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Article

MbNAC22, a Malus baccata NAC Transcription Factor, Increased Drought and Salt Tolerance in Arabidopsis

by
Kuibao Jiao
1,†,
Jiaxin Han
2,†,
Baitao Guo
1,
Yuqi Wu
1,
Lei Zhang
1,
Yuze Li
1,
Penghui Song
1,
Deguo Han
2,
Yadong Duan
1,3,* and
Xingguo Li
2,*
1
Institute of Rural Revitalization Science and Technology, Heilongjiang Academy of Agricultural Sciences, Harbin 150028, China
2
Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northeast Region), Ministry of Agriculture and Rural Affairs, National–Local Joint Engineering Research Center for Development and Utilization of Small Fruits in Cold Regions, College of Horticulture & Landscape Architecture, Northeast Agricultural University, Harbin 150030, China
3
Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150081, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2023, 13(5), 1374; https://doi.org/10.3390/agronomy13051374
Submission received: 8 April 2023 / Revised: 10 May 2023 / Accepted: 12 May 2023 / Published: 14 May 2023
(This article belongs to the Special Issue Decrypting the Mechanism of Protein Transcription in Plant Stress Resistance)
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Abstract

:
As an excellent grafting material, Malus baccata (L.) Borkh is native to Liaoning, Jilin, Heilongjiang and other regions in China, with a strong adverse environmental adaptability. As a typical transcription factor, the NAC gene acts as a regulator in many molecular pathways responding to abiotic stress. However, research of NAC in the Malus baccata has just begun. In the present research, a new NAC transcription factor, MbNAC22, was obtained from the seedlings of Malus baccata, and its function in drought and salt treatments was studied by heterologous expression. The open reading frame of the MbNAC22 gene is 768, encoding 255 amino acids (aa). Through confocal microscopy, MbNAC22 was found to be located in the nucleus. The heterologous expression of MbNAC22 in Arabidopsis showed that it enhanced the viability of Arabidopsis under drought and salt treatments. Under stresses, the chlorophyll content of the plants decreased, but the decline of the overexpressed-MbNAC22 Arabidopsis was relatively low. Through phenotypic observation and determination of stress-related physiological indicators, it was found that compared with WT Arabidopsis, overexpressed-MbNAC22 Arabidopsis had a higher tolerance to stresses. Under stresses, the overexpression of MbNAC22 positively regulated ion-transport-related genes (AtNHX1 and AtSOS1), the key genes of the ABA pathway (AtNCED3 and AtDREB2A), the proline synthesis gene (AtP5CS2) and the drought-induced gene (AtERD11), while the expression of the leaf senescence-associated gene (AtSAG21) and programmed cell death related gene (AtAEP1) was inhibited. Therefore, we speculate that MbNAC22 responds positively to drought and salt stresses by regulating the expression of stress-related genes.

