup> recirculation from the leaves to the roots through phloem by removing Na<sup>+</sup> from the rising stream of raw sap at the aerial parts. This system thus plays the role of controlling the Na<sup>+</sup> accumulation in the leaves and plant resistance to salt stress [73].

With regard to potassium, the phloem loading and its discharge contribute to the establishment of the osmotic potential gradients (and therefore hydric) created between the source organs (high concentration of sugars and ions in the phloem sap) and the well organs (lower concentrations). The osmotic gradient is initiated at the level of the source organs by the creation of an electrochemical potential due to the H+-ATPases activity of the fellow cells that are in direct electrical contact with the cells of the screened canals (making the phloem vessels) via plasmodesmata. This energization of the membrane allows the influx of sugars (essentially sucrose) and potassium into the cells. In summary, the available data indicate that control of K<sup>+</sup> transport in phloem tissues of source and well organs contribute to three main functions: (i) the phloem cells membrane potential regulation, tending to bring its value closer to that of equilibrium potential of K<sup>+</sup> (*EK*), (ii) the installation of the osmotic gradient responsible for the sap flow between the source and well organs, and (iii) well organs (including seeds and fruits) potassium supply.

The electrophysiological characterization of the potassium conductance of phloem cells is still poorly advanced because of the difficulty to obtain protoplasts. This difficulty is less with corn roots, whose stele is easy to separate from the cortex. Phloem cells can then be obtained by dissection in which potassium conductance has been identified. They are close to the IRKs in their selectivity and responses to inhibitors, but they show a small correlation. It means that they allow an entrance or output of potassium according to the membrane potential value. In *Arabidopsis*, the AKT2 gene from the Shaker potassium channel family could code this type of conductance [74,75].

In *Arabidopsis thaliana*, the use of plants expressing the GFP gene under the AtSUC2 gene promoter (active specifically in phloem fellow cells) made it possible to isolate protoplasts from these cells and to identify two potassium conductance—An outgoing conductance of the ORK type and an incoming conductance of the IRK type. However, conductance that may be specific to cells located either in the source regions or in the well regions has not yet been demonstrated.

#### **7. Adaptive Strategies of Plants to Na**+**: Exclusion and Inclusion**

The ability of a plant to compartmentalize Na<sup>+</sup> at the cellular level induces a difference of Na<sup>+</sup> management in the whole plant. We can distinguish two ways of plants responses to salt (exclusion and inclusion). "These strategies characterize behavior patterns that are not mutually exclusive" (Levigneron et al. reviewed in [76]). Excluder type plants are generally salinity-sensitive and are unable to control the level of cytoplasmic Na+. This ion is transported in the xylem, conveyed to the leaves by transpiration stream, and then partly "re-circulated" by the phloem to be brought back to the roots. These sensitive species, therefore, contain little Na<sup>+</sup> in the leaves and an excess in the roots. Includer plants, which are resistant to NaCl, accumulate Na<sup>+</sup> in the leaves where it is sequestered (in the vacuole, foliar epidermis, and old limbs). However, excluder plants also accumulate Na<sup>+</sup> in the vacuole of root and stem cells. Of course, these two types of behavior are extreme, and some species can incorporate behaviors characteristic of both types of strategy.

#### *7.1. K*<sup>+</sup> *and Na*<sup>+</sup> *Transport Systems in Plants*

The kinetic characteristics of K<sup>+</sup> transport systems were studied (since 1950) using the 42K and 86Rb tracers, in particular by Epstein et al. The incorporation rate analysis of the tracer into the excised barley roots, in terms of the external concentration, reveals complex kinetics that presents two phases [77]. This kinetics, which can be analyzed according to the Michaelis-Menten formalism, suggests the existence of two absorption mechanisms. The first mechanism corresponds to a high-affinity saturable system (Km <sup>≈</sup> 20 <sup>μ</sup>M), which allows the influx of K<sup>+</sup> from low concentrations in the area (less than

1 mM). The second mechanism corresponds to a low-affinity system (Km ≈10 mM) responsible for ions absorption from high concentrations. The second absorption mechanism differs from the first by the fact that it is not selective for K<sup>+</sup> (vis-a-vis of Na+), and its ability to transport K<sup>+</sup> depends on the nature of the accompanying anion [78]. Electrophysiological data obtained on roots suggest that H+-K<sup>+</sup> symports are responsible for high-affinity K<sup>+</sup> transport [79]. Low-affinity absorption is passive and involves channels.

For sodium, it is established that its initial entrance from the external environment into the cytoplasm of the roots cortical cells is passive [48], either via non-selective voltage-dependent cation channels (NSCCs) [2] or probably via some family members of sodium transporter [80,81] (Figure 2).

**Figure 2.** Na<sup>+</sup> transport at the level of the whole plant. Sodium ions can enter the cells of the root through non-selective channels (NSCCs) not formally identified at the molecular level, some of which appear to be inactivated by cyclic nucleotides (cAMP and cGMP; Maathuis and Sanders 2001), transporters HKT and high concentration of Na, KUP/HAK carriers. Na excretion cell roots to the soil solution or to the vessels of the xylem involve the antiport H+/Na<sup>+</sup> SOS1, whose activity is regulated by the SOS3 CBL protein associated with the SOS2 kinase of the HKT conveyors allow desalinization of xylem sap and phloem loading in Na<sup>+</sup> at the leaf level.

Several families of channels and transporters involved in K<sup>+</sup> and Na<sup>+</sup> transport have been identified at the molecular level in plants.

#### *7.2. Channels*

#### 7.2.1. Shaker Channels

These channels exist in plants, fungi, bacteria, and animals. The first members of this family were identified in animals.

These channels are formed with four subunits, which are organized around a central pore. The hydrophobic region of each subunit includes six transmembrane segments (TMS). A membrane loop (called P, for pore) between the fifth and sixth TMS participates in wall constitution of the central pore. Subunits can gather into homotetramers or heterotetramers. These channels are all voltage-regulated and active on the plasma membrane. They are very selective of K<sup>+</sup> beside Na+. In higher plants, several Shaker channels have been cloned and characterized. There are nine members in *Arabidopsis*, with different functional properties, expression patterns, and localization [82]. The first two Shaker channels identified in plants are AKT1 and KAT1, cloned in 1992 in *Arabidopsis* [83]. In a very interesting way, the characterization of these functional systems has shown that they act as inward rectifying channels [55,84], despite strong homology with voltage-dependent, highly selective K<sup>+</sup> channels, which act as outgoing channels. This observation has generated a lot of interest and triggered numerous studies on the structure–function relationship of these channels, with the main objectives of understanding the mechanisms of opening-closing of the pore and the regulation by the voltage.

There are three main functional types of Shaker channels—incoming rectification channels (KAT, AKT1, and ATKC1 families), outgoing rectification channels (SKOR family), and low rectification channels (AKT2 family). The fourth TMS, carrying positively charged residues (R or K), is the cause of the channel sensitivity to the voltage. The pore loop (P) studies by controlled mutagenesis have identified a motif (TxGYG) involved in ionic selectivity.

The role in the plant of several *Arabidopsis* Shakers was analyzed by reverse genetics. In a general way, these channels allow the massive exchanges of K<sup>+</sup> (influx or efflux), between the symplast and the apoplast (K<sup>+</sup> entrance of the cell for the incoming channels, exit for the outgoing channels), entry, and exit by low rectification channels. They play a role in the removal of K<sup>+</sup> from the soil solution (AKT1 and AtKC1 channels in Arabidopsis), long-distance K<sup>+</sup> transport in the xylem and phloem (SKOR and AKT2 channels), or in the transport of K<sup>+</sup> in the guard cells at the origin of stomatal movements (GORK, KAT1, and KAT2 channels) [85,86]. The shaker channels, outstanding the potassium conductance of the plasmalemma, participate in parallel to the regulation of cellular potassium concentration, to the control of the membrane potential, and to the osmotic potential regulation.

#### 7.2.2. KCO Channels

KCO (or TPK) is the second family of specific K<sup>+</sup> channels identified in plants. These channels are probably composed of either two subunits (family KCO-2P) or four subunits (family KCO-1P), which are organized around a central pore associating four domains (P for pore). In *Arabidopsis*, the KCO-2P family (two P domains per subunit) has five members, and the KCO-1P family (one pore domain per subunit) has only one member [85]. The first member of the KCO-2P family, KCO1, was discovered in silico via the use of the highly conserved GYGD motif in Shaker channels [87]. It has been expressed in insect cells, where it acts as a selective channel of K+. At the subcellular level, *AtKCO1* has been localized at the level of the tonoplast [61,88], suggesting that it plays a different role from that of the Shaker channels in transporting K<sup>+</sup> through intracellular membranes. Electrophysiological analysis of vacuolar currents on invalidated mutant kco1 suggests that KCO1 contributes to SV type currents, which are outgoing and slow vacuolar currents [61].

#### 7.2.3. Non-Selective Cationic Channels (NSCCs)

These channels, less selective of K<sup>+</sup> than Shaker, have been characterized in different cell types [89] NSCCs include CNGCs and GLRs, which are still poorly characterized. Obviously, all transporters have not significant role in potassium or sodium uptake, thus recent studies on GLRs showed that their expression throughout the plant, open up the possibility that GLR receptors could have a pervasive role in plants as non-specific amino acid sensors in diverse biological processes [90]. There has been no progress in elucidating their role in potassium and sodium uptake for the last two decades.

An indication of the CNGCs involvement in Na<sup>+</sup> influx is that the addition of similar cyclic nucleotides in the environment inhibits Na<sup>+</sup> influx and non-selective cation channel activity [91]. In animals, CNGCs are non-selective cationic channels involved in signal transduction in response to different stimuli. They are permeable to Ca2+, Na+, and K<sup>+</sup> [92]. Activation of these channels leads to membrane depolarization and cytoplasmic calcium concentration enhancement, thereby activating the signaling pathways dependent on this ion. These channels have a similar structure to the Shaker-type voltage-dependent potassium channels, a hydrophobic domain formed by 6 TMSs (named S1 to S6), and a P domain forming the pore between the fifth and the sixth TMS. In their hydrophilic N- and

C-terminal ends, they have respectively a calmodulin binding domain (CaMBD) and a cyclic nucleotide binding domain (CNBD).

In plants, a family of ion channels homologous to CNGCs animal channels was identified in the late 1990s. The first cDNA encoding a channel belonging to this family was cloned in barley by screening an expression library by searching for calmodulin-interacting proteins and was named *HvCBT1* [93]. The second member of the family was isolated from tobacco by the same approach [94]. This cDNA, named *NtCBP4*, has 61.2% identity with *HvCBT1*. In *Arabidopsis*, 20 family members, named CNGC-1 to 20, have been identified in silico by sequence analogy [95].

CNGCs are a class of nonselective cation channels that are permeable to monovalent and divalent cations such as Na+, K+, and Ca2<sup>+</sup> [89,96,97]. Although their down-regulation can prevent Na<sup>+</sup> uptake, it can potentially be concomitantly harmful to the plants, as the uptake of other elements will be compromised. However, in rice root, the downregulation of the rice (*Oryza sativa*) *OsCNGC1* contributed to the superior tolerance of the cultivar FL478 to salt stress [25], as it could avert toxic Na<sup>+</sup> influx, in contrast to the sensitive cultivar, in which the gene was up-regulated by salinity stress. Also, *Arabidopsis thaliana* null mutants, *Atcngc10*, were found to have enhanced growth under salt stress compared to wild-type plants [98]. Furthermore, Atcngc3 T-DNA insertion mutants showed an increase in tolerance to high levels of NaCl and KCl [99]. With regard to the correlation between CNGC down-regulation and stress tolerance, Mekawy et al. (2015) evaluated the relative tolerance of two rice cultivars, Egyptian Yasmine and Sakha 102. They observed that the greater tolerance of Egyptian Yasmine was partially attributable to the down-regulation of OsCNGC1, with the concomitant up-regulation of plasma membrane protein 3 (PMP3), a plasma membrane protein involved in the inhibition of excess Na<sup>+</sup> uptake at the level of the root [100].

Also, some observations show that, in *Arabidopsis*, the *AtCNGC1 and AtCNGC2* channels introduced into yeast expression plasmids appear to complement a defective yeast mutant for K<sup>+</sup> transport [95]. In tobacco, over-expression of *NtCBP4* confers transgenic plants nickel tolerance and tin hypersensitivity that decrease Ni2<sup>+</sup> accumulation and increase Pb2<sup>+</sup> accumulation [94]. Subsequently, it has been shown that NtCBP4 is expressed on the plasma membrane of tobacco cells [94]. The hypothesis is that *NtCBP4* would be a transport system (perhaps permeable to Ca2<sup>+</sup>) allowing Pb2<sup>+</sup> entry into the cell.

The data in planta on the function of a CNGC were obtained indirectly following genetic analysis on an altered *Arabidopsis* mutant in response to a pathogen [101]. This study has made it possible, for the first time, to highlight the involvement of a CNGC ion channel in a signaling pathway. In general, CNGCs are probably involved, like their homologs in animal cell signaling [89,102]. They would be permeable to monovalent and/or Ca2<sup>+</sup> cations and regulated by cyclic nucleotides and calmodulin. In plant CNGCs, the cyclic nucleotide-binding domain and the calmodulin-binding domain are both located in the C-terminal cytoplasmic region, where they overlap slightly [102].

#### *7.3. Transporters*

The KUP/HAK/KT family. A transporter belonging to a new family of K<sup>+</sup> transport systems has been identified in *Escherichia coli* (KUP1) [103] and in yeast *Schwanniomyces occidentalis* (SoHAK1) [104]. The *SoHAK1*expression in a mutated strain of *S. cerevisiae* for K<sup>+</sup> uptake systems restored growth onto a low K<sup>+</sup> concentration environment [104], SoHAK1 seems to be a high-affinity K<sup>+</sup> transporter. The homologs in plants, named KUP, HAK, or KT (for "K<sup>+</sup> uptake," "High-Affinity K<sup>+</sup> transporter," and K<sup>+</sup> Transporter, respectively), form a large family containing at least 17 members in rice [105]. The structure of these transporters is poorly known. The hydrophobicity profiles suggest that they have 12 TMSs and a long cytoplasmic loop between the second and third segments.

