*Article Linum usitatissimum AccD* **Enhances Seed Fatty Acid Accumulation and Tolerance to Environmental Stresses during Seed Germination in** *Arabidopsis thaliana*

**Rui Du, Xinye Li, Huan Hu, Yu Zhao, Mingxun Chen and Zijin Liu \***

National Yangling Agricultural Biotechnology & Breeding Center, Shaanxi Key Laboratory of Crop Heterosis and College of Agronomy, Northwest A&F University, Yangling 712100, China; durui000@nwafu.edu.cn (R.D.); lixinye0324@163.com (X.L.); huhuan@nwafu.edu.cn (H.H.); nwafu\_zy@163.com (Y.Z.); cmx786@nwafu.edu.cn (M.C.)

**\*** Correspondence: liuzijin@nwafu.edu.cn

**Abstract:** Flax (*Linum usitatissimum* L.), as an important oil-producing crop, is widely distributed throughout the world, and its seeds are rich in polyunsaturated fatty acids (FAs). Previous studies have revealed that *Arabidopsis thaliana* ACETYL-CoA CARBOXYLASE (AtACCase) is vital for FA biosynthesis. However, the functions of *L. usitatissimum AccD* (*LuAccD*) on FA accumulation and seed germination remain unclear. In the present study, we cloned the LuAccD coding sequence from the flax cultivar 'Longya 10', identified conserved protein domains, and performed a phylogenetic analysis to elucidate its relationship with homologs from a range of plant species. Ectopic expression of *LuAccD* in *A. thaliana* wild-type background enhanced seed FA accumulation without altering seed morphological characteristics, including seed size, 1000-seed weight, and seed coat color. Consistently, the expression of key genes involved in FA biosynthesis was greatly up-regulated in the developing seeds of *LuAccD* overexpression lines. Additionally, we demonstrated that LuAccD acts as a positive regulator of salt and mannitol tolerance during seed germination in *A. thaliana*. These results provide important insights into the functions of *LuAccD*, which facilitates the oil quantity and abiotic stress tolerance of oil-producing crops through genetic manipulation.

**Keywords:** *LuAccD*; fatty acids; salt stress; mannitol stress; seed germination

#### **1. Introduction**

Flax (*Linum usitatissimum* L., 2n = 30) is a versatile annual plant with global production areas of approximately 12 million acres mainly in Kazakhstan, Russia, Canada, and China; primarily cultivated for its seed oil (oilseed flax) and stem fiber (fiber flax) [1]. Oilseed flax generally contains approximately 50% oil which is composed of five major fatty acids (FAs): palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) [2,3]. Among them, the percentage of C18:3 in the flaxseed oil ranges from 40% to 60%, which is significantly higher than that of *Zea mays* (~1%), *Glycine max* (~8%), and *Brassica napus* (~11%) [3]. As polyunsaturated FAs, C18:2 and C18:3 cannot be biosynthesized in the human body and are the precursors for long-chain polyunsaturated FAs, inclusive of arachidonic acid and eicosapentaenoic acid. These long-chain polyunsaturated FAs have a significant role in the prevention of a variety of diseases, including cancers, inflammatory, cardiovascular, and autoimmune diseases [4–6]. Therefore, together with a high amount of proteins (up to 18.29%), fiber (27.3%), vitamin B1, and lignans, particularly secoisolariciresinol diglucoside (294–700 mg/100 g) [7–9], flax serves as a predominant source which offers a wide range of nutritional and therapeutic applications. In the past decade, China has become the largest importer with the import of \$31,108 million, which is equivalent to 26.8% of total global flax import in the year 2020 [10]. However, oilseed flax is mainly grown in the arid and semi-arid regions of the Northern and Northwestern China, which is one of the areas

**Citation:** Du, R.; Li, X.; Hu, H.; Zhao, Y.; Chen, M.; Liu, Z. *Linum usitatissimum AccD* Enhances Seed Fatty Acid Accumulation and Tolerance to Environmental Stresses during Seed Germination in *Arabidopsis thaliana*. *Plants* **2023**, *12*, 3100. https://doi.org/10.3390/ plants12173100

Academic Editor: Wei Ma

Received: 25 July 2023 Revised: 15 August 2023 Accepted: 27 August 2023 Published: 29 August 2023

**Copyright:** © 2023 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 (https:// creativecommons.org/licenses/by/ 4.0/).

more vulnerable to global climate change [11]. Unpredictable environmental stresses, such as drought and salinity–alkalinity, pose a threat to biological diversity and the quality of oilseed flax. Therefore, identifying the key genes involved in seed FA accumulation and response to adversities in *L. usitatissimum* would provide potential targets for molecular breeding in oil-producing crops including *L. usitatissimum*.

In plants, FA biosynthesis starts with the provision of carbon from glycolysis. After glycolysis, pyruvate dehydrogenase catalyzes the conversion of pyruvate to acetyl-CoA, the initial substrate for de novo FA biosynthesis which occurs in the plastids [12]. Acetyl-CoA carboxylase (ACCase) converts acetyl-CoA and bicarbonate into malonyl-CoA, which is the first committed step in FA biosynthesis [13,14]. In the plastids of dicots and nongraminaceous monocots, ACCase mainly comprises four distinct subunits, namely biotin carboxylase, biotin carboxyl carrier protein, α-subunit of carboxyltransferase (CTα), and β-subunit of carboxyltransferase (CTβ) [15,16]. Studies have shown that increased activity of *A. thaliana* AtACCase in the tuber amyloplasts of *Solanum tuberosum* led to an increase of more than five times in the triacylglycerol content [17]. The mutation of *A. thaliana ACC1* (*AtACC1*), an essential gene encoding ACCase, significantly decreased the contents of long-chain FAs in leaves under cold treatment [18]. Overexpression of *AtACC1* in *B. napus* not only altered seed FA compositions, with the largest effect being an increase in C18:1, but also caused an increase of approximately 5% in seed oil content [19]. Meanwhile, overexpression of each subunit of *Gossypium hirsutum* ACCase effectively increased seed oil content in the transgenic plants of *G. hirsutum*. Among them, the oil content of *GhBCCP1* transgenic seeds was significantly increased by 21.92%, while that of *GhBC1* and *GhCTβ* transgenic seeds was elevated by ~17% [20]. The latest study showed that the interaction between α-CT and CARBOXYLTRANSFERASE INTERACTORs was enhanced by light, which in turn attenuates carbon flux into triacylglycerol accumulation in *A. thaliana* leaves [21]. Homologous expression of *NtAccD*, located in the plastid genome, raised the ACCase level and FA content in the resultant transgenic leaves in *Nicotiana tabacum* cv. *Xanthi* [22]. Semi-quantitative RT-PCR and quantitative real-time PCR (qRT-PCR) results showed that the expression level of *EgAccD* is positively correlated with the *Elaeis guineensis* productivity [23]. The functions of *AccD* genes from *A. thaliana* and other plants have been well characterized, but the roles of *AccD* from *L. usitatissimum* in the regulation of seed FA accumulation and in response to salt and osmotic stresses remain unclear.

For the sessile crops, environmental factors are crucial in determining crop growth and development. Of these, drought and salt are the most prevalent and detrimental constraints to agricultural production [24–27]. Previous studies have demonstrated that drought can negatively affect the yield potential, oil content and FA compositions, and fiber quality traits of flax [26,28,29]. Meanwhile, soil salinity–alkalinity can result in delayed germination, low seedling survival, irregular growth, and lower yield of flax [27]. In addition, drought can result in osmotic stress by altering water potential and cell turgor, and salt can induce osmotic stress and ion toxicity [30]. It is worth noting that the hyperosmotic signal caused by drought and salt stresses promotes the accumulation of phytohormone abscisic acid (ABA), which in turn triggers a series of adaptive responses in plants [31]. Therefore, ABA biosynthesis and signal transduction are of great importance for plants to resist abiotic stresses.

In this study, we cloned the *LuAccD* gene from the flax cultivar 'Longya 10 and found that overexpression of *LuAccD* in *A. thaliana* wild-type plants significantly increased the accumulation of seed total FAs by boosting the transcription levels of several key genes involved in FA biosynthesis. We also demonstrated that LuAccD enhances tolerance to salt and mannitol stresses during seed germination via mediating the ABA biosynthesis and ABA-responsive pathway in *A. thaliana*.

#### **2. Results**

#### *2.1. Sequence Analysis of LuAccD Protein*

The protein sequence of AtAccD was applied to BLASTP in the Phytozome (https:// phytozome-next.jgi.doe.gov/, accessed on 12 October 2020) database and one homologous polypeptide of Lus10002473 was identified from the *L. usitatissimum* genome, namely LuAccD. As shown in Figure 1A, LuAccD and AtAccD had 330 and 488 amino acids, respectively. A 58.4% identity in amino acid sequence was matched between LuAccD and AtAccD, and their carboxyltransferase domains shared 61.5% identity (Table S2). Phylogenetic analysis indicated that LuAccD presents a relatively distant relationship with AccD from other crops we selected (Figure 1B). These results suggested that LuAccD may have a similar function as AtAccD in some ways.

**Figure 1.** Protein sequence alignment and phylogenetic analysis of AccD. (**A**) Sequence alignment of amino acids from LuAccD and AtAccD. The asterisks represent strictly conserved amino acids. The crotonase-like superfamily domain, which was predicated by the NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 20 October 2022), is highlighted in blue in the sequences. (**B**) Phylogenetic analysis of AccD proteins from *L. usitatissimum*, *A. thaliana*, and other crops. Numbers indicate the phylogenetic confidence of the tree topology and denote the bootstrap values on neighbor-joining analysis. Br: *Brassica rapa*, Bj: *Brassica juncea*, Bn: *Brassica napus*, Bo: *Brassica oleracea* var. *oleracea*, At: *Arabidopsis thaliana*, Cs: *Camelina sativa*, Gr: *Gossypium raimondii*, Gm: *Glycine max*, Pf: *Perilla frutescens*, Lu: *Linum usitatissimum*. The accession numbers of AccD are listed in parentheses.