1. Introduction

Drought and salt are natural factors that inhibit plant nutrition and reproductive growth, as well as reduce plant yield and quality [1,2]. When a plant is under drought stress, its own morphology will change, such as increasing the root–shoot ratio [3], decreasing the leaves and thickening the epidermis [4]. Salt stress leads to stomatal closure, leaf growth restriction and even premature cell death. Therefore, in the long process of evolution, plants have evolved a complex self-regulation mechanism to adapt to drought and salt stresses, in which transcription factors play an irreplaceable role. WAKY, bZIP and NAC, as activators or repressors in molecular regulatory mechanisms, regulate plant vegetative growth, reproductive development and the response to adverse conditions [5]. NAM, ATAF and CUC2, respectively, provide initial letters for naming NAC TFs [6]. NAC is one of the plant-specific transcription factor families containing NAC domain, which is involved in the regulatory mechanism of response to abiotic stress [5]. The N-terminal has a NAC domain composed of 5 sub-domains (A–E), containing about 150–160 amino acids [7]. It may be related to the formation of dimer and DNA binding [8]. The C-terminal has a transcriptional regulatory region, which usually acts as a functional domain and has transcriptional activation or transcriptional inhibition functions [9]. NAC TFs recognize and combine downstream genes containing CGT [A/G] and CACG (NAC recognition sequence, NACRS) sites, and regulate their expression to improve plant stress resistance [10]. For example, ANAC055/019/072 regulates the drought resistance of Arabidopsis by binding to the cis-elements CATGT(G) and CACG of the early drought response gene (ERD1) [11]. BES1/BZR1 can be combined with E-box and BRRE (CANNTG, CGTGT/CG) to participate in the drought stress response to brassinolide [12].
At present, NAC TFs have been found in many species, such as mung bean [13], Populus bonatii [14], kiwifruit [15], Vitis amurensis [16], Passiflora edulis [17], Linum usitatissimum [18] and Panax ginseng [19], all of which have identified NAC TFs. NAC TFs have been proven to regulate plant responses to adverse environments. For example, GmNAC12 can positively regulate the tolerance of soybean to drought stress through overexpression and gene knockout technologies [20]. In addition, GmNAC15 can positively respond to the adaptability of plants to salt stress, and overexpressed-GmNAC06 promotes the development of plant lateral roots, improves the scavenging ability of ROS, and carries out osmotic adjustment to enhance the salt tolerance of soybean [21,22]. Overexpression of TaNAC48 can reduce the content of ROS in plants, thus improving the drought resistance of plants. In addition, TaNAC48 may participate in the positive feedback regulation of the ABA signal pathway by inducing the expression of TaNCED3, and the higher expression level of TaNCED3 promotes the accumulation of TaDREB1A and TaCOR47 to achieve the purpose of drought resistance regulation [23]. Under drought stress, the water loss rate of overexpressed-ZmNAC48 decreased, stomatal closure increased and the expressions of NCED3 and ABA3 were higher than those of WT. Therefore, it is speculated that transgenic plants improve drought resistance by increasing ABA accumulation [24]. NAC TFs are key regulators in the vegetative growth and reproductive development of plants. For example, ATAF1 and ANAC019 genes in Arabidopsis are negative and positive regulatory factors of Botrytis cinerea, respectively [25,26]. In addition, genome-wide expression analysis showed that NAC genes were involved in lignin accumulation, lateral root development, seed germination, flower organ formation and plant senescence [27]. Therefore, research on NAC transcription factors is of great significance.
Malus baccata is a deciduous tree belonging to the Malus Mill. of Rosaceae, which is widely planted in northeast China, Mongolia and Siberia regions. Malus baccata is an excellent ornamental tree species with an elegant appearance, luxuriant flowers and leaves. In addition, the tender leaves can be used for tea, and the wood can be used for carving. Most importantly, Malus baccata is an excellent apple grafting rootstock, which can be used to cultivate apple varieties with an excellent resistance to adversity. Not only is it extremely cold-resistant, but it also has a strong resistance to drought and salt stresses. In recent years, our research team has successively cloned MbNAC25 and MbNAC29 genes, studied their functions in cold and salt stresses, and found that they are positive regulators of cold and salt stresses [28,29]. In this research, MbNAC22 was obtained from the seedlings of Malus baccata, and bioinformatics analysis was carried out. In addition, through phenotypic observation, physiological index determination and expression analysis of related stress genes, it was found that MbNAC22 positively regulated the viability of plants under drought and salt treatments. This research provides strong evidence for NAC TFs to play a positive role in resisting the natural environment.

2. Materials and Methods

2.1. Plant Materials, Growth Conditions and Treatments

The Malus baccata seedlings were cultured in a MS medium containing 0.5 mg/L IBA and 0.5 mg/L 6-BA for 25 days, and then transferred to a MS medium containing 1.2 mg/L IBA for rooting. Subsequently, the 60-day-old plantlets of Malus baccata in good growth state were treated with hydroponics, and the growth conditions were as described by Han et al. [30]. The hydroponic seedlings were treated with stresses (4 °C/cold, 200 mM NaCl/salt, 20% PEG6000/simulated drought and 37 °C/heat) when they grew white new roots [29]. The collection time points of the samples were 0, 1, 3, 6, 9 and 12 h. Arabidopsis was cultivated in an environment with a daily temperature of 25 °C and grown in light for 2/3 days [31].

2.2. Cloning and Bioinformatic Analysis of MbNAC22

The MbNAC22 was isolated from the cDNA of Malus baccata (primer-MbNAC22-F/R: 5′-ATGGAGAGGATTAATTTTGTGAAGA-3′/5′-CTACTGTTTTCTTCTAATTAAACCT-3′). The target gene was ligated with a Cloning vector using pEasy-T5 Zero Cloning Kit (CT501), and escherichia coli containing MbNAC22 was obtained [32]. The MbNAC22 was translated by DNAMAN5.2, and the physical and chemical properties of the edited protein were analyzed by ProtParam (http://web.expasy.org/protparam/, accessed on 11 January 2022). The homology of the MbNAC22 protein was analyzed by BLAST in the NCBI (National Center for Biotechnology Information) database (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 31 January 2022), and multi-sequence alignment and phylogenetic tree construction were performed using MEGA 7.0 [2].

2.3. Subcellular Localization of MbNAC22

Using primer 5′-cgGGATCCATGGAGAGGATTAATTTTGTGAAGA-3′ (BamH I)/5′-gcGTCGACCTACTGTTTTCTTCTAATTAAACCT-3′ (Sal I), a MbNAC22 gene fragment with a restriction endonuclease site was cloned. The purified product was connected to linearized plasmid pSAT6-GFP-N1 to obtain the 35S-MbNAC22-GFP vector [28]. The 35S-MbNAC22-GFP and the 35S-GFP vectors were transferred into GV3101 Agrobacterium, respectively. The cells to be detected were obtained by injecting Agrobacterium into the abaxial epidermal cell of Nicotiana tabacum leaves. The subcellular localization of the MbNAC22 protein was observed by confocal microscopy. In addition, nuclear-localized mCherry was constructed using the method of Wang et al. to indicate the nucleus [33].