In plants, the first gene of the HAK/KT/KUP family, named *HvHAK*, was cloned in barley by qRT-PCR, with corresponding primers to conserved regions of *E. coli* KUP1 transporters and SoHAK1 [58]. In *Arabidopsis*, the first members identified in the HAK/KT/KUP family were cloned by complementation of a yeast mutant [106] or by the search for homologous sequences to KUP1 and HvHAK in the data banks [60]. Overexpression of AtKUP1 and AtKUP2 cDNAs induces an 86Rb<sup>+</sup> influx in yeast or in *Arabidopsis* growth cells [60,106]. For AtKUP1, the absorption kinetics in terms of concentration shows a Michaellian style in the low concentration range (less than 100 μmol. L−1), raising the kinetics associated with the mechanism I in roots [60,106]. This similarity suggested that KUP-type systems are responsible for active K<sup>+</sup> transport with high affinity in plant cells. However, the analysis of absorption kinetics by the AtKUP1 system as a function of K<sup>+</sup> concentration also reveals low-affinity transport activity [60]. In other words, the AtKUP1 system alone can generate biphasic absorption kinetics, which evokes the kinetics observed in the roots (mechanism I plus mechanism II). The duality of transport kinetics by AtKUP1 could reflect two different modes of operation for this system. No current was detected by heterologous expression of AtKUP1 in the Xenopus oocyte, and the transport mechanisms unable to be determined [60]. However, the K<sup>+</sup> influx generated by *HvHAK1* and *AtKUP1* proteins in yeast is inhibited by the presence of Na<sup>+</sup> in the environment [58,106]. The localization of *AtKUP1* gene expression analyzed by northern blot, has led to variable results in which, the mRNA is undetectable in the roots but present in the aerial parts [60], mainly expressed in roots [58,106] or undetectable throughout the plant [107]. These variations could be associated with differences in plant growth conditions. This would mean that the accumulation of *AtKUP1 mRNA* is highly dependent on environmental conditions.

By a classic genetic approach based on the search for altered mutants in absorbent hairs growth, the authors of reference [108] have isolated another family member, named *TRH1* or *AtKUP4*. The trh1 mutant shows a decrease in 86Rb<sup>+</sup> uptake. The phenotype of absorbent hair growth of the mutant plants is not restored when they are grown in an environment containing 50 mM of K+. The high-affinity K<sup>+</sup> transporter function of *TRH1* has been demonstrated by the complementation of yeast mutant *trk1*. TRH1 is expressed in the roots and in the aerial parts. It could be involved in the absorbent hair formation by allowing the influx of K<sup>+</sup> necessary for the growth and the elongation of these cells.

In general, all these HAK/KT/KUP transporters are not sufficiently characterized at the functional level, because of difficulties in expressing them in a heterologous system (a few rare members, however, express themselves in the yeast *S. cerevisiae* and/or in *E. coli* bacteria). In plants, they are present in many cell types and seem to be found on both the plasma membrane and the vacuolar membrane [105].

#### 7.3.1. HKT Transporters

HKT transporters have homologs in fungi (TRK) and bacteria. Their predicted global structure, based on sequence analyses, is similar to that of potassium channels (at 2 TMS) that exist for example in bacteria. The hydrophobic region of the HKT polypeptides comprises four repeats of the (1 TMS/1 P/1 TMS) module. In the functional protein, the four loops are arranged to form a central pore [109].

All HKT transporters characterized so far in plants are permeable to Na+, and some are also permeable to K+. The role of these transporters in planta of K<sup>+</sup> transport has not yet been clarified. Several studies have demonstrated the role of these systems in planta in the transport of Na<sup>+</sup> and revealed that HKTs are involved in the tolerance of plants to salinity.

The protein sequence of *TaHKT1* has about 20% homology with the TRK systems identified in yeast and its structure would integrate 10 to 12 hydrophobic regions likely to correspond to TMS.

The *TaHKT1* expression in the Xenopus oocyte causes an activated current by the addition of K<sup>+</sup> or other cations to the external medium. The intensity of this current increases when the pH of the external medium is lowered. However, the analysis of transgenic plants overexpressing *TaHKT1* did not make it possible to highlight a contribution of this system to the absorption function of K<sup>+</sup> by the root [110]. Subsequent analyses revealed a sensitivity of the transport to the presence of Na<sup>+</sup> in the area. These data suggested that *TaHKT1* would rather function as a high-affinity Na+: K<sup>+</sup> symport for K<sup>+</sup> (ca = 10 μM), energized by the electrochemical gradient of Na<sup>+</sup> across the membrane [111], which is completely unexpected energy coupling mechanism in plants. Moreover, this type of operation is limited to conditions of low external concentration of Na+.

When the Na<sup>+</sup> concentration is higher, the transport of K<sup>+</sup> by *TaHKT1* would be blocked and this system would function as a low-affinity Na<sup>+</sup> transporter (*Km* close to 5 mM) [111]. The physiological significance of this result remains unclear since in vivo K+-transport analyses in higher plants have never revealed Na+-K<sup>+</sup> symport activity (e.g., the addition of Na<sup>+</sup> in the medium does not stimulate K<sup>+</sup> uptake).

The only member of the HKT family in *Arabidopsis*, orthologue of the wheat *TaHKT1* gene, has been identified and designated as *AtHKT1*. The expression of this gene in yeast strains lacking the Na<sup>+</sup> efflux system aggravates their sensitivity to Na+, but it does not suppress K<sup>+</sup> transport deficiency in *trk1* and *trk2* mutants that have difficulty to absorb potassium [112]

When expressed in the *Xenopus* oocyte, *AtHKT1* exhibits strictly selective Na<sup>+</sup> transport activity, without any permeability to K+. Similarly, *AtHKT1*expression does not complement a type of *E. coli* mutant unable to absorb K+, which helps to show that *AtHKT1* carries only Na+.

*AtHKT1* is expressed in the vascular tissues of the root and the aerial parts, at the level of the phloem and the xylem parenchyma [113].

While the *AtHKT1* gene is unique in *Arabidopsis*, it is interesting to note that the HKT family in rice has 7–9 members, depending on the cultivars [114]. The analysis of the polypeptide sequences of the transporters encoded by these genes shows a rather significant difference between the members—apart from two pairs of highly homologous transporters (OsHKT3/OsHKT9 and OsHKT1/OsHKT2, 93 and 91% identity, respectively), the percentage of identity between the different transporters is between 40 and 50%. Nipponbare (japonica), *Ni-OsHKT2*, and *Ni-OsHKT5* probably do not encode functional transporters due to large deletions or the presence of "stop codons" in the reading frame. However, *OsHKT2* is identified in another cultivar (*indica*) and codes for a functional transporter, Po-OsHKT2 [115].

Localization studies by analysis of transformed plants with a promoter (GUS fusion) has shown that these two HKTs are expressed at the vascular tissue level. Specifically, all of the available data (including in situ hybridization analyses) reveal that *OsHKT1* is localized in foliar vascular tissue but also in the root cortex and endoderm [32], whereas *OsHKT8* is mainly localized at the level of the xylem parenchyma, in the roots and in the leaves [116].

The most detailed data at the functional level concerns OsHKT1. This system is one of the closest counterparts in rice of the first HKT characterized, TaHKT1 (wheat), which is a transporter of K<sup>+</sup> and Na<sup>+</sup> (*OsHKT1* and *TaHKT1* have 67% identity). *OsHKT1* has been characterized by three different teams, leading to conflicting results. Expressed in the Xenopus oocyte, OsHKT1 is described as a cationic transport system, with little discrimination with respect to the different alkaline cations [117], or as a very selective transporter of Na<sup>+</sup> [115]. Expressed in yeast, it is described either as a K<sup>+</sup> permeable transport system [117] or as a Na<sup>+</sup> transport system blocked by K<sup>+</sup> [114]. OsHKT1 expression in *S. cerevisiae* yeast mutants deficient for K<sup>+</sup> transport did not allow growth on medium poor in K<sup>+</sup> (0.1 mM KCl). The growth inhibition test on *S. cerevisiae G19* yeast strains, highly sensitive to Na<sup>+</sup> following the disruption of ENA genes (which code for Na<sup>+</sup> excretory ATPases), revealed that the cells expressing OsHKT1 exhibited more sensitivity to Na<sup>+</sup> than those expressing TaHKT1 in the presence of 50 and 100 mM NaCl.

#### 7.3.2. CHX Transporters (Monovalent Cation H<sup>+</sup> Exchanger)

These transport systems have been identified in plants on the basis of their homology with systems previously characterized in other organisms, such as bacteria, yeasts or algae. Only transporters involved in sodium compartmentalization in the plant vacuole are now relatively well known.

As in unicellular organisms, transports through the tonoplast is activated by an H+-ATPase pump that establishes a proton gradient [118]. The operation of the CHXs is electron-based and thus does not disturb the potential difference across the membrane. These systems are probably involved in both monovalent cation homeostasis and cytoplasmic and/or vacuolar pH regulation [119].

From a biochemical point of view, tonoplast antiport Na+/H<sup>+</sup> activity, which may be involved in sodium vacuolar compartmentalization, was initially demonstrated by the Blumwald group in several species [120]. This Na+/H<sup>+</sup> antiport activity was associated with a 170 kDa vacuolar protein identified in *Beta vulgaris*, whose accumulation is increased by NaCl treatments [121]. Antibodies planned against this protein inhibited the Na+/H<sup>+</sup> antiport activity. This protein was, therefore, a good candidate for the antiport activity detected on the tonoplast but its coding gene remains unknown.

From the molecular point of view, an *Arabidopsis* cDNA, named *AtNHX1*, related to the yeast ScNHX1 protein, constituted the first characterized system. Only this tonoplast antiport Na+/H<sup>+</sup> of Arabidopsis antigen has yet clearly been involved in sodium vacuolar compartmentalization [5,120,122,123]. The expression of this plant cDNA complements defective yeasts in the Na+/H<sup>+</sup> transporter present in the vacuolar membrane [123]. In *Arabidopsis*, *AtNHX1* overexpression confers to transgenic plant tolerance to external Na<sup>+</sup> concentrations above 200 mM [5]. *AtNHX1* is expressed in all plant tissues and is found on the internal system tonoplast and on the membranes (RER, Glogi). Systematic sequencing of the *Arabidopsis thaliana* genome has identified 35 genes that can encode proteins being similar to antiport Na+/H+. Constitutive overexpression of *AtNHX1* improves salinity tolerance also in tomato [124], *Brassica napus* [125], and soybean [126]. Fukuda et al. have identified an *AtNHX1* homologue in rice, *OsNHX1*. OsNHX1 expression is induced into the roots and into the aerial parts during salt stress. The authors found that OsNHX1overexpression enhances the salinity tolerance of transgenic cells and plants [127].

Within the *CHX* family, some members may be good candidates for K<sup>+</sup> transport. This is the case in *Arabidopsis* for AtKEAs that resemble the K+/H<sup>+</sup> bacterial antigens KefB and KefC. However, no experimental data for these systems are available, except for expression data in *Arabidopsis* tissues. Of the 28 KEA genes in this plant, 18 are specifically expressed during the microgametogenesis phase or in sporophytic tissues, suggesting that CHXs are involved in the regulation of potassium homeostasis in the pollen growth phase and germination [128]. Two CHXs have been characterized in more detail. *AtCHX17* appears to be preferentially expressed in roots under stress conditions, such as high salt concentrations, low external pH, low external K<sup>+</sup> concentration, and/or basic acid treatment [125]. The analysis of the mutant *AND-T atnhk17* suggests that this gene has a function in potassium homeostasis since the mutant plants accumulate less potassium than the wild ones. When expressed in yeast, *AtNHX17* co-localizes with markers of the *Golgi apparatus* and complements the pH sensitivity of a *kha1* mutant yeast strain [129], suggesting a role in potassium homeostasis and pH regulation under stress conditions. Loss of function mutants of this gene showed alteration in the ultrastructure of the chloroplast with a sharp decrease in chlorophyll level in the leaves, and an increase in cytosolic pH in the guard cells. The growth of *atnhx23* mutants was enhanced by the addition of high concentrations of potassium in the environment but altered by the addition of NaCl [130]. All these data suggest that *AtNHX23* is an antiport K<sup>+</sup> (Na+/H+) active at the level of the chloroplast envelope and involved in potassium homeostasis and perhaps in regulating the pH of the stroma.

The Na+/H<sup>+</sup> antiport systems of the plasma membrane are still poorly characterized. The only information relates to the SOS1 protein in *Arabidopsis*, which has a homologous sequence with antiport Na+/H<sup>+</sup> and would be involved in sodium efflux at the plasmalemma level [62]. Evidence has been provided that SOS1 does have antiport Na+/H<sup>+</sup> activity [62].

The sodium hypersensitive *Arabidopsis* mutant *sos1* exhibits, when cultivated in presence of moderate NaCl concentrations (40 mM), higher Na<sup>+</sup> content in its roots than those observed in the plant control of wild-type genotype. Moreover, using the reporter gene system, the authors have highlighted the localization of SOS1 in epidermal cells at the root end. These results suggest the involvement of *SOS1* in Na<sup>+</sup> efflux from the roots in the environment. In addition, it is interesting to note that SOS1 overexpression in *Arabidopsis* significantly improves plants tolerance to salinity. AtSOS1 is, therefore, an important determinant of salt sensitivity in plants. *AtSOS1* activity is controlled by *AtSOS2* and *AtSOS3*. AtSOS3 (a Ca2<sup>+</sup> affine protein belonging to the CBL family) directly interacts with AtSOS2 which a serine/threonine protein kinase is [131]. The interaction of *AtSOS3 and AtSOS2* triggers *AtSOS2* protein kinase activity, which phosphorylates and activates SOS1. Moreover, CBL/CIPK perceive cytosolic Ca2<sup>+</sup> signals resulting from salt stress and have important roles in regulating salt stress response and ion homeostasis [132].