#### *2.2. LuAccD Increases the Seed FA Accumulation in A. thaliana*

Studies have revealed that the loss of *AtAccD* function results in embryo lethality of *A. thaliana* [32]. To preliminarily investigate the functions of *LuAccD* on the accumulation of seed FAs, we introduced the overexpression construct of *35S: LuAccD–6HA* (Figure 2A) into the *A. thaliana* wild-type (Col-0) plants. We obtained six independent T3 homozygous *Col-0 35S: LuAccD–6HA* transgenic lines (#1, #2, #4, #5, #6, and #11) and identified them by the analysis of PCR-based DNA genotyping (Figure 2B). Meanwhile, qRT-PCR results showed that the *LuAccD* expression is not detected in the Col-0, but highly present in the six transgenic lines (Figure 2C). Therefore, we selected *Col-0 35S: LuAccD–6HA#2* and *Col-0 35S: LuAccD–6HA#4* for follow-up experiments. The phenotype analysis showed that there are no significant differences in the seed coat color, seed size, and 1000-seed weight between Col-0 and *Col-0 35S: LuAccD–6HA* transgenic plants (#2 and #4) (Figure S1). However, the contents of seed total FAs and all major FA compositions were both significantly elevated in *Col-0 35S: LuAccD–6HA* plants compared to those in Col-0 (Figure 2D,E). These results suggested that ectopic expression of *LuAccD* promotes FA accumulation without affecting other measured agronomic traits in *A. thaliana* seeds.

**Figure 2.** Overexpression of *LuAccD* increased the accumulation of seed FAs in *A. thaliana*. (**A**) Schematic illustration of the constitutive expression cassette of *LuAccD*. RB, right border; LB, left border; NOS-pro, nopaline synthase promoter; NOS-ter, nopaline synthase terminator; Basta, glyphosate; 35S-pro, CaMV 35S promoter. (**B**) PCR-based DNA genotyping of *Col-0 35S: LuAccD–6HA* transgenic plants. Cas, cassette. (**C**) Transcript levels of *LuAccD* in the wild-type (Col-0) and *Col-0 35S: LuAccD–6HA* developing seeds at 12 days after pollination measured by qRT-PCR. *AtEF1αA4* was used as an internal control. The values are presented as the mean ± SD (n = 3). (**D**) Comparisons of seed total FA content between Col-0 and *Col-0 35S: LuAccD–6HA* transgenic plants. (**E**) Comparison of the major seed FA compositions between the Col-0 and *Col-0 35S: LuAccD–6HA* transgenic plants. Values represent means ± SD and error bars denote SD. Three independent experiments were carried out and each biological replicate contains three technical replicates. Asterisks (\*) indicate significant differences in the FA contents between *Col-0 35S: LuAccD–6HA* and Col-0 plants (two-tailed paired Student's *t*-test, *p* ≤ 0.05).

#### *2.3. LuAccD Increases the Expression Levels of Genes Contributing to Seed FA Accumulation*

To further investigate how LuAccD controls seed FA accumulation at transcription level, several key genes inclusive of *AtBCCP1* (*BIOTIN CARBOXYL CARRIER PRO-TEIN ISOFORM1*), *AtBCCP2*, *AtMCAT* (*MALONYL COA-ACP MALONYLTRANSFERASE*), *AtKASI* (*3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE I*), *AtKASII*, *AtSSI2* (*SUP-PRESSOR OF SA INSENSITIVE 2*), *AtFAD2* (*FATTY ACID DESATURASE2*), *AtFAD3*, and *AtPDAT2* (*PHOSPHOLIPID: DIACYLGLYCEROL ACYLTRANSFERASE2*), were selected for expression analysis. The expression levels of these genes were assessed by qRT-PCR using the developing seeds at 12 days after pollination (DAP) between Col-0 and *Col-0 35S: LuAccD–6HA#4* transgenic plants. The transcript levels of *AtBCCP1*, *AtBCCP2*, *AtMCAT*, *AtKASI*, *AtKASII*, *AtSSI2*, *AtFAD2*, *AtFAD3*, and *AtPDAT2* in the developing seeds of *Col-0 35S: LuAccD–6HA#4* transgenic plants were significantly higher than those of the Col-0 at 12 DAP (Figure 3). These results demonstrated that LuAccD contributes to seed FA accumulation by up-regulating the expression of *AtBCCP1*, *AtBCCP2*, *AtMCAT*, *AtKASI*, *AtKASII*, *AtSSI2*, *AtFAD2*, *AtFAD3*, and *AtPDAT2* during seed development in *A. thaliana*.

**Figure 3.** Expression analysis of genes contributing to FA accumulation in the wild-type (Col-0) and *Col-0 35S: LuAccD–6HA#4* developing seeds at 12 days after pollination. Results were normalized against the expression of *AtEF1αA4* as an internal control. Values are means ± SD (n = 3). Asterisks (\*) represent significant differences between Col-0 and *Col-0 35S: LuAccD–6HA#4* transgenic plants determined by two-tailed paired Student's *t*-test (*p* ≤ 0.05).

#### *2.4. LuAccD Promotes Seed Germination under Salt and Mannitol Stresses in A. thaliana*

To determine the effects of LuAccD in response to abiotic stresses, seed germination of Col-0 and *Col-0 35S: LuAccD–6HA* plants were observed on MS agar medium containing 150 mM NaCl or 300 mM mannitol. As shown in Figure 4, Col-0 and *Col-0 35S: LuAccD–6HA* lines displayed similar germination rates and seedling growth on the medium without stress treatment (Figure 4). However, the seed germination rate of *Col-0 35S: LuAccD–6HA* lines was higher than that of Col-0 under the stress of 150 mM NaCl or 300 mM mannitol (Figure 4). Therefore, we indicated that LuAccD positively regulates the resistance of salt and mannitol stresses during seed germination in *A. thaliana*.

**Figure 4.** Response of wild-type (Col-0) and transgenic plants overexpressing *LuAccD* to NaCl and mannitol in seed germination. (**A**) Germination phenotype of seeds from the different lines grown on 1/2 MS plates or 1/2 MS plates with 150 mM NaCl or 300 mM mannitol for 7 days after sowing. Bar = 2 mm. (**B**) Germination rates of seeds from the different lines grown on 1/2 MS plates or 1/2 MS plates with 150 mM NaCl or 300 mM mannitol. Seed germination percentages were quantified every day from 1st day to the 7th day after sowing, and the embryonic axis protrusion was considered as seed germination. Date are the means ± SD (n = 3). Error bars denote SD. The value of each biological replicate was the average calculated over three technical replicates. For each technical replicate, we recorded the germination rates of 150 seeds from the same batch.

#### *2.5. LuAccD Inhibits Expression Levels of Several Genes Contributing to ABA Biosynthesis and Signal Transduction*

To better understand how LuAccD influences seed germination in response to salt and mannitol stresses, we assessed the expression of five ABA-related genes in Col-0 and *Col-0 35S: LuAccD–6HA#4* transgenic seeds at 12 h after sowing. As illustrated in Figure 5, there were no significant differences in the expression levels of *AtNCED3* (*NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3*), *AtAAO3* (*ABSCISIC ALDEHYDE OXIDASE 3)*, *AtABI3* (*ABSCISIC ACID INSENSITIVE 3*), *AtEM1* (*EARLY METHIONINE-LABELED 1*), and *AtEM6* between Col-0 and *Col-0 35S: LuAccD-6HA#4* transgenic seeds under the normal condition. The treatment of 150 mM NaCl or 300 mM mannitol remarkably induced the expression of these genes in both Col-0 and *Col-0 35S: LuAccD–6HA#4* germinating seeds. But the expression levels of these genes were always lower in *Col-0 35S: LuAccD–6HA#4* transgenic lines than those in Col-0 (Figure 5). These results suggested that overexpression of *LuAccD* inhibits the expression of *AtNCED3*, *AtAAO3*, *AtABI3*, *AtEM1*, and *AtEM6*, which weakens the ABA biosynthesis and ABA signal transduction, thereby resulting in the low sensitivity of transgenic plants to salt and mannitol stresses during seed germination.

**Figure 5.** Comparison of relative transcript levels of ABA-related genes between wild-type (Col-0) and *Col-0 35S: LuAccD–6HA#4* transgenic plants. Total RNA was extracted from germinating seeds grown on the 1/2 MS or 1/2 MS containing 150 mM NaCl or 300 mM mannitol at 12 h after sowing. The expression levels of genes were calculated relative to that of the internal control *AtEF1αA4*. Values represent means ± SD (n = 3). Asterisks (\*) represent significant differences between Col-0 and *Col-0 35S: LuAccD–6HA#4* lines (two-tailed paired Student's *t*-test, *p* ≤ 0.05).

#### **3. Discussion**

Flax is an important oil-producing crop that has attracted interest due to its high content of C18:3. Flax with improved tolerance to stresses can also be used to expand cultivation into currently undeveloped and marginal lands [33]. Therefore, it is desirable to generate elite flax germplasm with a high oil content and resistance to environmental stresses, including salt and drought stresses. In this study, we found that *LuAccD* promotes the seed FA accumulation and facilitates seed germination under salt and mannitol stresses in *A. thaliana*.