2.4. QRT-PCR Analysis of MbNAC22

The RNA of the stress treated sample was extracted, and reversely transcribed into the first strand cDNA for analysis of the expression pattern of the MbNAC22 gene. MbActF1: 5′-ACACGGGGAGGTAGTGACAA-3′ and MbActR1: 5′-CCTCCAATGGATCCTCGTTA-3′) were used as the internal reference control primers, as well as qRT-PCR primers MbNAC22-qF/R: 5′-CGACTGGGTTCTTTG-3′/5′-CGAGTTCTTGGGTTT-3′. Refer to Liang et al. for the operation method of qRT-PCR [34]. The expression of MbNAC22 was calculated using 2−ΔΔCT method [35].

2.5. Vector Construction and Plant Transformation

Using ClonExpress® II One Step Cloning Kit, the MbNAC22 fragment with the enzyme cleavage site was connected to the pCAMBIA2300 vector that had been digested by Sal I and BamH I. After successful sequencing of a single colony, agrobacterium GV3101 (AC1001) containing MbNAC22 fragment was obtained [36]. The wild-type (WT) Arabidopsis ecotype Columbia was transformed by inflorescence impregnation method. Overexpressed-MbNAC22 Arabidopsis and pCAMBIA2300-Arabidopsis (VL) were obtained by screening the medium containing kanamycin (50 mg L−1) [37]. The RNA of Arabidopsis that had been preliminarily screened by kanamycin was extracted, reverse-transcribed, and qRT-PCR was performed using primers (MbNAC22-qF/R) to analyze the expression of MbNAC22 to verify whether the transformation was successful. T3 generation homozygous Arabidopsis was used for the further research.

2.6. Observation of Morphological Changes and Determination of Related Physiological Indicators

According to the expression of MbNAC22 under stresses, drought and salt were selected as the stresses for subsequent research. When Arabidopsis grows 10–12 leaves, stress treatments can be applied. Arabidopsis with basically the same growth was selected for the experiment. Each strain (WT, VL, S2, 6 and 7) was divided into three groups, one group grew normally as the control group, and the other two groups were treated with drought and salt stresses, respectively. The drought treatment group had the same conditions as the control group, except that they did not receive water for 7 days (without water 7 d). The salt stress treatment group was continuously irrigated with saline water containing 200 mM NaCl for 7 days (200 mM NaCl for 7 d). After relieving drought and salt stresses and returning to normal growth for 7 d, morphological changes in Arabidopsis were observed and survival rates were calculated.
The chlorophyll and proline (Pro) were extracted by acetone [28] and ninhydrin [34] reaction method, respectively. The activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were determined with test boxes (YX-W-A500, YX-W-A501 and YX-W-A502). The method of Ma et al. was used to measure the electrolyte leakage (EL) [38].

2.7. Analysis Stress-Related Genes

The cDNA of T3 generation Arabidopsis was obtained after stress treatments. Under salt treatment, qRT-PCR was used to analyze the expression of ion-transport-related genes (AtNHX1 and AtSOS1), the leaf senescence-associated gene (AtSAG21) and the programmed cell death related gene (AtAEP1). The expressions of the key genes of the ABA pathway (AtNCED3 and AtDREB2A), the proline synthesis gene (AtP5CS2) and the drought-induced gene (AtERD11) under drought stress were analyzed. The qRT-PCR primers: AtActin-F/R: 5′-CTGGATTCTGGTGATGGTGTGTCT-3′/5′-GAACCACCGATCCAGAC ACTGTAC-3′; AtNCED3-F/R: 5′-CCAGCTCTTCATTTCCCTAAGCAAT-3′/5′-AACACTAGGAT CAGCCGTTTTAGGA-3′; AtDREB2A-F/R: 5′-GAAAGAGAAACAGAAGGAGCAAGGG3′/5′-CATTTAGGTCACGTAGAAGCTCA-3′; AtERD11-F/R: 5′-GCATTGAAATTGAGTCGCATG AGTT-3′/5′-CGTAAACATCGAGGACTTTGGCTAG-3′; AtP5CS2-F/R: 5′-CACCCATAAGGAT CTTCCTGTCTT-3′/5′-CAATTCTCAACAGCCTCTGTCC-3′; AtSOS1-F/R: 5′-TCGTTTCAGCC AAATCAGAAAGT-3′/5′-TTTGCCTTGTGCTGCTTTCC-3′; AtNHX1-F/R: 5′-TGAGCCTTCA GGGAACCAC-3′/5′-AAAGCCACGACCTCCAAAGA-3′; AtAEP1-F/R: 5′-TTACAAACAGAG ACTTTGCACG-3′/5′-CCGGTGCCTTTTGATACTTATG-3′; AtSAG21-F/R: 5′-CTGTGATGAAG AAGA AGGGAGT-3′/5′-TCTGTAATAACCGGTTTTGGGA-3′ [39].