#### *7.4. Ion Transporters Mediating Role in Salt Tolerance*

In Arabidopsis roots, *AtCNGC3* is thought to be involved in Na<sup>+</sup> fluxes. It has been reported that a null mutation in *AtCNGC3* would reduce the net Na<sup>+</sup> uptake during the early stages of NaCl exposure (40–80 mM). However, longer exposure of wild type (WT) and mutant seedlings to NaCl (80–120 mM), induces the accumulation of similar Na<sup>+</sup> concentrations in both plants [99].

These results indicate the involvement of *AtCNGC3* in Na<sup>+</sup> uptake during the early stages of salt stress. In salt-tolerant rice varieties, *OsCNGC1* is negatively more regulated than in salt-sensitive varieties subjected to salt stress conditions [133]. *Arabidopsis thaliana AtHKT1;1*, facilitates the influx of Na<sup>+</sup> into heterologous expression systems [134]. Apparently, there is a determinant of salt stress tolerance that controls the influx of Na<sup>+</sup> into the roots, resulting in lower accumulation of Na<sup>+</sup> in *athkt1* mutants than in WT plants [135]. Horie et al. demonstrated that *OsHKT2;1*, regulates the influx of Na<sup>+</sup> into root cells [32]. Plants lacking the *OsHKT2;1* gene, when exposed to 0.5 mM Na<sup>+</sup> in the absence of K+, exhibit lower Na<sup>+</sup> accumulation and reduced growth [32]. *OsHKT2;2*/*1*, a new isoform of HKT isolated from the rice plant roots that is no more than an intermediate between *OsHKT2;1* and *OsHKT2;2*, was supposed to confer salt tolerance to the Nona Bokra rice cultivar by allowing the absorption of K<sup>+</sup> in roots under salt stress [136]. It has now been shown that OsHKT2;2/1 regulates the influx of Na<sup>+</sup> into the roots of plants exposed to salt stress [137]. Note that the constitutive overexpression of *AtNHX1* in Arabidopsis improves salt tolerance [138]. Besides, overexpression of NHX1 in various transgenic plants, such as Brassica [139], cotton [17], maize [140], rice [141], tobacco [142], tomato [143], and wheat [144], exposed to NaCl concentrations ranging from 100 mM to 200 mM improve their tolerance to salt stress. The induction of NHX1 and NHX2 in response to salt stress depends on ABA [145,146]. It is widely known that, during salt stress, NHX activity increases, which promotes salt stress tolerance in many plants [147]. AtCHX21, expressed in the endodermal cells of the roots and its mutants, subjected to salt stress, accumulate less Na<sup>+</sup> in their xylem and leaves sap, indicating that CHX21 could be involved in the transport of Na<sup>+</sup> through the endoderm in the stele [148]. Under moderately saline conditions, SOS1 most likely occurs in the xylem load of Na+, due to the fact that Na<sup>+</sup> accumulates to a lesser extent in sos1 mutants [149]. In high salinity conditions, the xylem load of Na<sup>+</sup> is probably a passive process because a high concentration of cytosolic Na<sup>+</sup> in xylem parenchyma cells and a comparatively depolarized plasma membrane would favor the movement of Na<sup>+</sup> in the xylem [150]. Plants can recover xylem Na<sup>+</sup> from root cells to avoid high concentrations of Na<sup>+</sup> in aerial tissues [151]. This recovery has been observed in the basal regions of the roots and shoots of plants such as maize, beans, and soybeans [2,65]. In *Arabidopsis*, the *HKT1* mutation renders the mutants hypersensitive to salt stress and causes a greater accumulation of Na<sup>+</sup> in the leaves [152–154]. Inactivation lines have higher levels of Na<sup>+</sup> but low levels of K<sup>+</sup> in shoots. These results show that AtHKT1 is involved in the recovery of Na<sup>+</sup> from xylem while directly stimulating the load of K+. This is one of the mechanisms to maintain a higher K+/Na<sup>+</sup> ratio in shoots during salt stress in plants [155]. Synergistic effects of *SOS1*, *HKT1;5*, and *NHX1* have been proposed to regulate Na<sup>+</sup> homeostasis in *Puccinellia tenuiflora*, a halophytic plant [156]. The NaCl stress–induced vacuolar compartmentalization of its xylem load has been attributed to regulation by the differential expression of *NHX1* and *HKT1;5*. The NaCl stress-induced expression of SOS1 and NHX1 in the roots would also have been more effective in excluding Na<sup>+</sup> and Cl- in the intertidal population of *Suaeda salsa* [157]. The genetic or environmental variation of salt tolerance among halophyte populations is related to the differential expression of Na<sup>+</sup> efflux channels. Detailed structural analysis of HKT1;5 was performed in *Triticum sp*. [158]. Variations in its amino acid sequences result in a change in Na<sup>+</sup> affinity and a subsequent change in salt tolerance in two species of *Triticum*. Comparative analysis of antioxidant mechanisms in *Cynodon dactylon* (salt-tolerant grass) and *Oryza sativa* (salt-sensitive plant) was corroborated by the high expression levels of SOS 1 and NHX1 transporters in Cynodon [159]. Salt tolerance in barley has been attributed to the regulation of Na<sup>+</sup> loading in root xylem elements [160]. This is controlled by a cross between reactive oxygen species (ROS), nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase), Ca2+, and K+.

#### **8. Calcineurin B–Like Proteins (CBL) and CBL-Interacting Protein Kinases (CIPK) and Salt Tolerance in Plants**

Calcium serves as a pivotal messenger in many adaptation and developmental processes. Cellular calcium signals are detected and transmitted by sensor molecules such as calcium-binding proteins. In plants, the calcineurin B-like protein (CBL) family seems to be a unique group of calcium sensors and plays a key role in decoding calcium transients by specifically interacting with and regulating a family of protein kinases (CIPKs) [161]. Several CBL proteins appear to be targeted to the plasma membrane by processes of dual lipid modification by myristoylation and S-acylation. Additionally, CBL/CIPK complexes have been identified in other cellular localizations, suggesting that this network may confer spatial specificity in Ca2<sup>+</sup> signaling.

Molecular genetics analysis of loss-of-function mutants involves various CBL proteins and CIPKs as important components of abiotic stress responses, hormone reactions, and ion transport processes. The event of CBL and CIPK proteins appears not to be restricted to plants, raising the question about the function of these Ca2<sup>+</sup> decoding components in non-plant species.

#### *8.1. Organization of the CBL–CIPK Network*

CBL proteins have been initially identified from *Arabidopsis thaliana* [162]. Bioinformatics and comparative genomic analysis in plants have provided details about the sequence specificity, conservation, function, and complexity, and ancestry of CBL and CIPK proteins families from lower plants to higher plants. Bioinformatics research reports showed that *Arabidopsis thaliana* has 10 CBLs and 26 CIPKs [163], while in other plants, *Populus trichocarpa* has 10 CBLs and 27 CIPKs [164], *Oryza sativa* has 10 CBLs and 31 CIPKs [165], *Zea mays* has 8 CBLs and 43 CIPKs [165], *Vitis vinifera* has eight CBLs and 21 CIPKs [166], *Sorghum bicolor* has 6 CBLs and 32 CIPKs [166], *Glycine max* has 52 CIPKs [62], and *Brassica rapa L*. (Chinese cabbage) has 17 CBL genes [167].

All CBL proteins share a rather conserved core region consisting of four EF-hand calcium-binding domains that are separated by spacing regions encompassing an absolutely conserved number of amino acids in all CBL Proteins [161].

In contrast to CNB from animals and fungi, CBLs do not interact with a PP2B-type phosphatase that appears to be absent in plants.

Instead, CBL proteins interact with a group of serine-threonine protein kinases that evolutionary belong to the superfamily of CaM-dependent kinases (CaMKs) and form a phylogenetically separate cluster within the group of SNF1 related kinases. Therefore, this group has also been indicated as *Snf1* related kinase group 3 (*SnRK3*; [168]. As in other kinases of the CaMK group, the kinase domain in CIPKs is segregated by a domain called "junction domain" from the less-conserved C-terminal regulatory domain. Within the regulatory region of CIPKs, a conserved NAF domain (designated according to the prominent amino acids N, A and F) mediates binding of CBL proteins and simultaneously functions as an auto-inhibitory domain [169]. Binding of CBLs to the NAF motif removes the auto-inhibitory domain from the kinase domain, thereby conferring auto-phosphorylation and activation of the kinase [170]. Additional phosphorylation of the activation loop within the kinase domain by a yet unknown kinase further contributes to the activation of CIPKs [171].

Kinases related to CIPKs, like the AMP-activated protein kinase (AMPK), are dephosphorylated by type 2C protein phosphatases (PP2C) [172]. Interestingly, CIPKs can associate with PP2Cs like ABI1 and ABI2 via a C-terminal protein-phosphatase interaction (PPI) domain [173]. Currently, it is not known if PP2Cs may dephosphorylate CIPKs or if phosphorylation of PP2Cs by CIPKs occurs in vivo. Alternatively, the generation of CIPK/PP2C complexes could serve the formation of signaling kinase/phosphatase modules allowing for rapid alternating phosphorylation/dephosphorylation of target proteins.

In this regard, crystallization studies of CBL4 in complex with the regulatory domain of CIPK24 suggest that either CBLs or PP2Cs may mutually exclusively interact with the regulatory domain of CIPKs, and that formation of a trimeric complex is unlikely [174]. Therefore, it is tempting to speculate that PP2C interaction with the PPI domain of CIPKs leads to competitive replacing of the CBL protein, which binds to the NAF and partly to the PPI domain. Dissociation of the CBL protein would release the otherwise masked auto-inhibitory domain of the CIPK resulting in inactivation of the kinase. Alternative Ca2<sup>+</sup>-dependent binding of CBL proteins to the CIPK would favor phosphorylation of a given substrate by out-competing the PP2C from the complex. However, as the target stowage domains are still unknown for CIPKs and PP2Cs, such models can currently not consider the influence of substrate binding.

Interestingly, the PPI domain was shown to be structurally related to the kinase-associated domain1 (KA1) of the kinase KIN2/PAR-1/MARK subfamily [174,175]. Moreover, SnRK1, the SNF1 homologous in plants, also contains such a structural domain [175]. Although the function of this domain is not known, this finding may point to a mechanism of protein regulation that is conserved from animals to plants [174,175].

#### *8.2. Mechanisms of CBL-CIPK Pathway*

Structural characteristics of CBL and CIPK proteins provide the basis for their interaction. The crystal structure of the complex of Ca2+-CBL4 with the C-terminal regulatory domain of CIPK24 was first resolved [174]. It reveals how the CBL-CIPK complex decodes intracellular Ca2<sup>+</sup> signals provoked by extracellular stimulation [176]. The CBL protein harbors four elongation factor hands (EF-hands), and each EF-hand contains a conserved α-helix-loop-α-helix structure responsible for Ca2<sup>+</sup> binding [163]. The EF-hands are organized in fixed spaces that are 22, 25, and 32 amino acids distant from EF1 to EF4 in turn [177,178]. The loop region is characterized by a consensus sequence of 12 residues DKDGDGKIDFEE [163]. Amino acids in positions 1 (X), 3 (Y), 5 (Z), 7 (−X), 9 (−Y), and 12 (−Z) are responsible for Ca2<sup>+</sup> coordination [176]. EF1 contains an insertion of two amino acid residues between position X and position Y. Variation of amino acids in these positions causes the change of Ca2+-binding affinity [163]. Amino acid residues of CBL4 at positions X, Y, Z, and <sup>−</sup>Z bind Ca2<sup>+</sup> depending on side-chain donor oxygen, while backbone carbonyl oxygen atom and water facilitation are used at positions −Y and −X, respectively [176].

The CIPK protein consists of two domains, one is the conserved N-terminal kinase catalytic domain, which comprises a phosphorylation site-containing activation loop, and the other is the highly variant C-terminal regulatory domain harboring NAF/FISL motif and a phosphatase interaction motif (PPI) [170]. The NAF motif, named by its highly conserved amino acids Asn (N), Ala (A), Phe (F), Ile (I), Ser (S), and Leu (L), is necessary for binding CBL protein. This motif is necessary for sustaining the interaction between CIPK24 and CBL4 and is able to attach the C-terminal regulatory domain of CIPK24 to cover its activation loop for keeping the kinase in an auto-inhibited state (Figure 3) [179]. Attachment of Ca2<sup>+</sup> by EF-hands leads to the modification of molecular surface properties of CBL4 [176] and supports CBL4 interact with CIPK24 via the NAF motif. The interaction triggers the conformational changes of CIPK24 and exposes its activation loop [180]. Once the activation loop is free, the auto-inhibited CIPK24 is phosphorylated by an unknown upstream kinase and activates CIPK24. Subsequently, the activated CIPK24 phosphorylates the Na+/H<sup>+</sup> exchanger SOS1 on the PM to exclude the excess Na<sup>+</sup> from the cell (Figure 3a) [180]. Abscisic acid-insensitive 2 (ABI2), a member of protein phosphatase 2C (PP2C), was identified as a CIPK24-interacting phosphatase [179]. The salt-tolerant phenotype of *abi2* indicated that ABI2 is a negative regulator of CIPK24 in the SOS pathway. Up to now, the blocking mechanism of ABI2 in the CBL4-CIPK24 pathway is not yet elucidated. It is assumed that ABI2 might function in the process of dephosphorylating SOS1 (Figure 3b) or CIPK24 (Figure 3c) [179].

**Figure 3.** Mechanism of Calcineurin B-like protein 4 (CBL4)-CBL–interacting protein kinase (CIPK24) signaling pathway. **(a)** The Ca2+-binding CBL4 interacts with the NAF motif of CIPK24 and changes the conformation of CIPK24. CIPK24 exposes its activation loop and then is phosphorylated by an unknown upstream protein kinase. Activated CIPK24 phosphorylates and stimulates salt overly sensitive 1 (SOS1), subsequently. **(b)** Abscisic acid-insensitive 2 (ABI2) binds to the phosphatase interaction (PPI) domain of CIPK24 and dephosphorylates SOS1 which was phosphorylated by CIPK24. **(c)** Activated CIPK24 is dephosphorylated by ABI2, and its activity is inhibited. (Adapted from Mao et al. (2016)).