The previous study showed that *AtAccD* is essential for FA biosynthesis [32]. Consistently, we found that ectopic expression of *LuAccD* significantly promotes the accumulation of seed total FAs and major FA compositions in *A. thaliana* (Figure 2D,E). The high percent identity of carboxyltransferase domains, which play an important role in FA biosynthesis [34], was observed between LuAccD and AtAccD (Figure 1A). Therefore, we inferred that LuAccD exhibits a conserved role with AtAccD in regulating the FA accumulation of *A. thaliana* seeds. Inconsistently, the overexpression of LuAccD in Col-0 did not alter the 1000-seed weight (Figure S1). This might be ascribed to the fact that other seed components affecting seed weight, such as storage proteins, offset the higher seed total FA content in *LuAccD* transgenic seeds. The exact explanation needs to be supported by further experimental results. Notably, seed coat color, seed length and width were also not altered (Figure S1). These results indicated that *LuAccD* can be regarded as a valuable potential for flax molecular breeding.

The collaborative expression of genes participating in FA biosynthesis is important for the oil accumulation in seeds [35–37]. Overexpression of *LuAccD* induced the transcript levels of several genes involved in oil biosynthetic processes, including FA biosynthesis and modification, and triacylglycerol deposition, which, in turn, contributes to oil accumulation in seeds (Figure 3). Of these enzymes, *BCCP1* and *BCCP2*, like *AccD*, also encode the subunit of ACCase, which functions as a sensor or gating system that controls the overall flux of FA biosynthesis [38,39]. MCAMT converts malonyl-CoA and ACYL CARRIER PROTEIN (ACP) into CoA and malonyl-ACP, which is a key building block for the FA biosynthesis [40]. Therefore, the up-regulated expression of *AtBCCP1*, *AtBCCP2*, and *AtMACT* by LuAccD should increase the overall flux of seed FAs at the early stage of the FA biosynthetic pathway in *A. thaliana*. Additionally, three separate condensing enzymes, or 3-ketoacyl-ACP synthases (KASI–KASIII), are essential for the production of C18 FAs. Among them, KASI participates in the conversion of acetyl-ACP to palmitoyl-ACP, whereas KASII mainly utilizes palmitoyl-ACP as the substrate to produce stearoyl-ACP [41]. Studies have shown that the deficiency of KASI leads to disrupted embryo development before the globular stage and noticeably decreases seed total FA content (~33.6% of the wild-type) in *A. thaliana* [42]. SSI2 (FAB2) encodes a stearoyl-acyl carrier protein desaturase that converts C18:0 into C18:1 [43]. FAD2 catalyzes the conversion of C18:1 to C18:2 which is further desaturated by FAD3 to form C18:3 [44–46]. Thus, the highly up-regulated expression of *AtKASI*, *AtKASII*, *AtSSI2*, *AtFAD2*, and *AtFAD3* in *Col-0 35S: LuAccD–6HA* would accelerate the accumulation of FAs in seeds at the middle stage of the biosynthetic pathway. *PDAT2* encoding a phospholipid: diacylglycerol acyl-transferase promotes triacylglycerol production [47]. Therefore, ectopic expression of *LuAccD* in *A. thaliana* could trigger multiple transcriptional regulatory events that affect FA accumulation in seeds.

Seed germination is a critical checkpoint for crop survival under adverse conditions, and ABA plays a critical role in affecting seed germination and seedling establishment, especially under abiotic stresses [48–51]. In our study, we found that overexpression of *LuAccD* in Col-0 weakens the sensitivity of the transgenic seeds to salt and mannitol during germination (Figure 4). At the cellular level, the transcript levels of five stress-response genes, *AtNCED3*, *AtAAO3*, *AtABI3*, *AtEM1*, and *AtEM6*, were higher in Col-0 germinating seeds than in the *Col-0 35S: LuAccD–6HA* under the NaCl or mannitol stress (Figure 5). *AtNCED3* encodes 9-cisepoxy carotenoid dioxygenase which functions in osmotic stress-induced ABA biosynthesis in *A. thaliana* [52]. It is highly induced by salt and drought stresses, and its inactivation is responsible for enhanced germination upon salt stress [53–55]. *AtAAO3* encodes an enzyme that catalyzes the final step of ABA biosynthesis [56], and the *Oryza sativa OsAAO3* mutation exhibited earlier seed germination [57]. AtABI3 as a major downstream component of ABA signaling has been long recognized as a master regulator of seed dormancy and ABA inhibition of seed germination [50]. The higher percentage of seed germination was observed in Atabi3 mutant compared to wild-type when exposed to ABA, mannitol or NaCl treatments [58]. *AtEM1* and *AtEM6* encoding the late embryogenesis abundant proteins are ABA-responsive marker genes, which are induced by ABA, salt and osmotic stresses [59–61]. Owing to these

results, we concluded that the lower expression of *AtNCED3*, *AtAAO3*, *AtABI3*, *AtEM1*, and *AtEM6* caused by the overexpression of *LuAccD* in *A. thaliana* attenuates the ABA biosynthesis and ABA signal transduction, thereby resulting in low sensitivity of *A. thaliana* to salt and mannitol stresses during seed germination.

#### **4. Materials and Methods**

#### *4.1. Plant Materials and Growth Conditions*

All *A. thaliana* materials used in this study were in the Columbia ecotype (Col-0) background, and were grown in a growth chamber at 22◦C with a 16/8 h light/dark cycle, which has been reported in detail previously [62].

#### *4.2. Gene Cloning and Plasmid Construction*

The protein sequence of AtAccD (ATCG00500) was used for protein blast against the *L. usitatissimum* reference genome (https://phytozome-next.jgi.doe.gov/pz/portal.html, accessed on 12 October 2020). One identified highly conserved sequence Lus10002473 was named *LuAccD*. The template cDNA was synthesized from total RNA extracted from germinated seeds of oil flax cultivar 'Longya 10'. The full-length CDS of *LuAccD* without the stop codon was amplified using specific primers by PCR and was cloned into the pGreen-35S–6HA vector, forming the *35S: LuAccD–6HA* fusion vector. Primer information for the plasmid construction is given in Table S1.

#### *4.3. Analysis of Protein Sequence and Phylogenetic Tree*

The protein sequence of LuAccD was obtained from Phytozome (https://phytozomenext.jgi.doe.gov/pz/portal.html, accessed on 12 October 2020). Multiple sequence alignment of AtAccD and LuAccD proteins was carried out using MUSCLE website (https: //www.ebi.ac.uk/Tools/msa/muscle/, accessed on 15 October 2022). The NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 20 October 2022) was used to predicate the conserved domain of LuAccD. The phylogenetic tree was constructed by the Neighbor-Joining (NJ) method using MEGA 7.0 software with 1000 bootstrap replications and the *p*-distance model.

#### *4.4. Generation of A. thaliana Transgenic Plants*

The construct of *35S: LuAccD–6HA* was transformed into the *Agrobacterium tumefaciens* strain GV3101, which was then introduced into Col-0 via the floral dip method [63]. The T1 transgenic plants were selected by Basta® (Bayer, Langenfeld, Germany) on soil and identified by using PCR in DNA level. T2 and T3 seeds were screened on 1/2 MS medium (pH 5.7, 1% sucrose, 1% agar) containing 10 μg/mL glufosinate-ammonium, and positive seedlings were transferred to soil. The T3 generation homozygous plants were used for subsequent experiments after cultivation under similar conditions.

#### *4.5. RNA Extraction and qRT-PCR Analysis*

The total RNA samples were isolated using the MiniBEST Plant RNA extraction kit (Takara Bio, Dalian, China). RNA reverse reaction was carried out with the PrimeScript RT kit (Takara Bio, Dalian, China). qRT-PCR was performed using an SYBR Green Mix (Takara Bio, Dalian, China) on a Quant Studio 7 real-time system. The relative expression values were normalized to that of the internal control *AtEF1αA4*. Statistical data were obtained from three biological replicates. For each biological replicate, two technical repetitions were performed. Primer information for qRT-PCR is given in Table S1.

#### *4.6. Microscopic Observation of A. thaliana Seed Traits*

The *A. thaliana* seeds were harvested from the siliques at the basal part of the major inflorescences. The mature seeds were imaged under an SZ61 stereomicroscope (Olympus, Tokyo, Japan), and their length and width were determined with ImageJ 1.48v software. The 1000-seed weight was measured by using a 0.0001 precision test analytical balance

(BSA124S-CW, Sartorius, Beijing, China). Three independent biological replicates and three technical replicates were performed. For seed size measurement, each technical replicate contains 300 seeds.

#### *4.7. Measurement of Seed FAs*

Isolation and determination of FAs were performed according to previously described [64]. In brief, seed FAs were methylated in the 2.5% (*v*/*v*) H2SO4 solution diluted with methanol at 80 ◦C for 2 h. After cooling to room temperature, the solution was added with 2 mL of 0.9% (*w*/*v*) NaCl and 2 mL of hexane in due order, and the organic phase was analyzed by gas chromatography using GC-2010 plus instrument (Shimadzu, Kyoto, Japan) with a flame ionization detector and a 30 m (length) × 0.25 mm (internal diameter) × 0.5 μm (liquid membrane thickness) column (Supelco wax-10, Supelco, Shanghai, China). Methyl heptadecanoate was used as an internal standard. The initial column temperature was maintained at 160 ◦C for 1 min, increased by 4 ◦C min−<sup>1</sup> to 240 ◦C, and held for 16 min at the final temperature. The peak for each FA composition was identified by their unique retention time, and their concentrations were calculated against the internal control.