2.8. Statistical Analysis

Drought and salt stress treatments were performed in triplicate, respectively, with consistent results. The data were processed with GraphPad Prism software (v8.0.2.263). The statistical difference was clarified through analysis of variance (ANOVA) by t-test, and * p ≤ 0.05 and ** p ≤ 0.01 * were used to indicate the significant differences. Three technical replicates were performed on each measured sample, representing the data in the form of mean ± standard error (± SE; n = 3). Three biological replicates were conducted on WT, VL and three MbNAC22 transgenic Arabidopsis strains (S2, 6 and 7).

3. Results

3.1. Sequence Analysis of MbNAC22

The MdNAC22 (NM_001294071.1) gene sequence of Malus domestica was used to obtain MbNAC22. The total length of the CDS of the MbNAC22 gene was 768 bp, and it was found to encode 255 amino acids (aa) through DNAMAN5.2 translation. Using ProtParam to analyze the protein properties, it was found that the theoretical isoelectric point, the average of hydropathicity (GRAVY), the aliphatic index and the molecular weight of MbNAC22 were 9.21, −0.666, 65.69 and 28.887 kDa, respectively. The instability index (II) was computed to be 50.29. Therefore, we speculate that the MbNAC22 protein was an unstable hydrophilic protein. The MbNAC22 protein mainly contains 6 amino acids, namely Ser (9.4%), Pro (8.2%), Arg (7.8%), Val (7.5%), Leu (6.7%) and Lys (6.7%). Sequence analysis showed that MbNAC22 belonged to the NAC transcription factor family, which contains a typical NAC conserved domain between 16 and 137 (aa) (Supplementary Figure S1).
Using DNAMAN5.2 and NCBI for homology analysis, the results showed that the N-terminal of the MbNAC22 protein had a complete NAC conservative domain composed of A-E 5 subdomains, which was consistent with the structural characteristics of the NAC family (Figure 1A). In addition, the construction of an evolutionary tree using MEGA 7.0 also revealed that MbNAC22, MsNAC83 (Malus sylvestris) and MdNAC22 (Malus domestic) belong to a branch with the highest homology and the closest genetic relationship. Secondly, the evolutionary relationship between PaNAC83 (Prunus avium) and PdNAC83 (Prunus dulcis) was also relatively close, while the homology with CiNAC83 (Carya illinoinensis), JcNAC83 (Jatropha curcas), PpNAC83 (Prunus persica), PmNAC83 (Prunus mume) and AtNAC83 (Arabidopsis thaliana) was relatively low, and the evolutionary relationship between PbNAC83 (Pyrus bretschneideri) was the farthest (Figure 1B).

3.2. Subcellular Localization of MbNAC22 Protein

Previous studies had shown that most NAC transcription factors were located in the nucleus to play a role. The abaxial epidermal cell of Nicotiana tabacum leaves was injected with vector containing Agrobacterium. The results are shown in Figure 2. The 35S-GFP empty vector can observe green fluorescent signals in the whole cell. However, cells containing MbNAC22 can only observe green fluorescent signals in the nucleus, which completely overlap the mCherry signal of the NLS-mCherry fusion protein; these signals were expressed. Therefore, MbNAC22 was a nuclear protein, which conformed to the typical functional characteristics of transcription factors.

3.3. Expression Pattern Analysis of MbNAC22

The expression pattern of MbNAC22 was studied by qRT-PCR. It was found that under the condition of CK, MbNAC22 was expressed in all parts of the Malus baccata, with higher expression in the new leaf and root, followed by the stem, and lowest expression in the mature leaf, with a difference of 8.38 times between the highest and lowest expression (Figure 3A). Based on these results, we found that MbNAC22 gene was highly expressed in the organs of nutrient transport and speculated that it was mainly involved in the vegetative growth of plants.
In order to study the expression change of MbNAC22 induced by abiotic stresses, four kinds of stresses were set to treat the seedlings of Malus baccata, and a total of six time points were set (0, 1, 3, 6, 9 and 12 h). Cold, drought, salt and heat treatments can rapidly induce the expression of MbNAC22, and there were expression peaks in the new leaf and root. In the new leaf, the peak time was 3, 9, 6 and 6 h, respectively. It was 8.32, 9.74, 9.27 and 6.68 times of the untreated (Figure 3B). In the root, the peak time was 6, 9, 3 and 1 h, respectively. It was 7.97, 10.0, 9.32 and 7.70 times of the untreated (Figure 3C). QRT-PCR data indicated that MbNAC22 was more susceptible to drought and salt stresses.