#### *8.3. Physiological Roles of CBLs and CIPKs in Plant Responses to Abiotic Stress Signals*

The physiological roles of CBL and CIPK were firstly uncovered in salt overly sensitive (SOS) pathway (Figure 4) [180]. The *Arabidopsis* mutants *sos1, sos2,* and *sos3* produced the same salt-sensitive phenotype under high-salt stress [181]. SOS3 and SOS2, also known as CBL4 and CIPK24 respectively, were demonstrated to synergistically up-regulate the activity of plasma membrane (PM)-located Na+/H<sup>+</sup> exchanger SOS1 in *Arabidopsis*, leading to the Na<sup>+</sup> efflux from cells in the high-salt environment [180].

**Figure 4.** Signaling pathways responsible for Na<sup>+</sup> extrusion in Arabidopsis under salt stress. Excess Na<sup>+</sup> and high osmolarity are separately sensed by unknown sensors at the plasma membrane level, which then induce an increase in cytosolic Ca2+. This increase is sensed by SOS3, which activates SOS2. The activated SOS3-SOS2 protein complex phosphorylates SOS1, the plasma membrane Na+/H+ antiporter, resulting in the efflux of Na<sup>+</sup> ions. SOS2 can regulate NHX1 antiport activity and V-H+-ATPase activity independently of SOS3, possibly by SOS3-like Ca2<sup>+</sup>-binding proteins (SCaBP) that target it to the tonoplast. Salt stress can also induce the accumulation of ABA, which, by means of ABI1 and ABI2, can negatively regulate SOS2 or SOS1 and NHX1.

It has been found that CBL-CIPK pathways work as regulators in nutrients transport systems, regulating sodium (Na+) [180], potassium(K+) [182], magnesium (Mg2<sup>+</sup>) [183], nitrate (NO3 −) [184], and proton (H+) homeostasis [185]. Recently, in some reviews, particular attention to the possible involvement of the CBLs and CIPKs in different ions sensitivity has been drawn [186,187].

As calcium sensor relieves in plants, calcineurin B–like (CBL) proteins provide an important contribution to decoding Ca2<sup>+</sup> signatures elicited by a variety of abiotic stresses. Currently, it is well known that CBLs perceive and transmit the Ca2<sup>+</sup> signals mainly to a group of serine/threonine protein kinases called CBL-interacting protein kinases (CIPKs).

In the year 2016, Cho et al. reported that the CBL10 member of this family has a novel interaction partner besides the CIPK proteins. Yeast two-hybrid screening with CBL10 as bait identified an *Arabidopsis* cDNA clone encoding a TOC34 protein, which is a member of the translocon of the outer membrane of chloroplasts (TOC) complex and possesses the GTPase activity. Bimolecular fluorescence complementation (BiFC) analysis verified that the CBL10–TOC34 interaction takes place at the outer membrane of chloroplasts in vivo and thus decreases its GTPase activity in *Arabidopsis* [188].

These findings indicate that a member of the CBL family, CBL10, can modulate not only the CIPK members but also TOC34, allowing the CBL family to relay the Ca2<sup>+</sup> signals in more diverse ways than currently known.

In tomato, the calcium sensor Cbl10 and its interacting protein kinase *Cipk6* define a signaling pathway in plant immunity [189]. Ca2<sup>+</sup> signaling is an early and necessary event in plant immunity. The tomato (*Solanum lycopersicum*) kinase *Pto* triggers localized programmed cell death (PCD) upon recognition of *Pseudomonas syringae* effectors AvrPto *or* AvrPtoB. In a virus-induced gene silencing screen in *Nicotiana benthamiana*, Fernando and al. identified two components of a Ca2<sup>+</sup>-signaling system, Cbl10 (for calcineurin B-like protein) and *Cipk6* (for calcineurin B-like interacting protein kinase), as their silencing inhibited Pto/AvrPto-elicited PCD. *N. benthamiana* Cbl10 and Cipk6 are also required for PCD triggered by other plant resistance genes and virus, oomycete, and nematode effectors and for host susceptibility to two *P. syringae* pathogens.

Tomato *Cipk6* interacts with Cbl10 and its in vitro kinase activity is enhanced in the presence of *Cbl10* and Ca2+, suggesting that tomato Cbl10 and Cipk6 constitute a Ca2+-regulated signaling module. Overexpression of tomato *Cipk6* in *N. benthamiana* leaves causes accumulation of reactive oxygen species (ROS), which requires the respiratory burst homolog *RbohB*. Tomato Cbl10 and Cipk6 interact with *RbohB* at the plasma membrane. Finally, Cbl10 and Cipk6 contribute to ROS generated during effector-triggered immunity in the interaction of *P. syringae* pv tomato DC3000 and *N. benthamiana*. The role of the Cbl/Cipk signaling module in PCD has been identified, establishing a mechanistic link between Ca2<sup>+</sup> and ROS signaling in plant immunity [189].

Xu et al. showed that the protein kinase CIPK23, encoded by the Arabidopsis Low-K+-sensitive 1 (*LKS1*) gene, regulates K<sup>+</sup> uptake under low K<sup>+</sup> conditions. Lesion of *LKS1* has reduced K<sup>+</sup> uptake and caused leaf chlorosis and growth inhibition, whereas overexpression of *LKS1* significantly enhanced K<sup>+</sup> uptake and tolerance to low K+. They demonstrated that *CIPK23* directly phosphorylates the K<sup>+</sup> transporter *AKT1* and further found that CIPK23 is activated by the binding of two calcineurin B-like proteins, CBL1 and CBL9 [55]. Further research on protein kinase *CIPK23* in *Arabidopsis* has revealed that CIPK23 is expressed in a variety of cell types and tissues and regulates distinct physiological processes including the opening/closing of stomata in the leaves, and the potassium uptake in the roots [190]. In addition, the authors showed that CIPK23 kinase interacts and functions with both CBL1 and CBL9 calcium sensors, providing a molecular link between intracellular calcium fluctuations and the regulation of transpiration and nutrient uptake. CBL1 and CBL9 can both recruit CIPK23 on the plasma membrane, suggesting that CIPK23-CBL complexes associated with the plasma membrane modulate the membrane on which the target proteins are located, including the AKT1 potassium channel [190,191] by proteins phosphorylation. Cheong et al provided more information on the mechanistic aspects of calcium signaling by plants. According to their finds, the combination of CIPK23 with a specific set of other components in the guard cells results in the regulation of the stomatal response to ABA, while CIPK23 and another set of components in the root tissues participate in the regulation of potassium absorption. Since CIPK23 is also present in other tissues, such as vascular tissues of roots, stems, and leaves, the authors hypothesized that CIPK23 could also be associated with other components of these tissues, for example during long-distance transport and distribution of K<sup>+</sup> throughout the plant. They showed that the other components that interact with CIPK23 include the CBL1 and CBL9 calcium sensors that functionally overlap in regulating stomatal movement and K<sup>+</sup> uptake. It is possible that other CBLs may also interact with CIPK23 in regulating K<sup>+</sup> nutrition. Such selective and overlapping interactions can encode unique responses that are different from any CBL–CIPK interaction. Among the CBLs that regulate a specific CIPK in the same process, some may play a more dominant role than others. For example, the functions of CIPK23 in stomatal response and K<sup>+</sup> absorption appear to be primarily regulated by CBL1 and CBL9, each functioning in other processes by regulating other CIPKs [190,192].

Hashimoto et al. have identified a novel general regulatory mechanism of CBL-CIPK complexes in that CBL phosphorylation at their flexible C-terminus probably induces conformational changes that enhance specificity and activity of CBL-CIPK complexes toward their target proteins. The phosphorylation status of CBLs does not appear to influence the stability, localization, or CIPK interaction of these calcium sensor proteins in general. However, proper phosphorylation of CBL1 is absolutely required for the in vivo activation of the AKT1*,* K<sup>+</sup> channel by CBL1-CIPK23 and CBL9-CIPK23 complexes in oocytes [190,193]. Moreover, the authors have shown that, by combining CBL1, CIPK23, and AKT1, the reconstituted CBL-dependent enhancement of phosphorylation of target proteins by CIPKs in vitro. In addition, they reported that phosphorylation of CBL1 by CIPK23 is also required for the CBL1-dependent enhancement of CIPK23 activity toward its substrate.

Recent studies have uncovered the crucial functions of CBL-CIPK complexes in an increasing number of biological processes like salt tolerance, potassium transport, nitrate sensing, and stomatal regulation [194]. CBL proteins determine the cellular localization of their interacting protein kinases in vivo and are essential for the activity of the resulting CBL-CIPK complexes toward their target proteins [55,184]. Despite the established importance of CBL-CIPK complexes in regulating the activity of ion channels and transporters like SOS1, AKT1, AKT2, and NRT1 [195], only very few target phosphorylation sites of CIPKs have been clearly identified. The occurrence of phosphorylation of CBLs by CIPKs appears not to be restricted to the model organism *Arabidopsis*.

In 2017, it was reported that *BdCIPK31*, a CIPK gene from *Brachypodium distachyon*, functions positively to drought and salt stress through the ABA signaling pathway [196]. Overexpressing *BdCIPK31* functions in stomatal closure, ion homeostasis, ROS scavenging, osmolyte biosynthesis, and transcriptional regulation of stress-related genes. In fact, it appears that transgenic tobacco plants overexpressing *BdCIPK31* presented improved drought and salt tolerance and displayed hypersensitive response to exogenous ABA [196]. Further investigations revealed that *BdCIPK31* functioned positively in ABA-mediated stomatal closure, and transgenic tobacco exhibited reduced water loss under dehydration conditions compared with the controls. *BdCIPK31* also affected Na+/K<sup>+</sup> homeostasis and root K<sup>+</sup> loss, which contributed to maintaining intracellular ion homeostasis under salt conditions. Moreover, the reactive oxygen species scavenging system and osmolyte accumulation were enhanced by *BdCIPK31* overexpression, which was conducive for alleviating oxidative and osmotic damages. Additionally, overexpression of *BdCIPK31* could elevate several stress-associated gene expressions under stress conditions [196].

In 2013, *TaCIPK14* and *TaCIPK29* were found to confer single or multiple stress tolerance in transgenic tobacco [197]. Transgenic tobaccos overexpressing TaCIPK14 exhibited higher contents of chlorophyll and sugar, higher catalase activity, while decreased amounts of H2O2 and malondialdehyde (MDA), and lesser ion leakage under cold and salt stresses. In addition, overexpression also enhanced the seed germination rate, root elongation and decreased Na<sup>+</sup> content in the transgenic lines under salt stress. Higher expression of stress-related genes was observed in lines overexpressing *TaCIPK14* compared to controls under stress conditions [197].

Under conditions of high salinity, *TaCIPK25* expression was markedly down-regulated in wheat roots [198]. Overexpression of *TaCIPK25* resulted in hypersensitivity to Na<sup>+</sup> and superfluous accumulation of Na<sup>+</sup> in transgenic wheat lines. The *TaCIPK25* expression did not decline in transgenic wheat and remained at an even higher level than that in wild-type wheat controls under high-salinity treatment. Furthermore, the transmembrane Na+/H<sup>+</sup> exchange was impaired in the root cells of transgenic wheat. These results suggested that *TaCIPK25* negatively regulated salt response in wheat [198].

#### **9. Conclusions and Perspectives**

The data available on the CHX family in *Arabidopsis* and other plants clearly highlight a novel and original mechanism involved in plants' tolerance to the salinity. This mechanism, which was previously not demonstrated in plants, allows detoxification of Na<sup>+</sup> in leaves by recirculation of this ion to the roots via the phloem. Plants face a dilemma regarding the transport of sodium. Sodium absorption is useful for lowering osmotic potential, being able to absorb water and maintaining turgor, but excess sodium is toxic. Many studies have focused on the toxic role of Na<sup>+</sup> in the plant during salt stress and the elucidation of the mechanisms of tolerance to this stress.

The role of Na<sup>+</sup> at lower concentrations is not well known. The current consensus is that the energization of the cell membrane is based solely on a proton gradient. However, the available data for some *CHXs* encourage us to continue to imagine that Na<sup>+</sup> (at non-toxic concentrations) can lead to symport systems and energize active K<sup>+</sup> uptake. Several indices seem to support this hypothesis, for example, AtNHX23 an antiport K<sup>+</sup> (Na+/H+) active at the level of the chloroplast envelope and involved in potassium homeostasis and perhaps in regulating the pH of the stroma. However, the Na+/H<sup>+</sup> antiport systems of the plasma membrane are still poorly characterized. The available information is only related to the SOS1 protein in *Arabidopsis*, which has a homologous sequence with antiport Na+/H<sup>+</sup> and would be involved in sodium efflux at the plasmalemma level. *SOS1* overexpression in *Arabidopsis* significantly improves plants' tolerance to salinity. AtSOS1 is,

therefore, an important determinant of salt sensitivity in plants. AtSOS1 activity is controlled by AtSOS2 and AtSOS3. AtSOS3 (a Ca2<sup>+</sup> affine protein belonging to the CBL family) directly interacts with AtSOS2, which is a serine/threonine protein kinase.

Studies on CBLs and CIPKs over the past decade have greatly advanced our knowledge of the function of single proteins in distinct physiological processes. Major advances in understanding this signaling system were through the identification of an increasing number of targets regulated by the CBL-CIPK complexes. The progress of the research on the CBL and CIPK families in different plant species other than *Arabidopsis thaliana* is still at an infant stage; in most cases, it is limited to interaction studies and expression analysis of these families.

The CBL-CIPK signaling model emphasizes the importance of future research that focuses on the molecular mechanisms underlying the regulation of transporters that allow us to better understand plant's response to abiotic stress such as salt stress and also establish a proficient method of identifying molecular targets for genetically engineered resistant crops with enhanced tolerance to various environmental stresses. Therefore, the most important challenge for future research is not only functional characterization but also the elucidating of the details of synergistic functions in this interaction network and revealing the molecular mechanisms of the complexes regulating target proteins.

**Author Contributions:** Conceptualization, H.-Y.L. and T.K.; T.K. wrote this manuscript. X.-W.L., F.-W.W., K.F.I.C., and M.N. participated in the writing and modification of this manuscript. Validation, X.-W.L. All authors read and approved the final manuscript.