#### *4.8. Determination of Seed Germination*

The *A. thaliana* seeds used for the germination analysis were harvested from plants grown under the same conditions at the same time and allowed to mature at room temperature for 3 months. The A. thaliana seeds were surface sterilized with 75% ethyl alcohol and were subsequently sown on 1/2 MS solid medium supplemented with or without 150 mM NaCl or 300 mM mannitol. The seeds were stratified at 4◦C for 2 days in darkness and were then placed in the climate chamber. The germination (emergence of radicles) rate was scored daily. After 7 days, the seedlings were photographed. Seed germination percentages were quantified every day from 1st day to the 7th day after sowing, and the embryonic axis protrusion was considered as seed germination. Date are the means ± SD (n = 3). Error bars denote SD. The value of each biological replicate was the average calculated over three technical replicates. For each technical replicate, we recorded the germination rates of 150 seeds from the same batch.

#### **5. Conclusions**

In this study, our results demonstrated that LuAccD exhibits a conserved role with AtAccD in promoting the seed FA accumulation in *A. thaliana*. Meanwhile, LuAccD could enhance the tolerance to salt and mannitol stresses during seed germination in *A. thaliana*. In this regard, LuAccD can be utilized as a potential target for the breeding of flax varieties with high FA content and stress tolerance.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12173100/s1, Figure S1. Morphological observation of mature *A. thaliana* seeds randomly selected from wild-type (Col-0) and overexpression transgenic plants carrying *LuAccD* (*Col-0 35S: LuAccD–6HA#2* and *#4*). Table S1. The primers used in this study. Table S2. Comparison of percent identity between the amino acid sequences of AtAccD and LuAccD.

**Author Contributions:** Z.L. conceived and designed the experiments. X.L. conducted the experiments and analyzed the data. R.D., H.H. and Y.Z. conducted parts of the experiments. R.D. wrote the draft of the manuscript, and M.C. and Z.L. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Key Research and Development Program of Shaanxi Province (grant no. 2022NY-158 and 2021LLRH-07), the PhD Start-up Fund of Northwest A&F University (grant no. Z1090121052), and a grant from the Yang Ling Seed Industry Innovation Center (Grants no. K3031122024 and K3031123009).

**Data Availability Statement:** All data included in this study are available upon reasonable request by contact with the corresponding author.

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

#### **References**


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## *Article* **Comparison of Salt Stress Tolerance among Two Leaf and Six Grain Cultivars of** *Amaranthus cruentus* **L.**

**Adrien Luyckx, Stanley Lutts and Muriel Quinet \***

Groupe de Recherche en Physiologie Végétale, Earth and Life Institute-Agronomy, Université Catholique de Louvain, 1348 Louvain-la-Neuve, Belgium; adrien.luyckx@uclouvain.be (A.L.); stanley.lutts@uclouvain.be (S.L.) **\*** Correspondence: muriel.quinet@uclouvain.be

**Abstract:** Amaranths (*Amaranthus* L.) are multi-use crop species renowned for their nutritional quality and their tolerance to biotic and abiotic stresses. Since the soil salinity of croplands is a growing problem worldwide, we tested the salinity tolerance of six grain and two leaf cultivars of *Amaranthus cruentus* L. The plants were grown for 53 days under hydroponic conditions at 0, 50 and 100 mM NaCl. We investigated the growth rate, photosynthetic activity, mineral content, pigments and biochemical compounds involved in oxidative stress. Although 100 mM NaCl always decreased biomass production, we highlighted Don Leon and K91 as tolerant cultivars under moderate salt stress (50 mM NaCl). Under salinity, sodium accumulated more in the shoots than in the roots, particularly in the stems. Sodium accumulation in the plants decreased the net photosynthetic rate, transpiration rate and stomatal conductance but increased water use efficiency, and it decreased chlorophyll, betalain and polyphenol content in the leaves. It also decreased the foliar content of calcium, magnesium and potassium but not the iron and zinc content. The physiological parameters responded differently to sodium accumulation depending on the cultivar, suggesting a different relative importance of ionic and osmotic phases of salt stress among cultivars. Our results allowed us to identify the morpho-physiological traits of the cultivars with different salt tolerance levels.

**Keywords:** abiotic stress; amaranth; orphan crop; plant physiology; pseudocereal; salinity

**Citation:** Luyckx, A.; Lutts, S.; Quinet, M. Comparison of Salt Stress Tolerance among Two Leaf and Six Grain Cultivars of *Amaranthus cruentus* L. *Plants* **2023**, *12*, 3310. https://doi.org/10.3390/ plants12183310

Academic Editors: Mingxun Chen, Lixi Jiang, Yuan Guo and Sylvia Lindberg

Received: 20 July 2023 Revised: 19 August 2023 Accepted: 11 September 2023 Published: 19 September 2023

**Copyright:** © 2023 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 (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

In a context of the increasing food demand and more adverse biotic and abiotic conditions for agricultural production, it is necessary to rely on robust crops that are able to thrive in stressful conditions, while limiting agriculture's contribution to global change [1]. Currently, soil salinity is one of the main abiotic stress threatening agricultural production worldwide [1].

Soil salinization, caused by an accumulation of soluble salts (mainly NaCl and Na2SO4) in the upper horizon of soil, is an expanding agronomic constraint for food production, especially in Asia, Africa, South America and Australasia in arid regions where precipitations are too low to leach excessive salts [2,3]. A soil is saline when its electrical conductivity is higher than 4 dS·m−<sup>1</sup> and sodic when the exchangeable sodium percentage is higher than 6% [4]. Salinization can be primary when it is of natural origin (weathering of saline rocks, saline bedrock, atmospheric deposition) or secondary when it is human-induced. In the latter case, saline or sodic soils can be the result of bad irrigation practices (e.g., with brackish water), sea level rising or an excessive use or bad management of mineral fertilizers [5]. Saline and sodic soils are estimated to cover more than 800 Mha, but we are lacking recent data [6,7]. Most of the salt-contaminated topsoils (>400 Mha, 0–30 cm) are saline (85%), whereas a much lower proportion is sodic or saline–sodic (15%) [6]. Salt-affected subsoils are twice as common as salinized topsoils (>800 Mha) and a higher proportion of them are sodic or saline–sodic (38%) [6]. Salt-affected soils are in expansion in part because of climate change and this issue is particularly exacerbating under arid and semi-arid climates and on irrigated croplands [5].

Salt stress in plants occurs in two phases, namely the osmotic and the ionic phases [8]. First, the osmotic stress is driven by the low hydric potential of saline soils that decreases water uptake. Osmotic stress has a direct effect on plant growth. Then, the ionic stress is caused by the toxicity of salt ions entering the plant tissues and the competitive effect with important nutrients. Tolerance mechanisms consist of, depending on the species, salt exclusion or salt accumulation involving tissue partitioning and subcellular compartmentation, and the synthesis of organic osmolytes for osmotic adjustment [8–10]. These tolerance mechanisms consume energy, which impedes growth and development [11].

Most major crops, which provide most of the world's calories, are salt-sensitive even though extensive research is in progress to increase their salt tolerance [12–14]. Therefore, the use of so-called "orphan" or "indigenous" crops and wild crop relatives (WCR) has been flourishing in the last few decades [15–18]. It is a large research field with different approaches, including the introgression of genes from orphan crops or WCR to major crops, breeding of under-domesticated crops or de novo domestication of wild species [19–22]. This array of approaches is promising for breeding for salinity tolerance [22,23]. The amaranth genus (*Amaranthus* spp.) contains several orphan crops and WCR that has gained increasing attention in the past few decades because of its tolerance to abiotic and biotic stress, including salinity.

*Amaranthus* is a subcosmopolitan genus of 50–70 herbaceous plant species, most of which are annual plants, in the Amaranthaceae family [24]. Several species of amaranths are used as crops, either as leafy vegetables for their nutritious leaves or as pseudocereals for their quinoa-like seeds rich in high-quality proteins. Although some species were staple crops in several Mesoamerican civilizations, they fell into disuse for several centuries after european colonization of the Americas. However, a surge in interest for amaranths has arisen in the last few decades because of their nutritive qualities [25–27] and their tolerance to several biotic and abiotic stresses. Among grain species, studies have been conducted on salinity [28–34] and drought [35,36] in *A. cruentus*, on salinity in *A. caudatus* [37,38] and salt and drought in *A. hypochondriacus* [39,40]. The plant response to abiotic stress has also been investigated in some leaf species, such as *A. tricolor* [41–43] or *A. hybridus* and *A. albus* [44]. Most of these studies show that amaranth is highly tolerant to drought and salinity, particularly at the vegetative stage. These works report variability among species and among cultivars of each species. It has been shown that gas exchange regulation, antioxidant defense, ion transporter regulation and osmotic adjustment are involved in the stress response. Amaranths express the NAD malic-type C4 photosynthetic pathway, which makes them more competitive in warm and/or dry environments by means of an higher water use efficiency compared to C3 plants [45]. *Amaranthus cruentus* L. (red amaranth), domesticated by the Aztecs in Mesoamerica, is used either for leaves (in Africa and South-East Asia but also in America [46]) or for seed production (mainly in America and Asia, but also in Africa [47]), usually with distinct cultivars [48–50]. However, this species, along with the two other grain amaranths (*A. hypochondriacus* L. and *A. caudatus* L.), are promising as dual-use crops, with leaves and seeds harvested on the same plants [51–53]. In these three 'grain' species, the nutritional quality of both leaves and seeds is high. Seeds are rich in proteins, more than most true cereals, and minerals [54,55], while leaves are also rich in proteins, minerals and vitamins [56–58]. Grain amaranth can produce seed yields of 2000–3500 kg·ha−<sup>1</sup> [59], whereas leaf amaranth can produce several dozens of tons per hectare of fresh leaves and young stems eaten as vegetables [60]. Despite their tolerance to salinity and their exceptional agronomic and nutritional value, amaranths have remained understudied crops. Indeed, the physiological mechanisms underlying abiotic stress tolerance are poorly known.