3.4. MbNAC22 Positively Regulates the Drought Tolerance of Malus baccata

The pCAMBIA2000-MbNAC22 vector driven by the CaMV 35S promoter was used to infect Arabidopsis and obtain the overexpressed-MbNAC22 plant. The overexpressed-MbNAC22 Arabidopsis was screened with kanamycin. With WT and VL as controls, 7 overexpressed-MbNAC22 strains (S1, 2, 4, 5, 6, 7 and 9) can be identified from 10 initially screened positive lines by qRT-PCR analysis (Figure 4A). Three homozygous T3 transgenic strains, S2, 6 and 7, were randomly selected for stress treatments.
The phenotypic differences among all Arabidopsis strains (WT, VL, S2, 6 and 7) were not significant in the untreated (Drought 0 d). After drought treatment (Drought 7 d), all strains were damaged to varying degrees, while overexpressed-MbNAC22 Arabidopsis had a low degree of damage (Figure 4B). After 7 days of the resumption of watering, it was found that drought stress reduced the survival rate of Arabidopsis. The survival rate of WT and VL decreased by 68.23% and 61.29%, respectively, while the survival rate of overexpressed-MbNAC22 Arabidopsis decreased by 11.93% on average. Overexpressed-MbNAC22 Arabidopsis had stronger viability (Figure 4C).
The physiological indexes of all Arabidopsis strains were basically the same in the untreated (Drought 0 d), which was consistent with their phenotypes. The proline content and EL of Arabidopsis increased after 7 d without water, while with chlorophyll it was the opposite. The contents of proline and chlorophyll in overexpressed-MbNAC22 Arabidopsis were significantly higher than that in WT and VL strains, while the EL was significantly lower than that in WT and VL strains. The overexpression of MbNAC22 may be involved in the antioxidant pathway of Arabidopsis. Drought stress induced an increase in antioxidant enzyme activity, and overexpressed-MbNAC22 Arabidopsis increased SOD, POD and CAT activities more significantly. It can be inferred from these data that the overexpressed-MbNAC22 Arabidopsis can better adapt to growth under drought (Figure 5).

3.5. Expression of Stress-Related Genes under Drought

The expression of stress-related genes was analyzed by qRT-PCR to determine the potential mechanism of MbNAC22 in plant response to drought. Without treatment, the expression of stress-related genes in all strains was relatively low. After 7 days without watering, the expression level of the key genes of the ABA pathway (AtNCED3 and AtDREB2A) (Figure 6A,B), the proline synthesis gene (AtP5CS2) and the drought-induced gene (AtERD11) increased significantly (Figure 6C,D). These results indicated that MbNAC22 can positively regulate the expression of stress-related genes after plants were subjected to drought stress, and may participate in multiple pathways of regulation of these downstream genes (Figure 6).

3.6. Expression of MbNAC22 in Malus baccata Enhances Salt Tolerancee

Under the control condition (Salt 0 d), all Arabidopsis strains grew well and no obvious difference was observed (Figure 7A). After salt stress (Salt 7 d), the overexpressed-MbNAC22 Arabidopsis grew better, suffered less damage and had a higher survival rate than WT and VL. The survival rates of the 3 transgenic lines were 81.87%, 82.18% and 80.13%, respectively, while the survival rates of WT and VL Arabidopsis were 27.78% and 25.75%, respectively (Figure 7B).
When 200 mM NaCL was not poured, it was found that the physiological index data of WT, VL and overexpressed-MbNAC22 Arabidopsis were highly similar. After pouring 200 mM NaCL for 7 d, the chlorophyll content of Arabidopsis decreased, while the EL, proline content and the activities of SOD, POD and CAT increased. The contents of proline and chlorophyll, as well as the activity of antioxidant enzymes in overexpressed-MbNAC22 Arabidopsis, were significantly higher than that of WT and VL strains, while the EL was significantly lower than that in WT and VL strains (Figure 8). From these data, we can infer that the overexpressed-MbNAC22 Arabidopsis was more resistant to salt stress.

3.7. Expression of Stress-Related Genes under Salt

MbNAC22 may improve plant salt resistance by participating in the salt stress response mechanism. The expression of stress-related genes was analyzed by qRT-PCR to determine the potential mechanism of MbNAC22 in plant response to salt. Under salt stress, the overexpression of MbNAC22 significantly induced the expression of ion-transport-related genes (AtNHX1 and AtSOS1) (Figure 9A,B). However, the expression of the leaf senescence-associated gene (AtSAG21) and the programmed cell death related gene (AtAEP1) in Arabidopsis was inhibited by MbNAC22 overexpression (Figure 9C,D). To sum up, MbNAC22 may improve the adaptability of plants to salt stress by participating in molecular pathways related to multiple salt stress response genes (Figure 9).