**Funding:** This research was supported by the Special Program for Research of Transgenic Plants (2016ZX08010-002), the National Natural Science Foundation of China (31601323), the National Key Research and Development Program (2016YFD0101005), and the Natural Science Foundation of Science Technology Department of Jilin Province (20170101015JC, 20180101028JC, 20190201259JC).

**Acknowledgments:** The authors are grateful to Prof. Li Haiyan for the critical discussion of this article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Physiological Responses of Selected Vegetable Crop Species to Water Stress**

#### **Eszter Nemeskéri \* and Lajos Helyes**

Institute of Horticulture, Szent István University, H-2100 Gödöll˝o, Hungary **\*** Correspondence: Nemeskeri.Eszter@mkk.szie.hu; Tel.: +36-28-522071; Fax: +36-28-410804

Received: 21 July 2019; Accepted: 11 August 2019; Published: 13 August 2019

**Abstract:** The frequency of drought periods influences the yield potential of crops under field conditions. The change in morphology and anatomy of plants has been tested during drought stress under controlled conditions but the change in physiological processes has not been adequately studied in separate studies but needs to be reviewed collectively. This review presents the responses of green peas, snap beans, tomatoes and sweet corn to water stress based on their stomatal behaviour, canopy temperature, chlorophyll fluorescence and the chlorophyll content of leaves. These stress markers can be used for screening the drought tolerance of genotypes, the irrigation schedules or prediction of yield.

**Keywords:** vegetable crops; stomatal conductance; canopy temperature; chlorophyll fluorescence; SPAD; water stress

#### **1. Introduction**

As a result of climate change, the increasing atmospheric CO2 enhances the photosynthesis capacity and improves water use efficiency therefore the amount of yield will increase in most of vegetable crops, however its advantage cannot be shown under limited water and nitrogen deficiency. The high temperature during reproductive growth is harmful for many important vegetable crops, such as tomatoes, peppers, beans and sweet corn, and yield reduction will probably occur [1]. The frequency of drought periods decreases vegetable yield and quality, however soluble solid content of produce may be increased by water deficiency in some crops [2,3]. Nevertheless, the occurrence of excess precipitation causes waterlogging in soils, the symptoms of which are similar to water deficit. Soil waterlogging impedes the oxygen supply and respiration of roots, water uptake and hydraulic conductance which results in stomatal closure [4,5]. Under these conditions the stomatal closure results in a reduction of net photosynthesis which is due to the decrease in stomatal conductance, chlorophyll fluorescence and chlorophyll content of leaves [6]. Excess water causes a decline in grain filling and grain weight of corn leading to decreased yield [7]. However, water stress commonly refers to water deficits not excess water.

The selection of the vegetable crops grown under field conditions for the investigation was based on their production in the world and Europe. During the last twenty years from 1997 to 2017 the growing area of tomatoes increased intensively, that of green peas increased moderately while the growing area of snap beans and sweet corn increased slowly in the world. During this time in Europe the growing area of tomatoes and snap beans decreased from 650.4 to 496.2 thousand ha while that of green peas increased slightly from 208 to 212 thousand ha and sweet corn's increased intensively (from 50.5 to 110.1 thousand ha) (FAOSTAT 2017 [8]. In Hungary, the production of green peas and sweet corn is performed in large field growing areas (19.5 thousand hectares and 34.5 thousand hectares, respectively) while snap beans are grown in smaller ones (1.6 thousand hectares) (FAOSTAT 2017) [8]. The other aspect of the selection was the sensitivity of plant species to water stress.

Corn, soybeans, beans and peas are considered to be moderately water stress sensitive while tomatoes belong to the extremely drought sensitive group [9,10]. The responses of plant species significantly depend on the intensity and duration of stress and their stages of development. The spring-sown green pea utilizes the precipitation well (if there is any) and requires a low temperature during vegetative growth but during the flowering and seed development periods it is sensitive to water deficiency. The crops require a warm temperature, even though they have different ripening times, snap beans have short (60 days), sweet corn has medium (75–90 days) and tomatoes have long ripening times (110–130 days), their generative stages of development coincided with dry June and July, thus they require irrigation. Irrigation scheduling and the amount of irrigation water are determined by the water stress tolerance and water use of the plant varieties. The evaluation of drought tolerance in field conditions is difficult because low soil moisture and high air temperature stress generally occur together, and it is difficult to evaluate the responses separately. Drought under field conditions promotes the evapotranspiration and affects the photosynthesis, which leads to reduced yield [11]. Use of remote-sensing methods makes the measurement of physiological responses of varieties to various strong water stresses easy. These non-destructive methods help the breeder to select drought tolerant genotypes and the growers to measure the water deficit of plants and decide the time of irrigation.

The selection for water stress tolerance in traditional breeding is based on the suitability of performance under a series of environmental conditions using extensive statistical methods. This progress could be improved by the introduction of traits which contribute to the prediction of yield in the drought-prone environments. In this study, the effect of water stress on the plants and those physiological traits which influenced the yield are mainly demonstrated. Information was gathered on the physiological responses of selected vegetable species to drought to analyse their use in breeding for high and stable yield.

#### **2. Water Stress during Growth of Vegetable Crops**

Sensitivity of plants to water stress such as snap beans and green peas differs with the stages of development. During the early stages of vegetative growth most crops are less sensitive to water scarcity [2,3,12,13], but during the generative stage the water deficiency results in changes of many physiological traits [2,14–16], causing the disturbance of fertility and reduction of yield. During flowering of legume plants water stress increases the ratio of flower drop [17], decreases the pod numbers and seed abortion in the pods [18,19] and increases the ratio of curved pods [20]. Under water deficiency, bean plants produce shorter shoots and smaller leaves and decrease the length of pods [21]. Semi-leafless pea varieties have reduced leaf area that is presumed to have a low water use and they have higher water use efficiency (WUE) than traditional varieties with normal leaves [22]. In sweet corn, ear differentiation begins at the six- or eight-leaf stage growth when the water deficiency decreases the length of ears and the numbers of ear rows [23], but during tasselling the water deficiency causes significant yield reduction [24,25]. Tomatoes are most sensitive to water deficiency at fruit setting and intensive fruit development periods [3], when the increasing water stress resulted in a 25 to 50% decrease in the yield [10,26–29]. During early flowering of tomatoes, water scarcity causes flower shedding and lack of fertilization [30], and during fruit setting, plants with small sized fruits are produced [10,31].

The effect of water stress on morphology and anatomy of plants has been studied by several researchers under controlled conditions [6,32–35], however, the changes in physiological responses have been less investigated under field conditions. The physiological characteristics that have an important role in the defence against drought can be measured by remote sensing techniques using non-destructive methods in open field conditions. The leaf photosynthetic activity of plants can be monitored with measurement of chlorophyll content using a portable chlorophyll meter and chlorophyll fluorescence while the measurement of stomatal conductance indicates the severity of water stress [2,3,14]. Spectral vegetation indices such as the green normalized vegetation index (GNDVI) and the normalized differential vegetation index (NDVI) were used for monitoring the growth of the plant to detect the water stress and for yield prediction [36–39]. Crop water stress index is determined by an infrared thermometry technique to indicate the change in canopy temperature of plants under water stress conditions. More physiological indices such as leaf water potential, relative water content, turgor potential, osmotic adjustment, difference between canopy and air temperature can also be used as a screening tools for testing the water stress tolerance of genotypes [40]. Studies have focused on the identification of drought tolerance-related traits using Quantitative Trait Locus (QTL)s and Marker Assisted Selection (MAS) techniques [41–44], however, the identification of the most relevant loci controlling drought tolerance and drought-related traits could be achieved by the integration of molecular genetics with physiology [45].

#### **3. Drought Tolerance**

Adaptive mechanisms promoting the survival of plants have been grouped into three categories; drought escape, drought avoidance and drought tolerance [46]. Drought escape is the ability of plants to accomplish their life cycle before the development of soil and plant water deficit. The varieties with early flowering and short maturity are able to escape drought [47], however, they are not drought tolerant in every case. The varieties with moderate drought sensitivity developed different defence strategies to avoid short- and long-term water stress which prevents the water loss in their cells and tissues. The essential defence mechanism against drought operating in the plants is the maintenance of the water status and the reduction of tissue water loss (Figure 1).

**Figure 1.** Defence mechanism against drought (Leonardis et al. [46]).

A well-developed deeply penetrating root system provides the water uptake and maintenance of water circulation inside the plant despite the low soil-moisture content. Nevertheless, in dry soil the lives of microorganisms are retarded when the activity of mycorrhiza living symbiotically with root nodules of legumes is low, which results in a decrease in the nitrogen uptake [48,49], therefore the growth of the plant is retarded. Long-term drought of soil accelerates the senescence of root nodules and production of reactive oxygen species (ROS) [50,51], therefore the nodule weight, root and shoot weight are decreased [52]. Water stress results in a change in the proportion of root weight as the ratio of root to shoot increases [53]. Under permanent low moisture content of soil, a 27–42% decrease in leaf weight and 12–27% decrease in specific leaf area of snap bean varieties were found [54]. Tomatoes are able to survive prolonged periods of low soil water content by the development of a deep root system [28,55]. In dry years, tomatoes inoculated with mycorrhiza easily endured the water scarcity, for example larger weight fruits and higher yield were produced by deficit irrigation than under non-irrigated conditions [56].

#### **4. Reduction of Water Loss**

Drought avoidance is the ability of plants to maintain high tissue water potential despite the deficiency of soil moisture. The mechanisms developed for the reduction of water loss are related to the duration of water stress.

During short-term water deficiency the leaf movement, deep penetrating roots with strong suction force and partial or total stomatal closure provide a decrease in the water loss. Leaf movement of plants not only protects from the photodamage caused by high irradiation but reduces the effective leaf area for transpiration [57]. Paraheliotropic movement of leaves occurs mainly in beans while leaf rolling is typical for maize. Fernandez and Castrillo [58] found that the extent of leaf rolling is linearly correlated with the water potential. During leaf rolling of maize the transpiration, stomatal conductance, intracellular CO2 concentration and net photosynthetic rate decreased [59]. Pastenes et al. [60] found that the degree of paraheliotropic leaf movement was larger in the water stressed plants because of lower water potential, however, it also occurred in the water supplied crops. Deep, thick and dense roots intensively promote the use of available water and the optimal development of aboveground parts. During short-term water stress (<7 nap), abscisic acid (ABA) is produced in the roots then it is transported into the leaves where ABA induces the stomatal closure and thus decreases the water loss [61,62]. Partial or total stomatal closure restricts the transpiration therefore the water and nutrient uptake is decreased, which results in a decrease in photosynthesis and growth of plants [63]. Stomatal responses of legume species are different; under water deficiency, beans have a rapid and complete stomatal closure generating the stomatal conductivity and photosynthesis decreases significantly, whereas in cowpeas (*Vigna unguiculata*), the stomata remain partially open and have a lower decrease in their net photosynthetic rate under the same conditions [64]. Under moderate water deficit conditions, the growth of snap beans was already retarded, and their leaf area decreased while the leaf area index (LAI) of sweet corn did not change [3,15]. Nevertheless, water scarcity did not influence the leaf area of tomatoes [65] but heat and water stress up to 6 days already significantly decreased the weight of shoots and roots of tomato seedlings under a controlled environment [66].

During long-term water deficiency, plants try to prevent the dehydration of cells of vegetative and generative organs with some morphological and physiological changes. Trichome density (leaf hairs) on the leaf protects the tissues from sunlight injury, decreases the water loss by evaporation and enhances the transpiration resistance [67]. Under water stress conditions, a lower number of trichomes was found only on the basal zone of leaves on both surfaces in comparison with irrigated plants [68]. However, according to Nobel [69], the length of trichomes can be more important than their frequency. The epicuticular wax layer of the leaf controls the water flow across the cuticle and protects from high radiation and prevents damage caused by UV light. Water stress induced the increase in the wax layer on the leaf surface of peas and the wax-rich varieties had significantly lower canopy temperature [70].

Drought tolerance is the ability of the plants to endure the long-term moisture deficit and survive the water loss. When the morphological changes seem to be insufficient to avoid the water deficiency, biochemical processes of plants are activated to maintain the osmotic adjustment and the structure of cell membranes in order to avoid cell dehydration. Decreasing the water potential of leaves induces the accumulation of different osmotic compounds such as sugars, amino acids and quaternary ammonium compounds. The osmotic pressure of cells is increased by the accumulation of osmotic compounds because water movement into the cells and tissues provides the maintenance of turgor [71]. It was found that peas and castor beans exposed to water deficit accumulate a significant amount of soluble sugars and proline [72,73], and the raffinose and sucrose level of leaves are significantly increased by water stress during flowering of snap beans [74]. Action of enzymatic and non-enzymatic antioxidants is intensified to alleviate the oxidative damages in the tissues. Concerted operation of numerous water soluble antioxidant compounds (ACW) contributes to the adaptation of plants to environmental

stresses. The level of ACW in the leaves is influenced by stomatal closure because it is related to ascorbic acid redox potential of guard cells [75]. In snap bean genotypes that have a high ACW level in leaves during the flowering and pod development periods, this provides a defence against water deficiency [74].

#### *4.1. Regulation of Water Circulation in Plants under Drought*

Many physiological processes are activated to mitigate the water loss of plants (Table 1). Transpiration is restrained as a result of stomatal closure and by decreasing leaf area. Stomata play an important role in the regulation of transpiration and CO2 uptake. Use of light energy gathered by photosynthesis determines the growth and biomass production of plants. In these processes, the stomatal characteristics such as stomatal size, number and ratio of stomata on abaxial and adaxial surfaces significantly affect the C assimilation and water use efficiency (WUE) [76,77]. The higher stomatal density on the abaxial surface of the leaf is related to a higher water use efficiency [78], while those existing on upper epidermis (adaxial surface) of the leaf influenced the water use of plants [15]. Nevertheless, the number of stomata on both epidermis of leaves changes significantly depending on the variety and water supply.


