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Article

Heterologous Expression of the Apple MdbZIP26 Gene in Arabidopsis thaliana Improves Resistance to High Salinity and Drought Stress

by
Ye Wan
1,2,†,
Yaqiong Wang
1,2,†,
Fan Wang
1,2,
Shuaishuai Feng
1,2,
Li Zhang
1,2,
Xiping Wang
1,2 and
Hua Gao
1,2,*
1
State Key Laboratory of Crop Stress Biology in Arid Areas, College of Horticulture, Northwest A&F University, Xianyang 712000, China
2
Key Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest China, Ministry of Agriculture, Northwest A&F University, Xianyang 712000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2022, 12(11), 2624; https://doi.org/10.3390/agronomy12112624
Submission received: 10 September 2022 / Revised: 12 October 2022 / Accepted: 18 October 2022 / Published: 25 October 2022
(This article belongs to the Special Issue Horticultural Plants Breeding for Abiotic Stress Tolerance)
Browse Figures
Versions Notes

Abstract

:
High salinity and drought seriously limit the production of many crops worldwide, including apple (Malus x. domestica Borkh). Members of the bZIP family of transcription factors play important roles in abiotic stress in various plants, but there have been few studies in perennial tree species. In our previous study, we conducted a genome-wide survey of bZIP family transcription factor genes in apple. Here, we focused on one of these genes, MdbZIP26, which is induced by high salinity, drought, and exogenous abscisic acid (ABA). The MdbZIP26 promoter contains several apparent cis-acting elements associated with abiotic stress response, such as ABRE/G-box, DRE, GT1, and GMSCAM4. The temporal and spatial expression patterns of MdbZIP26 were consistent with a role in abiotic stress response. Arabidopsis thaliana plants expressing MdbZIP26 showed enhanced tolerance to dehydration and salinity, and this was associated with altered expression of ABA/stress-regulated genes. Considered together, these results suggest that MdbZIP26 plays a role in the resistance of drought and high salinity stress in apple via ABA-mediated signaling.

1. Introduction

Apple (Malus x. domestica Borkh) is a major fruit crop in many regions of the world. Additionally, apples are an important source of nutrients and antioxidants, and are widely believed to promote human health [1]. However, the worldwide apple production is limited by salinity and drought stress, in which factors may be exacerbated by climate change [2,3,4]. Therefore, increasing the tolerance of apple to abiotic stress is an important goal to sustain production.
Drought or high salinity leads to the accumulation of stress-related hormone phytohormone abscisic acid (ABA) and expression of key transcriptional regulators [5]. These signaling components activate a diverse range of stress-responsive genes that finally lead to changes in molecular, physiological, and morphological processes that facilitate response to the stress [6,7]. ABA regulates many important aspects of growth and development in plants [8,9,10]. During vegetative growth, ABA is involved in various environmental stresses, especially drought and high salinity. Moreover, ABA promotes solute efflux-mediated stomatal closure in guard cells and regulates the expression of many genes [11,12].
In the core ABA signaling pathway, ABA is sensed by the ABA receptors of the Pyrabactin resistance1/PYR1-like/regulatory components (PYR/PYL/RCAR) family, which physically interact with protein phosphatase 2C (PP2C) to release its inhibition of subclass III sucrose non-fermenting-1 related protein kinase 2 (SnRK2). Then, the SnRK2 protein kinases phosphorylate bZIP transcription factors, leading to the activation of various downstream targets [13,14]. The bZIP proteins are identified by a conserved leucine-zipper domain, and comprise one of the largest transcription factor families in plants [15]. Previous studies have shown that bZIP proteins bind to ABA-responsive elements (ABREs) containing an ACGT core motif in the promoters of their target genes, thereby repressing or activating their expression. Therefore, bZIP transcription factors are also known as ABRE-binding factors (ABFs) or ABRE-binding proteins (AREBs) [2,16,17].
To date, numerous bZIP transcription factors have been found in a range of plant species, and many of them have been implicated in the response to abiotic stress. In Arabidopsis thaliana, the ABF genes, ABF2/AREB1, ABF4/AREB2, and ABF3, participate in drought response [18,19,20]. Moreover, in rice, OsbZIP16, OsbZIP23, OsbZIP72, and OsABI5 play important roles in ABA signal transduction and osmotic stress responses [21,22,23,24]. Furthermore, in wheat, overexpression of TabZIP96 enhanced the cold resistance of transgenic Arabidopsis thaliana and TabZIP15 enhanced the salt resistance of transgenic wheat [25,26]. In grape, VibZIP30 and VibZIP39 were upregulated in response to drought conditions [27,28]. However, only a few bZIP TFs in apples have been found, especially with respect to potential roles in abiotic stress response [29,30,31].
In a previous study, we carried out a genome-wide survey of bZIP family transcription factor genes in apple [30]. Here, we further characterized one of these, MdbZIP26, in terms of its expression in response to salinity and drought stress in apple, and its effect on the response to salinity and drought stress when expressed in Arabidopsis.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