4. Discussion

This research found that the NAC transcription factor can be found in the molecular mechanism of response to abiotic stress [40,41,42]. For example, transcriptome analysis showed that Pennisetum glaucum contained 151 NAC transcription factors, while 30 and 42 PgNACs were regulated by drought and salt stresses, respectively. This indicated that NAC TFs act as a regulator in the response mechanism of Pennisetum glaucum to natural stress [43]. CsATF1 improved the tolerance of plants to drought stress through the ABA-dependent pathway and reduced the accumulation of ROS in cucumber [44]. The mature genetic transformation system of most fruit trees had not been established successfully. Therefore, the transformation of Arabidopsis was still the primary method of verifying the gene function of woody plants [45,46]. In this study, the MbNAC22 gene was isolated from Malus baccata, and its function in drought and salt stresses was studied by transforming it into Arabidopsis. NCBI CD search analysis found that MbNAC22 was a typical NAC transcription factor, encoding 255 aa (Supplementary Figure S1). In addition, MbNAC22 had the closest evolutionary relationship with MsNAC83 and MdNAC22 (Figure 1).
Most NAC transcription factors were found to be localized in the nucleus and had trans-activated activity [47,48]. For example, the instantaneous expression of CmNAC60-GEP in N. benthamiana can prove that it was located in the nucleus [49], and SbNAC9 was also proved to be a nuclear protein [50]. In this study, it was confirmed that MbNAC22 was located in the nucleus, which was consistent with previous research results (Figure 2). It was speculated that MbNAC22 mainly plays its role of transcriptional regulation in the nucleus.
The expression of the NAC transcription factor was specific in different species. For example, MfNAC95 was expressed in the root, stem and leaf of the Medicago falcata, but the highest expression was in the root, and salt stress can up-regulate its expression [51]. In Picea wilsonii, PwNAC1 was detected in its pollen, root, stem, needle and seed, while PwNAC1 expression in its seed was the highest, followed by its stem [52]. In this research, it was found that the expression of MbNAC22 was tissue-specific, and it was highly expressed in the new leaf and root. In addition, when plants were exposed to cold, drought, salt and heat, MbNAC22 was rapidly induced to express, and the effects of drought and salt were more obvious (Figure 3).
Proline had the ability to regulate permeability and antioxidants, which was a natural abiotic stress protection agent in planting objects. Stresses can induce the production and accumulation of proline in large quantities [53,54,55]. Therefore, higher levels of proline can endow plants with stronger antioxidant capacity. In this study, the content of proline in overexpressed-MbNAC22 plants increased significantly, and the expression of the proline synthesis gene (AtP5CS2) also increased significantly, which promoted the synthesis and accumulation of proline and improved the adaptability of plants to drought and salt. ROS are involved in many signaling pathways and physiological processes in plants, but their high content leads to membrane lipid peroxidation [56,57]. Under drought and salt treatment, ROS accumulate in large amounts [58]. In order to regulate the oxidation balance in the body, plants remove ROS through the antioxidant enzyme system, which mainly relies on CAT, SOD and POD [59,60,61]. The heterologous expression of ThNAC7 in Arabidopsis improved the activity of antioxidant enzymes, strengthened the ability of ROS scavenging, and improved the survival ability under salt and osmotic stresses by inducing the transcription level of genes related to P5CS, SOD and POD [62]. In addition, the overexpression of ThNAC12 strengthened the ability of ROS scavenging by directly regulating the expression of ThPIP2;5 in Tamarix hispida, thus improving salt tolerance [63]. In this study, we measured the activities of SOD, POD and CAT to analyze whether MbNAC22 had a regulatory effect on the accumulation and elimination of ROS. The results showed that after stress treatments, the overexpressed-MbNAC22 plants had a stronger ability for scavenging ROS. Therefore, the overexpression of MbNAC22 gave greater protection to plants under drought and salt. In addition, the degree of damage of chlorophyll in overexpressed-MbNAC22 plants under stress was lower than that of WT and VL plants. Stresses can destroy cell walls and cell membrane as well as increase membrane permeability, while overexpressed-MbNAC22 plants suffer less damage, so they had lower EL (Figure 5 and Figure 8). These physiological index data indicate that MbNAC22 played a positive role in regulating drought and salt stresses. In addition, we detected the expression levels of stress-related genes in plants by qRT-PCR. The research found that the expression of ion-transport-related genes (AtNHX1 and AtSOS1), the key genes of the ABA pathway (AtNCED3 and AtDREB2A), and drought-induced genes (AtERD11) increased after stress treatment, while the expression of the leaf senescence-associated gene (AtSAG21) and programmed cell death related genes (AtAEP1) was inhibited (Figure 6 and Figure 9). Therefore, we speculate that MbNAC22 can improve plant protection under stresses by regulating drought and salt stress-related genes.
The data obtained through experiments provide strong evidence for the research progress of the MbNAC22 transcription factor in drought and salt tolerance. It also provides new research ideas and directions for the study of NAC TFs in other species. At the same time, it also provides a new basis for the selection of apple stress-resistant rootstocks and the cultivation of germplasm resources.

5. Conclusions

Under drought stress, on the one hand, MbNAC22 induced the up-regulated expression of AtNCED3 and AtDREB2A, thereby increasing the accumulation of ABA. A large amount of ABA can cause stomatal closure and reduce water transpiration, thus improving drought tolerance. On the other hand, MbNAC22 positively regulated the expression of AtERD11 and AtP5CS2, and increased the content of proline, thus protecting plants and reducing the damage of drought to plants. Salt stress can rapidly activate the MeJA synthesis pathway gene (JAM2/MYC2/LOX3/AOS1.1/AOS1.2/AOC4), induce the synthesis and accumulation of MeJA in plants, and thus activate the expression of MbNAC22. MbNAC22, as a transcription factor, may improve the salt tolerance of plants by up-regulating the expression of AtNHX1 and AtSOS1 genes, while inhibiting the expression of AtSAG21 and AtAEP1 under salt stress. In conclusion, this study showed that MbNAC22 has a positive regulatory effect on drought and salt stresses (Figure 10).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13051374/s1, Figure S1: Sequence analysis of MbNAC22 protein. (A) Amino acid sequences of MbNAC22. (B) Conserved domain of MbNAC22 protein.