To deepen our understanding of the salt response in *Amaranthus cruentus* at the vegetative stage, a screening of the salt tolerance of six grain and two leaf cultivars was conducted under hydroponic conditions at moderate and strong levels of salt stress (50 mM and 100 mM NaCl, respectively [61]). Plant growth and photosynthetic activity were monitored. Na and K were quantified in leaves, stem and roots. In addition, other mineral contents (Ca, Fe, Mg and Zn) were determined in leaves since Na accumulation in plants is known to affect mineral nutrition [41]. The pigments (chlorophylls, betalains) and biochemical compounds involved in oxidative stress (malondialdehyde, polyphenols, flavonoids and ascorbate) were investigated in the leaves due to their importance in stress response [62]. The aims were to identify (1) contrasted cultivars regarding their tolerance to salt stress and (2) the main physiological mechanisms explaining variability in salt tolerance among leaf and grain cultivars of *A. cruentus*.

#### **2. Results**

Plants were subjected to 0, 50 and 100 mM NaCl for 53 days. The eight cultivars differed by their origin, morphology, color and food purpose (Table S1). The plants tolerated the salt stress well. Indeed, the mortality rate was low. Complete senescence was observed at 100 mM NaCl only in two plants of Montana 5 and two plants of Don Leon, for a total of 10 plants per cultivar and condition.

#### *2.1. Biomass Production*

Under the control conditions, the mean leaf dry weight of all cultivars was 1.79 ± 0.43 g. The most productive cultivars were Alegria Disciplinada and Don Armando, whereas the less productive ones were K91, Montana 5 and Locale (Figure 1). Total dry weight was strongly correlated with leaf dry weight (r = 0.98), with leaves accounting for 62.5 ± 3.8% of the total dry biomass, whereas stem and roots accounted for 28.6 ± 4% and 8.8 ± 3.5%, respectively.

**Figure 1.** Effect of salinity (0, 50 and 100 mM NaCl) on the dry biomass production of eight *A. cruentus* cultivars after 53 days of growth. (**a**) Leaf dry weight; (**b**) stem dry weight; (**c**) root dry weight. Treatments followed by different letters for the same cultivar are significantly different (*p* < 0.05).

Salt decreased the dry weight of all organs (*p* < 0.001 for leaves, stem and roots) in all cultivars (Figure 1, Table S2). The leaf dry weight decreased by on average 57% at 100 mM NaCl (Figure 1a). The strongest effect was observed on Alegria Disciplinada (−82%), which was the most productive without salt. Red Amaranth and K91 were the least affected, with less than 50% of leaf weight loss. At 50 mM NaCl, salt caused an average decrease in leaf biomass of 27%, but with some variability among cultivars. Don Leon leaf dry weight was hardly affected by 50 mM NaCl, whereas Montana 5 leaf production dropped by 46%. Moreover, the leaf production of Don Armando, K91, Montana 5 and Red Amaranth was similar at 50 and 100 mM NaCl.

Stem dry weight decreased significantly at 50 mM NaCl only in Locale, Rouge, K91 and Don Armando, whereas it was not affected at 100 mM NaCl in Montana 5, Don Leon, Don Armando and Red Amaranth (Figure 1b). Root dry weight rarely decreased at 50 mM NaCl, but often strongly at 100 mM NaCl, by more than 70% in Alegria Disciplinada and even more than 80% in Don Leon (Figure 1c).

Salt stress slightly influenced the stem and root water content (*p* = 0.004 and *p* = 0.049, respectively), which were 95 ± 1% and 97 ± 3% in control conditions, respectively (Tables S2 and S3). The leaf water content was 88 ± 1% in control conditions. While no effect was observed at 50 mM NaCl, salt substantially decreased the leaf water content at 100 mM NaCl, which dropped to 81 ± 13%. This decrease was only significant in Montana 5 (−12.3%), Alegria Disciplinada (−16.2%) and Don Leon (−17.9%).

Based on the DW, the salt tolerance index (STI) was calculated (Table 1). At 50 mM NaCl, the most sensitive cultivar to salt was Montana 5, whereas Don Leon was the most tolerant. Cultivars behaved substantially differently at 100 mM NaCl, since Alegria Disciplinada was the most sensitive and Red Amaranth the most tolerant. Indeed, the tolerance index did not demonstrate any correlation at 50 mM and 100 mM NaCl (r = −0.11, *p* = 0.80).


**Table 1.** Salt tolerance index (STI) of the eight *A. cruentus* cultivars at 50 and 100 mM NaCl.

#### *2.2. Sodium Distribution in the Plant Organs*

Salt stress significantly increased Na concentrations in the leaves (*p* < 0.001), stems (*p* < 0.001) and roots (*p* < 0.001) but no differences were observed among different cultivars (Figure 2, Table S4). The sodium content in stems and roots was similar between 50 mM and 100 mM NaCl, whereas the accumulation was proportional to the stress intensity in leaves, especially in Locale, Rouge and Alegria Disciplinada. Salt stress caused an accumulation of Na in leaves up to 5.45 ± 2.74 mg·g−<sup>1</sup> DW in Montana 5 at 50 mM NaCl and up to 7.91 ± 1.61 mg·g−<sup>1</sup> DW at 100 mM NaCl in the same cultivar (Figure 2a). Rouge had the lowest accumulation of Na in leaves at 50 mM NaCl with 1.99 ± 0.36 mg·g−<sup>1</sup> DW; at 100 mM NaCl, the lowest accumulation was observed in K91 (3.95 ± 2.43 mg·g−<sup>1</sup> DW). Sodium accumulated in stems at a higher magnitude than in leaves (up to 8.38 ± 1.07 mg·g−<sup>1</sup> DW at 50 mM NaCl and up to 9.78 ± 1.17 mg·g−<sup>1</sup> DW at 100 mM NaCl, in both cases in Don Leon, Figure 2b). In contrast to stems, Na accumulated in roots at lower concentrations than in leaves (up to 4.90 ± 1.23 mg·g−<sup>1</sup> DW in K91 at 50 mM NaCl and up to 4.17 ± 0.60 mg·g−<sup>1</sup> DW in Red Amaranth at 100 mM NaCl, Figure 2c).

**Figure 2.** Effect of salinity (0, 50 and 100 mM NaCl) on the sodium content in (**a**) leaves; (**b**) stems and (**c**) roots of the eight *A. cruentus* cultivars. Treatments followed by different letters for the same cultivar are significantly different (*p* < 0.05).

#### *2.3. Overview of the Physiological Response of the Cultivars to Salt Stress*

In addition to plant growth, salinity affected the physiology of the eight cultivars. In order to identify the main physiological parameters involved in salt tolerance in *A. cruentus* and to differentiate the cultivars, principal component analysis (PCA) and correlation plots were used, as shown in Figures 3 and 4.

**Figure 3.** Principal component analysis (PCA) of the plant growth and mineral content of eight *A. cruentus* cultivars exposed to 0 mM, 50 mM and 100 mM NaCl. (**a**) Individual plot showing the eight cultivars position (LO, Locale; RO, Rouge; AD, Alegria Disciplinada; DA, Don Armando; DL, Don Leon; K9, K91; RA, Red Amaranth; M5, Montana 5) in the three salt treatments (red, 0 mM; yellow, 50 mM; blue, 100 mM). (**b**) Variable plot showing correlations between mineral content and biomass data (DW, dry weight; -L, leaf; -S, stem; -R, root).

**Figure 4.** Correlation graph of all measured parameters, grouped in four categories: mineral content, biomass production, pigments, biochemical activity in leaves and photosynthetic activity. Nonsignificant (*p* < 0.05) correlations are crossed out. Negative correlations are colored in shades of red, whereas positive correlations are in blue (see the color legend on the right). Na\_Shoot and Root\_ratio, ratio between the quantity of sodium in the shoot to the quantity in the roots; NaK, Na/K ratio; MDA, malondialdehyde.

Axis 1 and axis 2 of the PCA explained 55.4% of the variance (Figure 3). Axis 1 of the PCA separated plants in control conditions from those exposed to salt (Figure 3a). The sodium contents in leaves, stem and roots and Na/K ratios in leaves and roots were good positive predictors of salt-treated plants, whereas K and Mg content were negatively correlated with Na content (Figure 3b). Axis 2 was mainly explained by growth parameters. In contrast to NaCl treatments, there was no clear discrimination between cultivars.

Sodium accumulation in the plants affected several parameters, as shown on the correlation matrix (Figure 4). The sodium content in all organs (leaves, stems and roots) was negatively correlated with K content in all organs, Mg and Ca content in leaves, biomass of all organs, pigments (chlorophylls and betaxanthins but not betacyanins), photosynthetic activity (net photosynthetic rate, transpiration rate and stomatal conductance) and slightly with polyphenols in leaves. The Na/K ratio in leaves and roots, but not in stems, was also negatively correlated with all these parameters.

The impacted physiological, biochemical and mineral parameters will be further analyzed below.

#### *2.4. Mineral Content*

Salt decreased the K content in all organs (*p* < 0.001), but similarly decreased the content at 50 mM and 100 mM NaCl (Figure 5, Table S4). However, this decrease was proportionally higher in stems and roots compared to leaves (Figure 5). In leaves, the highest K reduction was observed in Red Amaranth (−59%) while the lowest was observed in Don Leon (−47%) (Figure 5a). In stems, the reduction in K content ranged from −47% (in Alegria Disciplinada) to −93% (in Red Amaranth), while it ranged from −39% (in K91) to −74% (in Locale and Don Leon) in roots (Figure 5b,c). As a result, salt stress caused an increase in the Na/K ratio in all organs (Figure 5d–f, Table S4). This increase was similar among the cultivars for leaves and roots but depended on the cultivar in stems (Figure 5d–f, Table S4).