**Table 1.** Physiological traits relevant for response to drought.

#### 4.1.1. Stomatal Characteristics

More stomata (134–195 stomata mm<sup>−</sup>2) were observed on the abaxial surface of tomato leaves but it was significantly less (40–62 mm−2) on the adaxial surface of leaves [76]. A significant difference can be demonstrated in stomatal density of leaves between snap beans, green peas and sweet corn grown under non-irrigated and deficit irrigated (50% water deficiency) conditions (Table 2). On the basis of 3 year experiments, on the lower epidermis of leaves the stomata density was significantly higher for snap beans under moderate and severe water stress and it was similarly high for sweet corn only in severe water deficiency, but no difference could be shown for green peas in comparison with the optimal water supplied plants [14,15,88]. On the upper epidermis of leaves more and larger sized stomata can be found for snap beans exposed to drought while there were fewer similar sized stomata for the green peas compared to the irrigated plants (Table 2). However, under water scarcity, significant differences in stomata number and size can be detected between the varieties. Under non-irrigated conditions, the size of stomata on the upper (adaxial) surface of leaves of green-podded bean varieties was smaller by 5–12%, but more of them were found than on optimal water supplied plants. Nevertheless, yellow-podded snap bean varieties had 13–18% larger sized stomata on the adaxial surface of leaves of plants exposed to water deficiency in comparison with the irrigated plants [15]. A larger stomatal density was observed for late ripening green pea varieties [14] and late ripening sweet corn hybrids under water scarcity [88] than for the short duration ones. Nevertheless, the distribution and size of stomata can be different on both areas and surfaces of the same leaf. Various number and sized stomata were detected on different areas of leaves of tomatoes; on the abaxial surface of leaves and their apical and middle areas, larger sized (32–34 μm) and more stomata were found than that on the adaxial surface. The stomata on the apical areas of leaves responded sensitively to water deficiency in that they showed fewer and larger sized stomata on the adaxial surface of leaves than for well-watered plants [68]. A significant correlation between the stomatal density and stomatal conductance (*r*<sup>2</sup> = 0.958) was established in tomatoes. According to this correlation, 130 stomata mm−<sup>2</sup> was associated with high stomatal conductance (0.1 mol H2O m−<sup>2</sup> s−1) [76]. Others [89] found that the relationship between stomatal density and WUE was positive and the size of stomata correlated negatively with the WUE for grass peas.



\* Based on average of years [14,15,88], μ = micron, I0 = non-irrigation, DI = deficit irrigation, WI = optimal water supply.

#### 4.1.2. Canopy Temperature-Transpiration

Under high photosynthetically active radiation (PAR) water deficit combined with high temperature results in an increase in leaf temperature and air temperature oscillation (±3–4 ◦C) due to the opening and closing of stomata [53]. Stomata closure triggers the decrease in the transpiration which contributes to the increase in canopy temperature of plants. One of the tasks of transpiration is to keep the temperature of plants at a favourable level for life processes. Decreasing transpiration causes the temperature of plants to increase. If soil water content is sufficient for the plant stand, the difference between canopy temperature and air temperature is zero or negative, but if the plants suffer from water stress this value is positive. An increase of 1 ◦C in canopy temperature related to a 10% decrease in the transpiration [31]. Size and stomatal density of genotypes are different thereby the transpiration varies in intensity which correlates with the difference of the canopy temperature of plants. Changes in canopy temperature have often been used to signal water stress [90] to evaluate the

drought tolerance of bean genotypes and the difference in canopy temperature and air temperature was used for the real time irrigation [91–94]. During the daytime the canopy temperature rises along with the daily air temperature and radiation as the available soil water changes. The lowest value of crop water stress index (CWSI) of maize was measured at 10:00 and 11:00 and it was the largest between 12:00 and 13:00 [95]. Under water deficiency, the canopy temperature of both snap beans and tomatoes was higher than the air temperature from 09:00 to 15:00 however, that of tomatoes was higher than the air temperature only at 12:00 and 13:00 [96]. Under water stress conditions, between 09:00 and 15:00, the canopy temperature of snap beans was higher by 3.8 ◦C than the air temperature while it was lower by 1.6 ◦C in well-watered plants [93]. When the amount of available moisture in the soil for the plants decreases, then the transpiration is limited depending on the air temperature, which results in an increasing canopy temperature. Under moderate water deficiency, at 25–50% available soil water the canopy temperature of snap beans almost coincided with the air temperature (Figure 2a) that denoted the need for irrigation [93]. Nevertheless, the available soil water below 25% was not able to satisfy the water demands of plants. In this case the cooling of the canopy was not shown by transpiration and the temperature on the foliage surface was higher than the air temperature by 2.5 ◦C on average, indicating the plants suffered from water stress (Figure 2b) [93].

**Figure 2.** Relationship between air and canopy temperature for snap beans under water deficit (**a**) and severe water stress (**b**). The thick line shows the increase in leaf temperature compared to air temperature (broken line) Source: Helyes et al. [93].

Tomatoes seemed to better use deep soil moisture with deep, strong suction force roots than the shallow rooted snap beans. Under water stress conditions, the canopy temperature of tomatoes was only higher than the air temperature by 1.8 ◦C, while it was significantly lower (0.6 ◦C) under optimal water supply conditions [92]. Air temperature had a small impact on the canopy temperature of tomatoes grown under regular irrigation and cut-off stand (i.e., irrigation was stopped 30 days before harvest) (*r*<sup>2</sup> = 0.60; *r*<sup>2</sup> = 0.55), however, the canopy temperature of water stressed plants increased with rising air temperature (*r*<sup>2</sup> = 0.59) (Figure 3) [31]. A close correlation between canopy temperature and leaf water potential of maize was established [80] and the lowest CWSI values were measured between 10:00 and 11:00 and the highest ones between 12:00 and 13:00 [95].

**Figure 3.** Relationship between the air and canopy temperature for the Kecskeméti jubileum tomato variety under rain-fed (thin line), cut-off (broken line) and regularly irrigated (thick line) conditions Source: Helyes et al. [31].

#### 4.1.3. Stomatal Conductance

Stomatal conductance indicates the speed of water vapour evaporation that depends on more plant-specific characteristics such as stomatal density, leaf age and size, guard cell and cell turgor [97]. Stomatal conductance is related to the photosynthetic assimilation rates ensuring an appropriate balance between CO2 uptake for photosynthesis and water loss through transpiration [98]. Variability in photosynthesis capacity can be explained by the CO2 diffusion through stomata and leaf mesophyll which was influenced by the mesophyll thickness and porosity and size of stomata. In drought-acclimated tomato plants the decrease in mesophyll CO2 conductance was due to an increased cell wall thickness [76]. Water stress significantly decreased the transpiration rate (37%) and stomatal conductance (26%) of maize [99]. Nevertheless, the extent of decrease in stomatal conductance depends on the growing period when the water deficiency occurred; at 7 days after anthesis of maize cultivars stomatal conductance decreased by 35% on average but at 21 days after anthesis this decrease was significantly larger (74%) under water deficiency than in well-watered cultivars [100]. In the case of tomatoes grown under non-irrigated conditions, stomatal conductance decreases from 14 to 73% depending on the weather and variety in comparison with the well-watered plants [3,32,87,101] (Table 3).


**Table 3.** Physiological traits related to water use and photosynthesis for vegetable crops under optimal water supply (OW) and water stress (WS) conditions.

\* SPAD = relative chlorophyll content of leaves; NDVI = normalized differential vegetation index.

Under water scarcity, stomatal conductance for both water and CO2 flow decreased by closing the stomata [104], thus it can be said that stomatal resistance increased. The extent of stomatal resistance mainly gives information about the speed of water vapour. Under severe water deficit conditions, stomatal resistance increased by 91% for snap beans, 34% for sweet corn and 12% for green peas in comparison with the well-watered plants (Table 3). The studies shown in Table 3 proved that snap beans responded more intensively to severe water stress than sweet corn and green peas. Flowering and pod development periods of legume crops are the most sensitive to water stress when stomatal resistance changes depending on the varieties and the degree of water stress. Under moderate water deficiency, the late ripening green pea varieties had high stomatal resistance (>3.0 s cm<sup>−</sup>1), while that of green-podded snap bean varieties was relatively low (0.8–1.2 s cm<sup>−</sup>1) and yellow-podded snap beans showed different values depending on the varieties (1.0–1.43 s cm<sup>−</sup>1) [14,15]. During tasselling, the late ripening sweet corn hybrids responded with higher stomatal resistance (3 s cm<sup>−</sup>1) to medium water deficiency than during the silking period [88].

#### *4.2. Photosynthesis in Drought*

The aspects of photosynthesis of selected vegetable crops which can be measured by remote sensing methods and used for the evaluation for drought tolerance of genotypes have to be taken into consideration. In the photosynthesis process the light capture and conversion of light energy to chemical energy is made by photosynthetic pigments in the photochemistry photosystems (PSI, PSII) of leaves. The light energy in the leaf that is not used for photosynthesis is either emitted as fluorescence or released as heat [105]. The efficiency of photosynthesis can be measured by the efficiency of PSII photochemistry or by the amount of photosynthetic pigments [106].

#### 4.2.1. Chlorophyll Fluorescence

Intense dry conditions of soil cause stomatal closure, reduced CO2 mesophyll conductance [107] and decreasing activity of PSII [108], which contributes to the decrease in photosynthesis. Photosystem II (PSII) is highly sensitive to light and drought [60] and the maximum quantum yield of PSII photochemistry (Fv/Fm) indicates an undisturbed or deficient operation of photosynthesis. Chlorophyll *a* fluorescence is considered to be suitable for the measurement of activity of photosynthesis because environmental stresses significantly affect the emission of chlorophyll fluorescence [109]. For example, UV-B radiation decreased the chlorophyll fluorescence of green peas [110] and ozone stress decreases the Fv/Fm and chlorophyll *a* concentration of leaves [111]. In higher plants, Fv/Fm fluorescence ranged from 0.78 to 0.84 [112], however this change depended on the variety and intensity of water stress.

In snap beans, the Fv/Fm ratio was relatively high (0.82–0.83) under optimal water supply conditions and it only decreased to 0.80 in the drought sensitive genotype under water stress conditions [102], which proved that chlorophyll fluorescence was not highly sensitive to water deficit.

In dry years, tomatoes grown under non-irrigated conditions had low photosynthetic activity (Fv/Fm = 0.662) and under moderate and optimal water supply conditions the Fv/Fm value ranged between 0.753 and 0.758 [3]. Likewise, the above-mentioned results from Nankishore and Farrell [32] showed a small decrease in Fv/Fm (5.1%) in tomatoes under drought (Table 3).

The maximum efficiency of PSII (Fv/Fm) of well-watered maize plants stayed constant while that of drought stressed plants stayed at control level during the first 2 days then decreased sharply as the soil became drier [103].

Use of Fv/Fm to evaluate the drought tolerance of crops is contradictory. Under controlled conditions, Fv/Fm for pot-grown grapevines decreased when water potential dropped but it seemed to be a good indicator to distinguish the moderate and severe drought stress in the field [113]. Drought stress affected the Fv/Fm parameter of the asparagus bean (*Vigna unguiculata* L.) [114]. Contrary to these results, no change was detected in the Fv/Fm for strawberries [115] and soybeans [116] grown under drought. Others [117,118] stated that PSII activity expressed by the Fv/Fm of drought tolerant tomato genotypes was less decreased under intensive water stress than sensitive ones. Likewise, Li et al. [119] found that Fv/Fm in drought tolerant barley varieties was higher than those of the drought sensitive group under drought stress. Under 4 day waterlogging conditions, the chlorophyll fluorescence (Fv/Fm) of flooding stress tolerant wax maize hybrids did not change significantly, while the photosynthesis efficiency of sensitive hybrids was relatively low and the Fv/Fm value decreased by 5.2% in comparison with the control [6]. The measurement of chlorophyll fluorescence as a rapid non-destructive method can be easily carried out in the field, thus it can be recommended for screening for drought tolerance [120].

#### 4.2.2. Photosynthetic Pigments

Environmental stresses change not only the activity of the photochemistry apparatus but the chlorophyll concentration in the leaf due to metabolic disturbance [121], whereupon the light absorption decreases. Water also absorbs the radiation in the infrared wavelength of the spectrum and as the water content of leaf decreases, the light absorption decreases and reflectance increases due to the radiative attributes of water [122,123]. Therefore, the water content of leaves and the amount of photosynthetic pigments in leaves both influence the light absorption by leaves. The light absorption of the leaf can be indirectly measured by portable chlorophyll meter. In this way the calculated SPAD values correlated with the chlorophyll content of leaves [124] expresses the efficiency of photosynthesis by the intercepted photosynthetic active radiation. The high SPAD value indicates the low water and chlorophyll concentration simultaneously in the leaf, resulting in a decrease in light absorption and increase in reflectance that is larger in extent in snap beans and smaller in green peas and tomatoes (Table 3). Iturbe-Ormaetxe et al. [125] stated that the decrease in chlorophyll *a* concentration of leaves was larger (−30%) than that of chlorophyll *b* (−20.8%) for green peas exposed to water stress than in well-watered plants.

#### **5. Relationship between Drought Stress Markers and Yield**

During reproductive periods of plants that are most sensitive to water deficiency, the changes in physiological responses can be used to screen the water stress tolerance of genotypes. During this time the water supply determines the yield production. Stomatal resistance and the relative chlorophyll content of leaves (SPAD) of the individual plants indicate the disturbance of water circulation and photosynthesis. Spectral vegetation indices indicate the absorption of solar energy of the canopy in the visible light spectrum. Health status and water deficit of vegetation can be monitored by different vegetation indices and it can also determine the need for irrigation [126–129]. The normalized differential vegetation index (NDVI) expresses the ratio of spectral reflectance on the canopy in the infrared and red region and it is used to monitor the effect of water stress on plant growth and forecast biomass [130,131].

The question is how closely the physiological traits are related to water circulation and photosynthesis and can be used to predict the expected yield. Nevertheless, the physiological traits measured during the generative stages of plant species are different (Table 4). On the basis of long-term experiments, stomatal resistance measured during flowering of snap beans and tomatoes correlated with the pod yield of individual plants and weight of tomato fruits under severe drought. A close correlation between the relative chlorophyll content of leaves (SPAD) and weight of tomato fruits and final yield was detected under both mild and severe water deficiency which can be used for selection of genotypes with water stress tolerance. During tasselling of sweet corn, the expected yield of plants can be less predicted by stomatal resistance (47%) and to a higher extent by spectral traits (58–68%) under moderate water deficiency. During flowering of green peas, stomatal resistance and chlorophyll content of leaves showed a close correlation with the expected yield only under severe drought (Table 4).