Author Contributions

Conceptualization, Y.D. and X.L.; formal analysis, K.J., J.H., B.G., Y.W., L.Z., Y.L., P.S., D.H., Y.D. and X.L.; funding acquisition, Y.D. and X.L.; methodology, K.J., J.H. and B.G.; methodology, Y.W., Y.L. and P.S.; supervision, X.L.; investigation, J.H., L.Z., Y.L. and D.H.; writing—original draft preparation, K.J. and J.H.; writing—review and editing, X.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Scientific Research Funds of Heilongjiang Provincial Research Institutes (CZKYF2023-1-C020), the National Natural Science Foundation of China (32172521) and the Collaborative Innovation System of Agricultural Bioeconomy in Heilongjiang Province.

Data Availability Statement

The original data in this study are available from the corresponding authors.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Figure 1. MbNAC22 homology analysis. (A) Multiple sequence alignment of the MbNAC22 protein by DNAMAN5.2. The red box is the NAC structure domain (A-E subdomain). (B) Evolution tree of the MbNAC22 protein by MEGA 7.0. The accession numbers are as follows: MsNAC83 (XP_050131946.1), MdNAC22 (ASF79344.1), PaNAC83 (XP_021800877.1), PdPaNAC83 (BBG93860.1), CiPaNAC83 (XP_042985431.1), JcPaNAC83 (XP_012088760.1), PpPaNAC83 (XP_007223645.1), PmPaNAC83 (XP_008222225.1), AtPaNAC83 (NP_196822.1) and PbPaNAC83 (XP_048430665.1). The red underline is the MbNAC22 protein.
Figure 1. MbNAC22 homology analysis. (A) Multiple sequence alignment of the MbNAC22 protein by DNAMAN5.2. The red box is the NAC structure domain (A-E subdomain). (B) Evolution tree of the MbNAC22 protein by MEGA 7.0. The accession numbers are as follows: MsNAC83 (XP_050131946.1), MdNAC22 (ASF79344.1), PaNAC83 (XP_021800877.1), PdPaNAC83 (BBG93860.1), CiPaNAC83 (XP_042985431.1), JcPaNAC83 (XP_012088760.1), PpPaNAC83 (XP_007223645.1), PmPaNAC83 (XP_008222225.1), AtPaNAC83 (NP_196822.1) and PbPaNAC83 (XP_048430665.1). The red underline is the MbNAC22 protein.
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Figure 2. Subcellular localization of MbNAC22 was observed by transient expression in the abaxial epidermal cell of Nicotiana tabacum leaves. (A,E) GFP fluorescence. (B,F) NLS-mCherry signals. (C,G) Images of cells to be detected under bright field. (D,H) Merged. Bar = 50 μm.
Figure 2. Subcellular localization of MbNAC22 was observed by transient expression in the abaxial epidermal cell of Nicotiana tabacum leaves. (A,E) GFP fluorescence. (B,F) NLS-mCherry signals. (C,G) Images of cells to be detected under bright field. (D,H) Merged. Bar = 50 μm.
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Figure 3. Expression of the MbNAC22 gene in Malus baccata. (A) Tissue-specific expression patterns of MbNAC22. Take the new leaf as control. (B) The expression levels of MbNAC22 in the new leaf and (C) root under different adverse treatments. Take stress treatments for 0 h as the control (CK), and select MbAct as the internal reference control. Calculate the mean value of the three measurements. (* p ≤ 0.05, ** p ≤ 0.01).
Figure 3. Expression of the MbNAC22 gene in Malus baccata. (A) Tissue-specific expression patterns of MbNAC22. Take the new leaf as control. (B) The expression levels of MbNAC22 in the new leaf and (C) root under different adverse treatments. Take stress treatments for 0 h as the control (CK), and select MbAct as the internal reference control. Calculate the mean value of the three measurements. (* p ≤ 0.05, ** p ≤ 0.01).
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Figure 4. Phenotypes and survival rates of different strains Arabidopsis under drought. (A) Relative expression level of MbNAC22 in Arabidopsis strains. (B) Phenotypes (Drought 0 d, 7 d and recover to normal growth). The white scale represents 3 cm. (C) Survival rates of different strains (control = Drought 0 d and Drought 7 d). Calculate the mean value of the three measurements. (** p ≤ 0.01).
Figure 4. Phenotypes and survival rates of different strains Arabidopsis under drought. (A) Relative expression level of MbNAC22 in Arabidopsis strains. (B) Phenotypes (Drought 0 d, 7 d and recover to normal growth). The white scale represents 3 cm. (C) Survival rates of different strains (control = Drought 0 d and Drought 7 d). Calculate the mean value of the three measurements. (** p ≤ 0.01).
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Figure 5. Evaluation of physiological indices responsive to drought stress. The contents of (A) chlorophyll and (B) proline; the activities of (C) SOD, (D) POD and (E) CAT; (F) EL. Each index of WT was used as the control. Calculate the mean value of the three measurements. (* p ≤ 0.05, ** p ≤ 0.01).