**Figure 5.** Effect of salinity (0, 50 and 100 mM NaCl) on the potassium content in (**a**) leaves; (**b**) shoot and (**c**) roots and the Na/K ratio in (**d**) leaves; (**e**) shoot and (**f**) roots of the eight *A. cruentus* cultivars. Treatments followed by different letters for the same cultivar are significantly different (*p* < 0.05).

Ca, Fe, Mg and Zn contents were measured in leaves only (Figure 6). A decrease in the Ca content was observed in response to salinity (*p* < 0.001, Figure 6a, Table S4), except in Montana 5. Magnesium content in the leaves of stressed plants decreased by more than 50% compared to the control plants (*p* < 0.001), but again there were no differences between 50 and 100 mM NaCl (Figure 6b, Table S4). Neither Fe (*p* = 0.24) nor Zn (*p* = 0.87) contents were affected by salinity (Figure 6c,d, Table S4).

**Figure 6.** Effect of salinity (0, 50 and 100 mM NaCl) on the (**a**) calcium, (**b**) magnesium, (**c**) iron and (**d**) zinc contents in leaves of the eight *A. cruentus* cultivars. Treatments followed by different letters for the same cultivar are significantly different (*p* < 0.05).

#### *2.5. Photosynthetic Activity in Relation to Sodium Accumulation in Leaves*

Salt decreased net photosynthesis (A, *p* = 0.0015), stomatal conductance (gs, *p* < 0.001) and net transpiration (E, *p* < 0.001), whereas it increased instantaneous water use efficiency (instWUE, *p* < 0.001) (Table S5). Figure 7 shows the photosynthetic parameters in relation to the sodium content in leaves.

The decrease in A was not always linked to Na content in the leaves, depending on the cultivar (Figure 7a). In Montana 5, the decrease in A was proportional to Na accumulation, whereas in most cultivars (Alegria Disciplinada, Don Armando, K91, Rouge), a sharp decrease occurred between 50 mM and 100 mM, despite the modest accumulation of Na in some cultivars (particularly in K91). In Red Amaranth, A was higher at 100 mM compared to 50 mM. In Locale and Don Leon, despite a significant accumulation of Na in the leaves at 100 mM compared to 50 mM NaCl, no decrease in A was observed.

The plant response was cultivar-dependent for E and gs (Figure 7b,c, Table S5). Both parameters strongly correlated (r = 0.976). They decreased proportionally with salt accumulation in Don Armando, Don Leon and Montana 5, whereas they did not differ much between 50 and 100 mM NaCl in Alegria Disciplinada, Locale and Rouge. A sharp decrease in gs and E, despite a modest accumulation of Na between 50 and 100 mM NaCl, occurred in Red Amaranth and K91.

**Figure 7.** Response of photosynthetic activity to foliar Na accumulation. (**a**) Net photosynthesis (μmol CO2 m−<sup>2</sup> s−1); (**b**) transpiration rate (mmol H2O m−<sup>2</sup> s−1); (**c**) stomatal conductance (mmol H2O m−<sup>2</sup> s<sup>−</sup>1); (**d**) intrinsic water use efficiency (μmol CO2 mmol H2O<sup>−</sup>1).

The response of instWUE to salt was less conspicuous compared to A, E and gs (Figure 7). Water use efficiency increased with salt accumulation in most of the cultivars, but only at 100 mM NaCl in Don Leon (Figure 7d). The increase was modest in Locale and Montana 5, whereas it was more marked in Rouge, K91 and Red Amaranth. In Alegria Disciplinada and Don Armando, instWUE increased at 50 mM NaCl but decreased at 100 mM and showed a high standard deviation value.

#### *2.6. Biochemical Compound Contents in Relation to Sodium Accumulation in Leaves*

Pigments (chlorophyll *a*, chlorophyll *b*, betaxanthins and betacyanins) and oxidative stress-related compounds (malondialdehyde (MDA), total flavonoids, total phenolics, ascorbate) were quantified (Figures 8 and 9, Table S6). Betacyanins, MDA and ascorbate were not affected by salt stress, while flavonoids (*p* = 0.018) were only slightly affected (Table S7). In contrast, the concentrations of chlorophyll *a* (*p* < 0.001), chlorophyll *b* (*p* < 0.001), betaxanthins (*p* < 0.001), and total phenolics (*p* < 0.001) strongly decreased with salt stress (Table S7). Since the content of chlorophyll *a* and *b* strongly correlated with one another (r = 0.938), only chlorophyll *a* is presented in Figure 8 and the data of chlorophyll *b* are shown in Table S6.

**Figure 8.** Response of foliar (**a**) chlorophyll *<sup>a</sup>* (mg·g−<sup>1</sup> FW), (**b**) betaxanthins (μmol·g−<sup>1</sup> FW); and (**c**) betacyanins (nmol·g−<sup>1</sup> FW) to foliar sodium accumulation.

The content in chlorophylls was strongly reduced by salt in all cultivars at 100 mM NaCl, but only in Alegria Disciplinada, Don Leon and Montana 5 at 50 mM NaCl (Figure 8a, Table S6). Salinity decreased the betaxanthin content by more than 50% in Don Armando, K91 and Montana (Figure 8a). As a result, the chlorophyll and betaxanthin contents in leaves regularly decreased with Na content increase in most cultivars, with the exception of K91, Don Armando and Red Amaranth (Figure 8a,b). In contrast, the concentration of betacyanins was not affected by the Na concentration in the leaves, except in K91 (Figure 8c).

Although it was not affected by salt stress and did not significantly vary among cultivars (Table S7), MDA response to sodium accumulation in the leaves was cultivardependent (Figure 9a). The response of total polyphenol and flavonoid content to sodium concentrations in the leaves was similar in most cultivars, with the exception of Don Leon and Alegria Disciplinada (Figure 9b,c). For most cultivars, the concentration of polyphenols decreased proportionally to the Na content in leaves. Some exceptions were nevertheless observed. For example, in Red Amaranth, the foliar Na content was roughly the same at 50 and 100 mM NaCl but a decrease in phenolics was observed (Figure 9b). In Don Armando and Don Leon, the Na concentration in the leaves of plants treated at 50 mM NaCl had no effect or even a positive effect on the phenolic content, respectively (Figure 9b). The ratio between oxidized and total ascorbate was similar whatever the Na content in all cultivars (Figure 9d).

**Figure 9.** Response of the foliar (**a**) malondialdehyde (MDA, nmol·g−<sup>1</sup> FW), (**b**) total phenolics (mg·g−<sup>1</sup> FW), (**c**) total flavonoids (μg·g−<sup>1</sup> FW) and (**d**) ratio between oxidized and total ascorbate to foliar sodium accumulation.

#### **3. Discussion**

In this study, we compared the tolerance of eight cultivars of *Amaranthus cruentus* to 50 and 100 mM NaCl in hydroponic conditions at the vegetative stage. Our results revealed different levels of tolerance and various physiological responses among the cultivars. Sodium accumulated in all plant organs regardless of the cultivar. Since the salt treatments were applied for four weeks, amaranth plants were subjected to both osmotic and ionic phases of salt stress. Indeed, it was previously observed that both phases of salt stress were detected a couple of days after stress imposition in amaranth [31].

#### *3.1. Variability in Salt Tolerance among Leaf and Seed Cultivars of A. cruentus*

Our results showed that *A. cruentus* plants were more affected by salt treatments than by the cultivars. This pattern had been observed in other studies screening genotypes for abiotic stresses. In a study of salinity resistance in 25 African rice cultivars (*Oryza glaberrima*), Prodjinoto et al. also found that plants were better discriminated by salt dose than by genotype [63]. Similarly, in a comparison of 12 Tartary buckwheat (*Fagopyrum tataricum*) cultivars, the response to water and heat stress was better explained by the differences between plants than the cultivar [64].

Despite this, differences between cultivars were highlighted in this study. Some cultivars such as Locale, Don Leon and K91 were tolerant to a moderate amount of salinity, given the similar biomass production at 0 and 50 mM NaCl. However, Montana 5, Red

Amaranth and Don Armando were nearly as tolerant at 50 mM than at 100 mM NaCl. We also observed that salt tolerance at 50 mM NaCl was not correlated with salt tolerance at 100 mM, meaning that a cultivar tolerant to a moderate salt dose is not always tolerant to a high salt dose in *A. cruentus*. In a previous study that investigated the salt tolerance of several leaf cultivars of *A. cruentus* after 2 weeks of NaCl treatment, Rouge was identified as salt-tolerant, while Locale was identified as more salt-sensitive compared to other *A. cruentus* cultivars [28]. Our results showed that Rouge accumulated less sodium in leaves and stems than Locale after 4 months of stress, which could explain its higher tolerance. However, both produced an equivalent shoot biomass in this experiment, despite being slightly higher in Rouge at 100 mM NaCl. Amaranth species are usually considered to be salt tolerant [28,29,39] and most cultivars tested in this study survived at 100 mM NaCl, demonstrating the value of growing *A. cruentus* in areas moderately affected by salt. The closely related species *Amaranthus hypochondriacus* was tested for drought and multi-salinity (NaCl, CaCl2, KCl, MgCl2, MgSO4) tolerance in field conditions in South Italy [39,40]. It was shown that this grain amaranth can be grown under conditions of moderate combined drought and saline stress, at the cost of a decrease in seed nutritional quality.

Genetic diversity is an important prerequisite for breeding for salt tolerance [65]. Here, we report a noticeable variability in the physiological response (photosynthetic activity and biochemical activity) of *A. cruentus* cultivars to moderate salt stress, paving the way for developing salt-tolerant lines.