**Table 4.** Correlation coefficients between physiological traits measured during flowering and yield under drought.

\* during tasselling <sup>y</sup> = fruit weight (g), gs =stomatal conductance, SR = stomatal resistance, NI = non-irrigation, DI = deficit irrigation Source: [2,3,14–16].

Other researchers used the normalized differential vegetation index (NDVI) for yield prediction; it was successful for castor beans [132], soybeans [133] and beans [134]. According to Spitkó et al. [38], a medium correlation (*r* = 0.5–0.6) was detected between NDVI and final yield at 15 days after flowering of maize but not during the flowering period. Different stress indices such as stress degree days (SDD) or crop water stress index (CWSI) can be used to evaluate the water stress tolerance of genotypes [25] for scheduling of irrigation [93] and maybe for prediction of yield. In the case of sweet corn, significant

negative correlation was detected between the CWSI and chlorophyll content of leaves (*r* = 0.802) but for the CWSI, a significant positive (*r* = 0.478) correlation was observed with the yield [25].

Helyes et al. [31] found a close correlation between the stress degree days (SDD) and yield of tomatoes. If the canopy temperature exceeded the air temperature (at noon), transpiration was reduced, which indicated water stress and resulted in yield reduction and quantity. Figure 4 shows the interrelation between the canopy and air temperature difference values and the yield. In our experiments the correlation was significant at *p* = 0.01 level with *r*<sup>2</sup> = 0.57 correlation coefficient. High yield per hectare can be achieved if the difference between the cumulative canopy and the air temperature is negative during the growing season.

**Figure 4.** Correlation between canopy and air temperature differences and yield Source: [31].

#### **6. Use of Physiological Characteristics**

The use of physiological traits in a breeding program, either directly by selection or stress markers, depends on their genetic correlation with the yield, heritability and genotype × environment interaction [11,135]. Under water stress, high heritability of stomatal resistance, photosynthesis rate and transpiration rate (*h*<sup>2</sup> = 0.91–0.99) was found for *Vigna mungo* that gives a possibility for successful selection of genotypes [35]. Under severe drought, stomatal conductance and relative chlorophyll content of leaves (SPAD) measured during flowering correlated with the expected yield therefore they are suitable for the selection of individual genotypes for green peas and tomatoes, while the use of these traits for the selection of sweet corn can be efficient only under moderate water stress (Table 4). In the case of snap beans, because the water deficiency has a significant effect on leaf area, the normalized differential vegetation index (NDVI) measured during flowering can predict the expected yield more efficiently than the SPAD value of the leaves of individual plants.

Application of remote sensing techniques makes monitoring the water status of plants and real time irrigation easy [39,136]. The trend in the canopy temperature and the difference between the leaf temperature and air temperature (SDD) can be considered to be the water stress markers of plants [92]. The relationships between the canopy temperature, air temperature and transpiration involving the atmospheric and soil conditions and plant characteristics [40], was used to develop the crop water stress index (CWSI), indicating the need for irrigation. During drought, the decrease in NDVI occurred to a larger extent in snap beans, while it was less in sweet corn and hardly changed in green peas in

comparison with optimal water supply conditions (Table 3). This explained why the NDVI was used as spectral indicator for irrigation scheduling mainly in snap beans [136].

In summary, some of the physiological traits influencing the decrease of water loss and biomass production of plants can be used to evaluate the water status of vegetable crops and the water stress tolerance of genotypes. During the generative period, under water deficit conditions, the changes in the stomatal conductance and chlorophyll content of leaves for individual plants is suitable for the estimation the productivity of genotypes. Nevertheless, leaf area of crops should be taken into consideration as they determine the transpiration and their chlorophyll density influences the intensity of photosynthesis and finally the yield. Water stress indices and spectral vegetation indices seemed to be more appropriate in the detection of perceived water deficiency than for the prediction of final yield.

**Author Contributions:** E.N. planned and wrote the first draft of the review. L.H. contributed to the writing and reviewed the final draft.

**Funding:** The publication was supported by the Ministry of Human Capacities grant Higher Education Institutional Excellence Program in framework of the water related research of Szent István University and grant number TUDFO/51757/2019-ITM FEKUTSTRAT and EFOP-3.6.3-VEKOP-16-2017-00008. The project is co-financed by the European Union and the European Social Fund.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Phytohormone-Mediated Stomatal Response, Escape and Quiescence Strategies in Plants under Flooding Stress**

### **Kazi Khayrul Bashar 1,\*, Md. Zablul Tareq 2, Md. Ruhul Amin 1, Ummay Honi 1, Md. Tahjib-Ul-Arif 3, Md. Abu Sadat <sup>1</sup> and Quazi Md. Mosaddeque Hossen <sup>1</sup>**


Received: 8 January 2019; Accepted: 16 January 2019; Published: 22 January 2019

**Abstract:** Generally, flooding causes waterlogging or submergence stress which is considered as one of the most important abiotic factors that severely hinders plant growth and development. Plants might not complete their life cycle even in short duration of flooding. As biologically intelligent organisms, plants always try to resist or survive under such adverse circumstances by adapting a wide array of mechanisms including hormonal homeostasis. Under this mechanism, plants try to adapt through diverse morphological, physiological and molecular changes, including the closing of stomata, elongating of petioles, hollow stems or internodes, or maintaining minimum physiological activity to store energy to combat post-flooding stress and to continue normal growth and development. Mainly, ethylene, gibberellins (GA) and abscisic acid (ABA) are directly and/or indirectly involved in hormonal homeostasis mechanisms. Responses of specific genes or transcription factors or reactive oxygen species (ROS) maintain the equilibrium between stomatal opening and closing, which is one of the fastest responses in plants when encountering flooding stress conditions. In this review paper, the sequential steps of some of the hormone-dependent survival mechanisms of plants under flooding stress conditions have been critically discussed.

**Keywords:** flood; plants; hormonal homeostasis; physiological activity

#### **1. Introduction**

Several oxygen limiting factors, such as flooding, waterlogging, and partial or full submergence are detrimental for normal growth and development of plants [1]. Sea, river belt and low land areas experience limited or reduced crop production due to the flooding stress. Plants try to adapt to these adverse conditions by applying several strategies, like the storage of energy, elongation of the petiole or internodes, maintenance of water level by regulating stomatal movements, the formation of adventitious roots, development of aerenchyma etc. [2–5]. Crop plants simultaneously activate various biochemical reactions, molecular and signaling pathways, and physiological processes to cope with this oxygen-limiting condition [1,6].

Phytohormone plays a central role in all morphological, anatomical, biochemical, molecular and signaling mechanisms for plant survival under oxygen-limiting stress conditions. Predominantly, ethylene, gibberellins (GA) and abscisic acid (ABA) play the most crucial roles during submergence stress conditions in plants [7]. Ethylene directly and/or indirectly induces GA expression that aids

plants in carrying out the escape and/or quiescence strategy. Petiole/internode elongation and storage of carbohydrates results from the escape and quiescence strategy, respectively in plants under submergence stress [8]. GAs are directly involved in escaping submergence stress in both DELLA (N-terminal D-E-L-L-A amino acid sequence) dependent and independent pathways [9]. Furthermore, ethylene and ABA are directly responsible for the stomatal closure under waterlogging or post-waterlogging stress [10]. On the other hand, GAs are responsible for the stomatal opening under the same stress conditions [11]. So, balanced expression of these three hormones is highly indispensable to maintain the ratio of stomatal opening and closing in plants under excess water stress conditions.

Based on the above facts, this review paper proposes flowcharts of sequential steps for stomatal closing and escaping and quiescencing strategies to offer the best possible explanations. This might help plant scientists to consider several factors during the development of waterlogging-tolerant plant varieties.

#### **2. Ethylene, GA and ABA Interactions in Plants under Submergence Stress**

Under submergence stress, ethylene, GA and ABA play influential roles in the survivability of the submerged plants, where ABA biosynthesis is reduced and GA signaling is induced for shoot elongation, especially in rice plants (variety: C9285) [12,13].

Plants have evolved two types of strategies, i.e., escape strategy and quiescence strategy, to survive under flooding stress. In the escape strategy, the rice plant elongates its internodes under slow progressive flooding conditions. On the other hand, rice plants reserve energy under deep transient flash flooding conditions to escape the unfavorable conditions, which is termed the quiescence strategy [14,15]. It is quite interesting that two different functions of two distinct gene families under the same subgroup of a transcription factor are involved in submergence tolerance in plants. This is a complex process, but one that is interesting to study, and required to unveil this mechanism by functional genomics. Under the *AP2/ERF* (*Apelata2/Ethylene response factor*) transcription factor (TF) subgroup, the *Snorkel (SK)* gene is responsible for internode elongation, whereas *Sub1A* (*Submergence 1A*) is related to shoot elongation restriction [16].

In low land rice varieties, the *ERF* transcription factor, *Sub1*, is considered as a major player in submergence tolerance [17,18]. As a quiescence strategy in rice genotypes, ethylene directly enhances *Sub1A* expression. *Sub1A* induces the over accumulation of GA signaling repressors, *Slender Rice-1* (*SLR1*) and *SLR1 Like-1* (*SLRL1*). *Sub1A,* a group under VII *AP2/ERF* transcription factor, restricts shoot elongation by suppressing the *SLR1* and *SLRL2* for saving energy that is necessary for growth and development under desubmergence conditions. Therefore, ethylene is indirectly responsible for the induction of these GA signaling repressors and the reduction of GA responsive gene expression under submergence stress through the *Sub1A*-dependent pathway. *Sub1A* may also play an important role in limiting ethylene production during submergence stress conditions, resulting in the restriction of ethylene-induced enhancement of GA responsiveness in submergence tolerant varieties [17]. The function of *Sub1A1* is also regulated by MPK3 (Mitogen-activated protein kinase3). MPK3-dependent phosphorylated *Sub1A1* binds to the G-box of the MPK3 promoter to regulate its activity [19].

On the other hand, ethylene can directly enhance GA responsive shoot elongation in rice genotypes as an escape strategy of submergence stress, which is not only a highly energy-consuming process, but also requires the continuous production of energy. Due to the lack of *Sub1A* in submergence-susceptible genotypes, plants' survivability in the desubmergence stage is very low or limited [20]. Thus, *Sub1A* acts as a limiting factor for ethylene-promoted GA responsive shoot elongation in tolerant genotypes during submergence conditions to store or save the energy required for normal physiological and biochemical activities [21].

Moreover, in rice, *Sub1A* actively participates in maintaining chlorophyll contents and carbohydrate reserves in photosynthetic tissues [22]. *Sub1A* increases brassinosteroid (BR) levels in rice plants under submergence stress. BR induces GA catabolic *SLR1* proteins which restrict shoot elongation under oxygen-limiting conditions. In addition, BR enhances the expression of *GA2ox7* (*GA 2 oxidase7*) as an early response (within 1 d after submergence), which is responsible for catabolic GA degradation of endogenous GA4 [23] (Figure 1). *OsAP2-39*, an *Apelata 2* (*AP2*) transcription factor, directly regulates the ABA biosynthetic gene *OsNCED1* (*9-cis-epoxycarotenoid dioxygenase 1*) and the GA repressing *EUI* (*Elongation of uppermost internode I*) gene. Over-expression of *OsAP2-39* has been shown to enhance drought resistance in rice by producing more ABA and degrading GA [24], which supports the antagonistic crosstalk between ABA and GA as a crucial mechanism to control plant growth and development under abiotic stress conditions [25]. From the above discussion, it can be said that *OsAP2-39* might have great scope to restrict GA signaling in plants under waterlogging or submergence conditions, and stomatal control through ABA signaling pathways under desubmergence conditions.

**Figure 1.** Ethylene-mediated escape and quiescence strategies in plants against submergence stress. Enhanced ethylene expression under submergence stress induces *Snorkel* and *Sub1A* genes for escape and quiescence strategies respectively. Ethylene suppresses ABA expression, which triggers GA1 expression for escape strategy in plants. ABA: Abscisic acid; AP2/ERF: Apelata2/Ethylene response factor; Brs: Brassinosteroids; DWF1/4: Dwarf 1/4; GA1: Gibberellins 1; GA2ox7: GA 2 oxidase7; GA3ox: GA 3 oxidase; Sub1A: Submergence1A; SLRL1: SLR1 Like-1.

In deep water rice, ethylene enhances the expression of *Snorkel1* (*SK1*) and *Snorkel2* (*SK2*) which are responsible for significant internode elongation via GA signaling pathways [26]. *Snorkel* genes are only present in lowland deepwater rice accessions for their internode elongation through downregulation of BR biosynthesis as an escape mechanism under submergence stress [1] (Figure 2).

**Figure 2.** Proposed model for rice internode elongation under submergence stress conditions. Modified figure [13]. *EIL1a* binds with the both promoters of *SK1/2* and *SD1* in rice under submergence stress conditions. *SK1/2* downregulates Brs that induce internode elongation through the accumulation of GAs. On the other hand, *SD1* induces internode elongation through the accumulation of GA4. Brs: brassinosteroids; EIL1a: Ethylene Insensitive 3-like 1a; GA: Gibberellins; SD1: Semidwarf1; SK1/2: Snorkel 1/2.

In rice, *Snorkel* dependent (variety: C9285) and independent (variety: T65) internode elongation for escaping the submergence stress conditions was discovered by [13]. In this escaping strategy the accumulated ethylene enhances *OsEIL1a* (*Ethylene Insensitive 3-like 1a*) in rice plants. In the *Snorkel* dependent escaping strategy, *OsEIL1a* binds to the promoter of *Snorkel1/2* to accumulate the transcript of *Snorkel1/2* [27]. This leads to the downregulation of BR to induce GA-mediated (mainly GA1) internode elongation [22,25]. GA response enhances the expression of cyclins transcription factor, which leads to rapid cell division in lotus (*Nelumbo nucifera*) under submergence stress conditions [28]. However, as a *Snorkel* independent escaping strategy, *OsEIL1a* binds to the promoter of *SD1 (Semidwarf1)* for DWH (deepwater rice-specific haplotype) mediated rapid amplification of *SD1* transactivation. The SD1 protein catalyzes the biosynthesis of bioactive GA species, GA4 that increases GA4 level in addition to GA1 after submergence. GA4 is more capable of internode elongation than GA1 [13]. So, *Snorkel* independent *SD1* mediated internode elongation in rice is comparatively faster than that of the *Snorkel* dependent pathway (Figure 2).