Figure 5. Evaluation of physiological indices responsive to drought stress. The contents of (A) chlorophyll and (B) proline; the activities of (C) SOD, (D) POD and (E) CAT; (F) EL. Each index of WT was used as the control. Calculate the mean value of the three measurements. (* p ≤ 0.05, ** p ≤ 0.01).
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Figure 6. qRT-PCR analyses of the related genes under drought stress in Arabidopsis leaves. (A) Relative mRNA expression level of AtNCED3, (B) relative mRNA expression level of AtDREB2A, (C) relative mRNA expression level of AtP5CS2, (D) relative mRNA expression level of AtERD11. Calculate the mean value of the three measurements. (** p ≤ 0.01).
Figure 6. qRT-PCR analyses of the related genes under drought stress in Arabidopsis leaves. (A) Relative mRNA expression level of AtNCED3, (B) relative mRNA expression level of AtDREB2A, (C) relative mRNA expression level of AtP5CS2, (D) relative mRNA expression level of AtERD11. Calculate the mean value of the three measurements. (** p ≤ 0.01).
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Figure 7. Phenotypes and survival rates of different strains of Arabidopsis under salt. (A) Phenotypes (Salt 0 d, 7 d and recover to normal growth). The white scale represents 3 cm. (B) Survival rates of different strains (control = Salt 0 d and Salt 7 d). Calculate the mean value of the three measurements. (** p ≤ 0.01).
Figure 7. Phenotypes and survival rates of different strains of Arabidopsis under salt. (A) Phenotypes (Salt 0 d, 7 d and recover to normal growth). The white scale represents 3 cm. (B) Survival rates of different strains (control = Salt 0 d and Salt 7 d). Calculate the mean value of the three measurements. (** p ≤ 0.01).
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Figure 8. Evaluation of physiological indices responsive to salt stress. The contents of (A) chlorophyll and (B) proline; the activities of (C) SOD, (D) POD and (E) CAT; (F) EL. Each index of WT was used as the control. Calculate the mean value of the three measurements. (* p ≤ 0.05, ** p ≤ 0.01).
Figure 8. Evaluation of physiological indices responsive to salt stress. The contents of (A) chlorophyll and (B) proline; the activities of (C) SOD, (D) POD and (E) CAT; (F) EL. Each index of WT was used as the control. Calculate the mean value of the three measurements. (* p ≤ 0.05, ** p ≤ 0.01).
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Figure 9. qRT-PCR analyses of the related genes under salt stress in Arabidopsis leaves. (A) Relative mRNA expression level of AtSOS1, (B) relative mRNA expression level of AtNHX1, (C) relative mRNA expression level of AtAEP1, (D) relative mRNA expression level of AtSAG21. Calculate the mean value of the three measurements. (** p ≤ 0.01).
Figure 9. qRT-PCR analyses of the related genes under salt stress in Arabidopsis leaves. (A) Relative mRNA expression level of AtSOS1, (B) relative mRNA expression level of AtNHX1, (C) relative mRNA expression level of AtAEP1, (D) relative mRNA expression level of AtSAG21. Calculate the mean value of the three measurements. (** p ≤ 0.01).
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Figure 10. The schematic diagram of MbNAC22 mechanism under drought or salt stresses is speculated.
Figure 10. The schematic diagram of MbNAC22 mechanism under drought or salt stresses is speculated.
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Jiao, K.; Han, J.; Guo, B.; Wu, Y.; Zhang, L.; Li, Y.; Song, P.; Han, D.; Duan, Y.; Li, X. MbNAC22, a Malus baccata NAC Transcription Factor, Increased Drought and Salt Tolerance in Arabidopsis. Agronomy 2023, 13, 1374. https://doi.org/10.3390/agronomy13051374

AMA Style

Jiao K, Han J, Guo B, Wu Y, Zhang L, Li Y, Song P, Han D, Duan Y, Li X. MbNAC22, a Malus baccata NAC Transcription Factor, Increased Drought and Salt Tolerance in Arabidopsis. Agronomy. 2023; 13(5):1374. https://doi.org/10.3390/agronomy13051374

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Jiao, Kuibao, Jiaxin Han, Baitao Guo, Yuqi Wu, Lei Zhang, Yuze Li, Penghui Song, Deguo Han, Yadong Duan, and Xingguo Li. 2023. "MbNAC22, a Malus baccata NAC Transcription Factor, Increased Drought and Salt Tolerance in Arabidopsis" Agronomy 13, no. 5: 1374. https://doi.org/10.3390/agronomy13051374

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