#### *3.2. Putative Physiological Role of Sodium in Amaranth*

Our results showed that the salt response of *A. cruentus* may differ according to the NaCl concentration. A previous study on *A. cruentus* leaf cultivars found that a low concentration of NaCl in hydroponic conditions could stimulate several parameters related to mineral content and oxidative status [34]. After two weeks of exposure, 30 mM NaCl had a positive effect on the plant growth and health compared to control conditions, whereas higher concentrations (60 and 90 mM) had detrimental effects [34]. After 4 weeks of exposure to 50 and 100 mM NaCl, the results of the present experiment differed substantially. Even though some parameters were not affected at 50 mM NaCl in some cultivars, or in some cases slightly up-regulated, generally, no positive effect of salt was observed on any parameter in our study. Previous works on *A. tricolor* demonstrated that similar NaCl concentrations (50–100 mM) had a positive effect on the nutritional quality (several minerals, macronutrients and phenolics) of leaves [41,42]. Often considered as a "functional nutrient" rather than as an essential nutrient in plants (e.g., possible substitution of K in some metabolic functions), Na is required in a small quantity in some NAD-ME-type C4 plants for pyruvate transport and conversion of some metabolic intermediates [66–69]. It was also demonstrated that Na is important in amaranth (*A. tricolor*) besides its putative role in C4 photosynthesis, for instance by stimulating N assimilation [70–73]. Further research using lower salt concentrations could determine the range of NaCl concentrations that stimulates *A. cruentus* growth and the nutritional quality of leaves.

#### *3.3. Impact of Sodium Accumulation on Photosynthetic Activity*

The eight cultivars investigated in this study could be distinguished based on the accumulation of Na in leaves, especially the accumulation difference between 50 mM and 100 mM NaCl, and its consequence on photosynthetic activity. The salt accumulation in leaves caused by salt stress was linked to an important decrease in stomatal conductance, transpiration rate and, to a lesser extent, carbon assimilation in some cultivars, whereas some others maintained their photosynthetic activity despite foliar sodium accumulation.

The significant water use efficiency increase in stressed plants was caused by a stronger decrease in the transpiration rate compared to the salt-induced decrease in net photosynthesis. Omamt et al. investigated the effect of saline stress on the WUE of various *A. cruentus*, *A. hypochondriacus* and *A. tricolor* genotypes [30]. They observed an increase of about 50% in instantaneous WUE in *A. cruentus* at 100 mM NaCl. However, the values recorded in

their study were 2–3 times higher than what we observed in the current study, even in control conditions. The same authors demonstrated that the decrease in photosynthetic activity was, at least in part, due to salt-induced stomatal closure and decrease in stomatal density [30]. An increase in WUE in *A. cruentus* cv. Locale was also observed by Gandonou et al. [29]. An increase in WUE is considered as a salt-tolerance mechanism, improving the capacity of the plant to limit water loss despite the salinity toxic effects [74,75]. Liao et al. reported an increase in WUE in maize in combined water and salt stress conditions [76]. These authors identified salt-induced osmotic adjustment in stomata as the main mechanism of stomatal conductance regulation, resulting in an increased salt tolerance in this C4 crop. In our study, the highest WUE under salinity was observed in K91 and Red Amaranth, suggesting that these cultivars were able to maintain photosynthesis and limit transpiration and thus showed a higher salt tolerance from the physiological point of view. In grasses, halophytism has been associated with C4 photosynthesis, which could be explained by the high WUE provided by this type of carbon fixation [77]. This could be also true in amaranths, since all species use C4 photosynthesis [45].

Another parameter often considered as a reliable physiological index for the tolerance to salt stress is the Na/K ratio, which significantly increased in all organs in our study [78]. In contrast to Na, K is an essential element used in different functions, such as in various steps of cell metabolism, photosynthesis and turgor pressure maintenance [79]. Maintaining a low Na/K ratio in plants and mainly in leaves is thus necessary. Red Amaranth and K91 showed the lowest Na/K ratios explained by their ability to restrict Na accumulation in leaves at 100 mM compared to 50 mM NaCl. Indeed, those cultivars were among the most tolerant at 100 mM NaCl.

#### *3.4. Foliar Biochemical Activity Response to Salt Stress*

Malondialdehyde is a by-product of lipid peroxidation, caused by reactive oxygen species, which are produced in response to stress, including salt stress [62,80,81]. In our experiment, foliar MDA content did not increase in response to salinity, suggesting low salt-induced oxidative stress on lipids in leaves of the selected *A. cruentus* cultivars. We observed that the total foliar polyphenol content (flavonoids also, to a lesser extent) decreased in plants exposed to NaCl. However, the phenylpropanoid pathway is often upregulated in response to many abiotic stresses because polyphenols have protective roles, for instance against oxidative stress [82,83]. Moreover, no difference in the ratio between oxidized and total ascorbate was observed between the control and stressed plants, although this metabolite is also involved in oxidative stress mitigation [84]. The low intensity of oxidative stress suggested by the low MDA content could explain why the plants exposed to salinity did not upregulate polyphenols and ascorbate production. Betalains, which include the two subfamilies of pigments betaxanthins and betacyanins, are also involved in plant tolerance to abiotic stress. Salinity-induced betalain accumulation has been described in two halophyte species of the Amaranthaceae family [85]. Sarker and Oba (2018) reported an increase in betacyanin and betaxanthin content in the leaves of *A. tricolor* exposed to 200 mM NaCl [86]. A photoprotective role of betacyanins was also described in *A. cruentus* [87]. Here, only the betaxanthin content significantly increased in response to stress.

#### *3.5. Differences between Leaf and Seed Cultivars*

There are two types of amaranth cultivars, depending on the harvested parts. African Rouge and Locale cultivars have been bred for leaf production, whereas the others are cultivated for their seeds. Even though the cultivar types are clearly distinguishable by several morphological traits (size and color of seeds, shape of leaves, branching pattern or inflorescence structure (see Table S1)), their physiological response to salinity stress did not differ at the vegetative stage. Only the transpiration rate was, on average, 1.5× higher in the grain cultivars than in leaf cultivars in the control conditions and at 50 mM NaCl, although it dropped at the same level at 100 mM NaCl. Comparing leaf and grain cultivars at the reproductive stage could reveal additional differences in response to salt stress. Although amaranths are usually used either for grain production or leaf production, the dual use of leaves and seeds on a single cultivar seems promising on the basis of several defoliation experiments [51–53,88–90]. To our knowledge, the tolerance of amaranth to defoliation under abiotic stress has not been investigated yet.

#### *3.6. Effect of Salt of the Nutritional Quality of Amaranth*

Amaranths are recognized for their exceptional nutritional value, both of their leaves and seeds [34,54,56,86,91]. Adverse environmental conditions could alter the nutritional quality of plants, although it was demonstrated that *A. tricolor* keeps its nutritional value under abiotic stress [86,92]. Although the effect of salinity on the nutritional quality was not a main objective of this work, we showed that salt stress caused a decrease in phenolic content in most cultivars. These metabolites, however, have nutritional and health benefits [93]. The decrease in the foliar content of several essential minerals (K, Ca, Mg) should also be highlighted. Given the outstanding nutritional value of this crop, understanding the effects of moderate salt stress on the nutritional quality of *A. cruentus* leaves is crucial and needs further research. A previous work on the two leaf cultivars demonstrated an increase in Mg, P, Fe, vitamin C, phenolic, α-tocopherol and carotenoid contents in leaves when exposed to 30 mM of NaCl, particularly in Rouge [34]. Salt stress and drought stress, the latter being in some respects similar to salt stress, can also improve the nutritional quality of amaranth leaves [42,94]. To our knowledge, the effect of abiotic stress, particularly salinity, on the nutritional quality of the seeds of grain amaranth has not been investigated extensively yet. More broadly, the effect of salt on the reproduction of amaranths requires further research.

#### **4. Materials and Methods**

#### *4.1. Plant Material and Growth Conditions*

Leafy cultivars Rouge and Locale were kindly provided by Dr. Christophe B. Gandonou (University of Abomey-Calavi, Cotonou, Benin) and selected based on previous works [28]. Since no information was available about the salt tolerance of grain cultivars, six grain cultivars differing by their origin were randomly selected and obtained from the Genebank of the Crop Research Institute (CRI, Prague, Cezch Republic) (see Table S1 for accession numbers).

Plants were cultivated in greenhouses (SeFy, UCLouvain) at 23–25 ◦C at day, 20–22 ◦C at night and 65% RH, under a 16 h photoperiod. When necessary, artificial light was provided by 650 W red-blue LumiGrow LED lights (minimum light intensity of 150 μmol m−<sup>2</sup> s<sup>−</sup>1). Seeds were sown in 2/3 peat compost (DCM, Amsterdam, The Netherlands) and 1/3 river sand (Mpro, Wavre, Belgium) (volume:volume). Two weeks later, seedlings were transplanted individually in 6 × 6 × 6 cm plastic pots in 2/3 peat compost +1/3 river sand. Two weeks later, they were transplanted in 15 L plastic tanks filled with Hoagland nutritive solution (5 mM KNO3, 5.5 mM Ca(NO3)2, 1 mM NH4H2PO4, 0.5 mM MgSO4, 25 μM KCl, 10 μM H3BO4, 1 μM MnSO4, 0.25 μM CuSO4, 1 μM ZnSO4, 10 μM (NH4)6Mo7O and 1.87 g L−<sup>1</sup> Fe-EDTA, and pH 5.5–6), with 1 seedling of each cultivar per tank (9 plants/tank). Tanks were randomly assigned to 0, 50 or 100 mM NaCl, with 9 replicates (27 tanks). The NaCl was added in the Hoagland solution and salt stress started 9 days after the transfer to plastic tanks. The nutritive solution was renewed once a week. The experiment took place over 53 days in November and December 2020.