Rumex plants showed *Snorkel* independent petiole elongation. In flood-tolerant *Rumex palustris,* ethylene reduces *RpNCED* expression which inhibits ABA biosynthesis. Thus, *R. palustris* elongates its petiole by degrading ABA into phaseic acid and enhancing GA1-mediated gene expression in an ethylene-mediated pathway under oxygen-limited condition [29]. *R. palustris* maintains gas exchange between the submerged tissues and the atmosphere by elongating shoots under long-term flooding stress condition [29,30]. In this stress condition, the accumulation of ethylene not only breaks down ABA into phaseic acid, but also downregulates ABA expression by inhibiting 9-cis-epoxycarotenoid dioxygenase expression. Elevated content of ethylene independently degrades ABA through ABA 8' hydroxylase pathway under submergence stress conditions [31]. Inhibition of ABA stimulates GA 3-oxidase to produce bioactive GA (GA1). The downstream function of GA is to mobilize food materials by the breakdown of starch and cell wall loosening, which ultimately elongates internode or leaf sheath to escape submergence or waterlogging stress conditions [30]. In rice, a similar type of ABA-dependent GA expression was also reported [32]. Both the quiescence strategy and escape

strategy are considered as the survival mechanisms of plants under submergence stress, but escape strategy is considered as a yield limiting factor in rice plants under de-submergence stress [33].

#### **3. DELLA-Dependent GA Expression under Submergence Stress**

Gibberellin-insensitive dwarf 1 (GID1), a soluble receptor for GA signaling, is involved in GA-mediated signaling pathways in plants under stress conditions, especially during abiotic stress conditions [34,35]. GA binds to GID1 and generates a GID1-GA complex which has the ability to interact with different growth repressors like DELLA or SLENDER1 (SLR1), a DELLA ortholog in rice [33]. Five types of DELLA are present in the model plant *Arabidopsis thaliana*, namely Gibberellin-insensitive (GAI), Repressor of GA1-3 (RGA), RGA-like1 (RGL1), RGL2 and RGL3 [36]. GID1-GA complex facilitates GA-mediated interaction between GID1 and DELLA protein, which is responsible for conformational changes in DELLA proteins. The *Sleepy1* (*SLY1*) gene contain the F-box domain, which is a positive regulator of GA signaling in *Arabidopsis*. *Sleepy1* (*SLY1*) in *Arabidopsis* and GID2 in rice are capable of recognizing this change of DELLA proteins where *SCF* (*SLY1*) (Skp1, Cullin, F-box), E3 ubiquitin ligase ubiquitinates DELLA protein, targeting DELLA for degradation through proteolysis by the 26S proteasome [34,35,37]. Thus GA expression is continued in plants by suppressing the expression of DELLA protein (Figure 3A).


**Figure 3.** DELLA-dependent and -independent GA signaling in plants under submergence stress conditions. (**3A**) DELLA-dependent escape strategy in plants; GID-GA complex makes conformational change in DELLA. After that SCF ubiquitinates DELLA protein which induces GA expression for petiole/stem elongation. (**3B**) DELLA-independent escape strategy in plants; Ca2+ accumulation enhances CDPK1 production for the phosphorylation of RSG to translocate it into the cytoplasm from nucleus. 14-3-3 inactivates RSG as a GA-mediated submergence escaping strategy. CDPK1: Ca2+-dependent protein kinase 1; DELLA: N-terminal D-E-L-L-A amino acid sequence; GA: Gibberellins; GA20ox1: GA 20 oxidase1; GID1: Gibberellin-insensitive dwarf 1; GID2: Gibberellin-insensitive dwarf 2; RSG: Repression of shoot growth; SCF: Skp1, Cullin, F-box; SLY1: Sleepy1.

#### **4. DELLA Independent GA Expression under Submergence Stress**

GAs can increase cytoplasmic Ca2+ very rapidly during various cellular processes occurring inside the cell. However, the mechanism of increasing GA-mediated cytoplasmic Ca2+ in a DELLA-independent manner is still unknown. The GA-mediated increase of Ca2+ and degradation of DELLAs are completely independent processes [38]. An elevated level of cytoplasmic Ca2+ activates Ca2+-dependent protein kinase (NtCDPK1) via a DELLA-independent GA pathway in tobacco [39]. NtCDPK1 is responsible for the translocation of Repression of shoot growth (RSG) from the nucleus to the cytoplasm [40,41]. RSG represses the expression of two important GA biosynthetic genes, *NtKO* and *NtGA20ox1*, which are considered as GA enhancing genes [42,43]. RSG binds to the promoter region of *NtGA20ox1* through GA-mediated approach while RSG binds to *NtKO* promoter in the independent manner of GA concentrations [43]. NtCDPK1 acts as a RSG kinase and phosphorylates RSG, which promotes 14-3-3 to bind with RSG at cytoplasm [40,44,45]. In rice plants, CDPK is induced under low oxygen stress for survivability under anaerobic conditions [46]. 14-3-3 proteins directly bind to the RSG in the cytoplasm and regulate RSG function negatively, making non-functional RSG [41,47]. So, inactivation of RSG by 14-3-3 proteins indirectly helps to express both *NtKO* and *NtGA20ox1* to continue GA biosynthesis for different cellular processes (Figure 3B).

#### **5. ABA in Plants under Waterlogging Stress**

Imbalanced conditions between leaf transpiration and root water uptake creates dehydration (physiological drought) stress in plants, that is noticed in plants under waterlogging stress with partially or fully damaged root tissues. The plant hormone ABA has the ability to modify root hydraulic properties [48]. For example, ABA downregulation and upregulation in tomato plants expresses lower and higher hydraulic conductance, respectively [49,50]. Depending on both the flooding duration and plant species, ABA differentially responds in leaves and in the roots of plants. *Malus sieversii* is considered as less tolerant to hypoxia than *Malus hupehensis,* showing a larger increase in ABA in both the leaf and root tissues [51]. In *Gerbera jamesonii,* ABA levels were increased in both leaf and root, where a transient increase in root follows a sharp decrease in the recovery period [52]. ABA is also increased in roots and leaves of *Triticum aestivum* L., but gradually decreases after reaching certain levels [53].

#### **6. Stomatal Regulation at Waterlogging Stress**

Stomata is a specialized epidermal pore-like structure consisting of two guard cells through which plants exchange both CO2 and O2 with the environment [54,55]. Stomatal conductance was insignificantly affected by flooding stress despite a significant reduction of photosynthesis in both flood-tolerant and flood-sensitive poplar genotypes [56]. A similar finding was also reported in maize plants where flooding resulted in a significant decrease in photosynthesis and ribulose-1,5-bisphosphate carboxylase activity without a noticeable reduction in the rates of stomatal conductance [57]. In GID1 mutated rice plant, increased chlorophyll content has been found, which is subsequently responsible for the increase of carbohydrate production and decrease of reactive oxygen species (ROS) accumulation under submergence stress [58]. Reduction in leaf transpiration is a common phenomenon of flooding stress, which affects the lowered stomatal aperture [59–61]. It is difficult to believe that flooding paradoxically causes leaf dehydration in plants [62–64]. Stomatal closing is operated by the turgor pressure and volume of guard cells. ABA-dependent signaling effluxes of anions, potassium ions and conversion of malate into starch trigger the reduction of turgor pressure, as well as changing the volume of guard cells close the stomata [65]. Under hypoxic stress, *hypoxia responsive universal stress protein 1* (*HRU1*) activates *AtrbohD* (*Respiratory burst oxidase homolog protein D*), following interaction between *ROP2* (*Rho of plants 2*) and *AtrbohD* in *Arabidopsis* under low oxygen stress conditions. NADPH oxidase *AtrbohD* is responsible for alcoholic fermentation and ABA-dependent stomatal closure in plants under abundant water conditions [66,67]. It has been

reported that barley plants swiftly close the stomata after flooding stress imposition [68]. A similar phenomenon was also observed in pea plants under flooding conditions, where a prompt closure of stomata from older leaves was recorded. Wilting of younger leaves was protected by older pea leaves by increasing ABA-dependent stomatal closure [69]. Unwilted younger pea leaves might result from the ABA transportation from older to younger leaves or de novo biosynthesis of ABA in the younger leaves [70]. Though *Populus deltoides* is considered as a waterlogging tolerant plant species, it showed a significant reduction in stomatal conductance at both waterlogging and de-waterlogging stress, with an exception at recovery period after 90 days of waterlogging stress [71].

#### *Hormonal Regulation in Stomatal Closing of Plants under Waterlogging Stress*

Several waterlogging related experiments showed a positive correlation between ABA accumulation and increase in ROS, in soybean roots [72], barley roots and leaves [73], maize leaves [74], bread wheat roots [75]. The enzymatic mechanisms responsible for ABA-triggered ROS generation in guard cells at the molecular level are little known at present. ABA initiates H2O2 generation by using the plasma membrane NADPH oxidase [76]. H2O2 activates plasma membrane Ca2+ channels, resulting in an increase in Ca2+ level in guard cells. [77]. Inhibition of inward K<sup>+</sup> channels in guard cells is the result of increased Ca2+ level in the cytoplasm of guard cells [78,79]. This results in reduced solute accumulation following a reduced amount of water entrance in the guard cells and ultimately leading to stomata closure [80]. In *Arabidopsis*, H2O2 can also stimulate NO (nitric oxide) production to induce stomatal closure [81]. Ethylene and ABA activate CuAO (copper amine oxidase) in *Vicia faba* [82,83]. Oxidation of putrescine by CuAO produces H2O2, follows stomatal closure in *V. faba* [82] (Figure 4). ABA is also responsible for stomatal closing in plants as a survival mechanism under post-waterlogging stress [84].

Moreover, H2O2 is also produced by extracellular calmodulin (ExtCaM) which is activated by heterotrophic G protein [85]. In rice, accumulated ethylene induces G protein for aerenchyma formation under flooding stress [86]. G protein induces H2O2 production for epidermal cell death in rice under submergence stress [87]. Inactivation of CTR1 (Constitutive triple response 1) is induced by the binding of ethylene to ETR1 (Ethylene receptor 1), ERS1 (Ethylene response sensor 1) and EIN4 (Ethylene insensitive 4), resulting in the activation of Galpha (G protein alpha subunit). Galpha promotes H2O2 production in plants via NADPH oxidases. ETR1 and ERS1 translocate the signals of H2O2 to EIN2, EIN3 and ARR2 (2-component response regulator), which are essential for stomatal closure functioning [88]. Activated G proteins may inhibit inward K+ channels via an elevated level of cytoplasmic Ca2+ in the guard cells [78,79]. From the above discussion, a proposed signaling pathway may work for stomatal closure in plants under waterlogging stress (Figure 4).

In *Arabidopsis thaliana,* flooding stress operates H2O2-mediated stomatal closure followed by an increase in antioxidant enzyme activities [89]. Improvement of anoxia tolerance was confirmed by applying exogenous ABA in different plants including maize, citrus, lettuce and *Arabidopsis* [90–94]. Waterlogged pea plant restricted its leaf ABA to translocate in shoot, while in non-waterlogged plants, ABA moves readily from shoots to roots [95,96]. This extra ABA in pea leaves was responsible for reducing leaf transpiration by the closing of stomata [96].

Stomata closing may occur in an ABA-independent manner. As for example, ABA was increased in citrus after 3 weeks of flooding, indicating that closing of stomata and increase of ABA are independent in citrus plants upon flooding stress, and not only that ABA was transported to younger leaves from older leaves, rather than, as expected, being transportated from roots to shoots [97].

**Figure 4.** Proposed model for hormone mediated stomatal closure under waterlogging and/or post-waterlogging stress conditions. Modified figure [84]. Ethylene and/or ABA are directly or indirectly enhance H2O2 production under waterlogging and/or post-waterlogging stress conditions. H2O2 is directly responsible for closing of stomata or indirectly increasing Ca2+ in the guard cell for stomatal closure by reducing the volume of the guard cell. ABA: Abscisic acid; AREB/ABF: (ABA)-responsive element binding proteins/ABRE(ABA-responsive element)-binding factors; ARR2: 2-component response regulator; CTR1: Constitutive triple response 1; CuAO: Copper amine oxisase; DREB/CBF: Dehydration-responsive element-binding/C-repeat binding factor; DST: Drought and salt tolerance; EIN: Ethylene Insensitive; ERS1: Ethylene response sensor 1; ETR1: Ethylene receptor 1; ExtCaM: Extracellular Calmodulin; GORK: Guard cell outward-rectifying K+; KUP6: K+ uptake transporter 6; MPK3/9: Mitogen-activated protein kinase3/9; MYB: **My**elo**b**lastosis; MYC2: **My**elo**c**ytomatosis; NAC: NAM (No apical meristem), ATAF (Arabidopsis transcription activation factor) and CUC (Cup-shaped cotyledon) transcription factor; P2C: Protein phosphatase 2C; ROS: Reactive oxygen species; SLAC1: Slow anion channel-associated 1; SRK2E: SNF1 (Sucrose nonfermenting 1)-related protein kinase 2; WRKY: W-tryptophan, R-arginine, K-lysine, Y-tyrosine.

#### **7. Conclusions**

From this above discussion, it can be concluded that ethylene directly or indirectly regulates the expression of gibberellins (GAs) and abscisic acid (ABA) in plants under flooding stress conditions. However, ABA plays a major role in stomatal closing, whereas escape and quiescence strategies are controlled by the expression of GA. Finally, it can be concluded that plants maintain their internal homeostasis by balancing hormonal cross talk under excess water stress.

**Author Contributions:** Conceptualization and writing-original draft preparation, K.K.B.; Writing-review and editing, K.K.B.; M.Z.T, M.R.A., U.H. and M.T.-U.-A.; Supervision, M.A.S. and Q.M.M.H.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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