#### *4.2. Biomass and Harvest*

Plants were harvested 53 days after sowing for destructive measurements, mineral and biochemical analyses. For three plants per cultivar and salt treatment, three young but well-expanded leaves were harvested in liquid nitrogen and stored at −80 ◦C for further biochemical analyses (see below). For five other plants per cultivar and treatment, the stems, leaves and roots were separated, weighted (for fresh weight), dried at 60 ◦C for 72 h, then weighed again (for dry weight). Water content was calculated as (fresh weight − dry weight)/fresh weight. Dry material was used for mineral analyses (see below). The salt tolerance index was calculated as the ratio between the mean total biomass production in salt conditions relative to the total biomass production in control conditions [78].

#### *4.3. Biochemical Analyses*

#### 4.3.1. Pigments

Chlorophyll *a* and *b* were quantified in the leaves of three plants per cultivar and treatment according to [95]. Briefly, 1.2 mL of 80% acetone (*v*/*v*) was added to 50 mg of finely ground (in liquid nitrogen) fresh leaves. After 60 min of incubation at 4 ◦C, tubes were centrifugated (10,000× *g*, 4 ◦C, 10 min). A second identical extraction was performed on the pellet; supernatants were combined. Absorbance of the supernatant was read at 663.2 and 646.8 nm (UV-1800 spectrophotomer, Shimadzu, Kyoto, Japan). Chlorophyll *a* and *b* contents were measured as follows: Chl *a* (mg/L) = 12.25 × Abs663.2 − 2.79 × Abs646.8 and Chl *b* (mg/L) = 21.50 × Abs646.8 − 5.10 × Abs663.2.

Betalains were extracted in deionized water overnight. Absorbance of the supernatant was read at 540 nm (betacyanins) and 475 nm (betaxanthins) (UV-1800 spectrophotomer, Shimadzu, Kyoto, Japan). Molar extinction coefficients of 62 × <sup>10</sup><sup>6</sup> cm<sup>2</sup> mol−<sup>1</sup> and <sup>48</sup> × <sup>10</sup><sup>6</sup> cm<sup>2</sup> mol<sup>−</sup>1, respectively, were used to quantify the pigment content [96,97].

#### 4.3.2. Phenolics

Total phenolics and flavonoids were quantified in the leaves of three plants per cultivar and treatment according to [98,99]. After grounding in liquid nitrogen, 1.4 mL of 80% methanol was added to 100 mg of fresh material, before centrifugation (20,000× *g*, 20 min, 4 ◦C). The supernatant was stored at −20 ◦C until quantification.

A volume of supernatant was added to an equal volume of 2% AlCl3 for total flavonoid quantification (adapted from [98]). After 10 min of incubation at room temperature in the dark, absorbance was read at 440 nm, with quercetin as the standard.

Total phenolics were quantified as follows: 200 μL of supernatant was added to 2.8 mL of deionized water and 200 μL of Folin–Ciocalteu reagent [99]. Three minutes later, 0.8 mL of 20% Na2CO3 was added before incubation in a water bath at 40 ◦C for 40 min. Absorbance was read at 760 nm with gallic acid as the standard.

#### 4.3.3. Malondialdehyde

Malondialdehyde, a marker of lipid peroxidation [100], was quantified in the leaves of three plants per cultivar and treatment according to [101]. It was extracted in 250 mg of finely ground fresh leaves with 4 mL of 5% trichloroacetic acid with 1.25% glycerol. After 5 min of incubation at 4 ◦C, tubes were centrifuged for 10 min at 4 ◦C, 12,000× *g*. Then, 2 mL of supernatant was added to 2 mL of a 0.67% aqueous solution of thiobarbituric acid. Samples were incubated for 30 min in a water bath at 100 ◦C. Absorbance was read at 532 and 600 nm. Malondialdehyde concentration (mM) was calculated as (A532nm − A600)/155 mM cm−<sup>1</sup> [101].

#### 4.3.4. Ascorbate

Ascorbate was quantified with some adaptations from [102], in the leaves of three plants per cultivar and treatment. Briefly, it was extracted in 250 mg of finely ground fresh leaves with 4 mL of 5% trichloroacetic acid (TCA). After 15 min of incubation on ice, samples were centrifugated (5 min, 4 ◦C, 10,000× *g*). Next, 200 μL of supernatant was added to 400 μL of phosphate buffer (0.2 M, pH 7.4). For oxidized ascorbate quantification, 0.4 mL of water was added. For total ascorbate quantification, 200 μL of 10 mM 2,2 -dithiothreitol was added, the samples were incubated 5 min at room temperature, then 200 μL of 0.5% N-ethylmaleimide was added. After 1 min of incubation at room temperature, 1 mL 10% TCA was added in all the tubes, then 0.8 mL 42.5% H3PO4 and 0.8 mL 4% dipyridyl were added. Finally, 400 μL of 3% FeCl3 was added while agitating. Tubes were incubated

60 min in a water bath at 37 ◦C. Absorbance was read at 525 nm. Ascorbic acid was used as the standard.

#### 4.3.5. Mineral Content

The concentrations of Na and K were quantified in the roots, stem and leaves of three plants per cultivar and treatment while the concentrations of Ca, Fe, Mg and Zn were quantified only in the leaves. For mineralization, 4 mL of 68% nitric acid was added to 50–100 mg of ground dry plant material. After one night of incubation, nitric acid was evaporated using a sand bath. Then, 1.5 mL of aqua regia (500 μL of 68% nitric acid and 1.5 mL of 37% hydrochloric acid) was added and incubated two minutes on the sand bath. The volume was adjusted to 10 mL with deionized water before filtration on Whatman Grade 1 paper. The concentration of Na, K, Mg, Ca, Fe and Zn was determined by atomic absorption spectroscopy (ICE 3300, Thermo Scientific, Waltham, MA, USA) after the required dilutions and addition of 1% LaCl3 for Na and K quantification.

#### *4.4. Photosynthetic Activity*

The portable photosynthesis system LCpro-SD (ADC Bioscientific Ltd., Hoddesdon, United Kingdom) was used for photosynthetic activity analyses. Measurements were performed on three plants per cultivar and treatment, 51 days after sowing (27 days after stress). Net photosynthesis (A), stomatal conductance (gs) and transpiration rate (E) were recorded in a young, well-expanded leaf after several minutes of stabilization, in conditions of ambient irradiance, carbon dioxide concentration, air humidity and temperature, similarly to [103]. Water use efficiency was calculated as A/E.

#### *4.5. Statistical Analyses*

All statistical analyses were performed in R, version 4.2.1 [104]. Normality of the data was verified based on histograms of residuals and homoscedasticity was verified with a Levene test. For each variable (plant growth, mineral content, physiological and biochemical parameters), analyses of variance (ANOVA 2) were performed with the salt treatment and cultivars as the fixed factors using the function "aov()" (base R). Detailed results of ANOVA 2 are presented in Tables S2, S4, S5 and S7. For each variable, Tukey's test from base R was used to perform multiple comparison tests to evaluate the differences between salt treatments within each cultivar. The package *ade4* was used to perform a principal component analysis to visualize the differences among the cultivars and salt treatments according to mineral and biomass data. Redundant variables with r > 0.8 were removed prior to analysis. Correlations between all the variables were quantified with the Pearson correlation coefficient (r) and significance tests were used to calculate the associated *p*-values with R package *corrplot*. Data are presented in figures and tables as the mean ± standard deviation, with adequate rounding.

#### **5. Conclusions**

Our results demonstrate a noticeable tolerance of *A. cruentus* cv. Don Leon and K91 at the vegetative stage under moderate salt stress (50 mM) in hydroponic conditions, since biomass production did not differ from the control conditions. Sodium accumulated in all organs regardless of the cultivar, mainly in stems. Different physiological responses to foliar sodium accumulation were observed among the cultivars, suggesting a predominance of the ionic phase when physiology was negatively affected in response to ion accumulation or of the osmotic phase when adverse effects on physiology were observed, despite no or a low sodium accumulation. Water use efficiency increased in response to salt because of an efficient decrease in stomatal conductance, despite a decrease in net photosynthesis. The lower transpiration rate of the leaf cultivars compared to the grain cultivars was the unique discriminating physiological trait between the two types of cultivars. Since salt stress did not increase neither MDA content nor the metabolites involved in protection against radical oxygen species such as polyphenols, betacyanins and ascorbate, we suggest

that the oxidative stress was limited. This work provides a basis for further investigation of the physiological mechanisms underlying the variations in salt tolerance in red amaranth, a plant with a promising future in resilient agro-ecosystems under global change.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12183310/s1, Table S1: Origin and main morphological characteristics of the eight *A. cruentus* cultivars used in this study; Table S2: Anova results of biomass production; Table S3: Water content of leaves, stem and roots of eight cultivars of *A. cruentus* exposed to 0 mM, 50 mM and 100 mM of NaCl; Table S4: Anova results of mineral content; Table S5: Anova results of photosynthetic activity; Table S6: Foliar content of chlorophyll *b* of eight cultivars of *A. cruentus* exposed to 0 mM, 50 mM and 100 mM of NaCl; Table S7: Anova results of biochemical data.

**Author Contributions:** Conceptualization, A.L., M.Q. and S.L.; methodology, A.L., M.Q. and S.L.; formal analysis, A.L.; investigation, A.L.; resources, M.Q. and S.L.; data curation, A.L.; writing—original draft preparation, A.L. and M.Q.; writing—review and editing, A.L., M.Q. and S.L.; visualization, A.L.; supervision, M.Q. and S.L.; project administration, M.Q. and S.L. All authors have read and agreed to the published version of the manuscript.

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

**Data Availability Statement:** Data are contained within the article or Supplementary Material.

**Acknowledgments:** The authors are grateful to Baudouin Capelle and Brigitte Vanpée for their technical assistance, and to the Genebank of the Crop Research Institute (Prague, Cezch Republic) for obtaining the seeds.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**


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