Apple (cultivar ‘Fuji’) plants were maintained in the apple germplasm resource orchard of Northwest A&F University, Yangling, Shaanxi, China (34°20′ N, 108°24′ E). Arabidopsis thaliana (accession Columbia-0) seeds were sterilized and planted on 1/2-strength MS medium (Sigma-Aldrich, St. Louis, MO, USA) plates containing 1% (w/v) sucrose and 0.8% (w/v) agar. Plates were maintained at 22 °C under a 16-h-light/8-h-dark photoperiod for germination and growth after stratification at 4 °C for 3 d. After 10 days, seedlings were transferred to soil and placed in a controlled environment room at 22 °C with a 16-h-light/8-h-dark photoperiod. Tobacco (Nicotiana tabacum cv. NC89) plants were grown in a greenhouse at 22 °C with a 16-h-light/8-h-dark photoperiod and 60% relative humidity. Six-week-old plants were used for Agrobacterium-mediated transient assays.

2.2. Dehydration, High Salinity Stress, and ABA Treatment of Apple Leaves

For stress and ABA treatment, 2-year-old apple plants growing in the same conditions were used, which had previously been planted in pots, all of which were derived from Baishui Apple Experimental Station of Northwest A&F University. For the ABA treatment, ABA (300 μM) was applied as a foliar spray, and leaves were sampled at 1, 12, 24, and 48 h post-treatment, as previously described [32,33]. For the salt treatment, salt stress was induced by irrigating seedlings with 2 L of 250 mM NaCl; plants irrigated with the same volume of water were used as controls. Leaves of treated and control plants were collected at 1, 12, 24, and 48 h, respectively [34,35]. For the drought treatment, samples were collected at 48, 96, 120, and 168 h after irrigation cessation and 48 h after rewatering [36,37].

2.3. Bioinformatic and Phylogenetic Analyses

The predicted, full-length amino acid sequence of MdbZIP26 was obtained from the Genome Database for Rosaceae (GDR; https://www.rosaceae.org/organism/Malus/x-domestica, accessed on 20 October 2017). In addition, amino acid sequences of bZIP TFs, previously reported from other plants, were obtained from the National Center for Biotechnology Information (NCBI; http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 21 October 2017). Multiple sequence alignments were performed using DNAMAN software, and the bZIP conserved domain, Pfam domain, and HNS domain were predicted using SMART software (http://smart.embl-heidelberg.de/, accessed on 24 October 2017) [38]. The phylogenetic tree was constructed by the Neighbor-joining method using MEGA4 software and the calibration parameter Bootstrap = 1000 [39]. Phosphorylation sites were predicted as previously described [18,40].

2.4. Promoter Cloning and Prediction of Cis-Elements

Genomic DNA was extracted from apple leaves using the CTAB method, and a ~2.5 kb MdbZIP26 promoter sequence was amplified by PCR (primers are listed in Supplementary Table S1). Putative cis-elements in the MdbZIP26 promoter were identified using PLANTCARE (http://bioinformatics.psb.ugent.be//webtools/plantcare/html/, accessed on 27 March 2018) [41] and PLACE (http://www.dna.affrc.go.jp/PLACE/signalup.html, accessed on 29 March 2018) [42].

2.5. Analysis of MdbZIP26 Promoter: GUS Activity

The 2.5-kb MdbZIP26 promoter fragment was inserted into the pCambia1391Z vector and sequenced in its entirety. This construction was introduced into Agrobacterium tumefaciens strain GV3101 and transformed into Arabidopsis thaliana using the floral dip method. The GUS staining assay was performed as previously described [43]. Samples were stained in GUS staining buffer and incubated at 37 °C for 24 h, followed by decoloring with 70% ethanol. Three samples representing each growth period were observed and photographed using a microscope.

2.6. Subcellular Localization

The full-length MdbZIP26 open reading frame with eliminated stop codon was amplified and ligated into the pCAMBIA1302-GFP vector. Tobacco leaves were used for transient Agrobacterium-mediated transformation and the fluorescence signal was observed after 48 h with a fluorescence microscope (BX51; OLYMPUS, Tokyo, Japan).

2.7. Generation of Transgenic Arabidopsis Thaliana Expressing the MdbZIP26 Gene

The full-length MdbZIP26 open reading frame was amplified from cDNA derived from apple leaves, using gene-specific primers (Supplementary Table S1). Then, the amplified fragment was inserted into the pCambia 2300 vector under control of the CaMV35S promoter. This construction was introduced into Agrobacterium tumefaciens strain GV3101 and transformed into Arabidopsis thaliana using the floral dip method. The seeds were harvested and sown onto MS medium containing kanamycin for selection of transformants.
Of the obtained 50 independent transgenic lines, three (designated as L1, L2, and L3) showing the strongest tolerance to both dehydration and high salinity were chosen for further studies, and homozygous T3 lines were identified.

2.8. Osmotic Stress and ABA Treatment of Transgenic Arabidopsis Thaliana

Seeds from wild-type and the three transgenic lines were sown in quadrants onto MS medium or MS medium supplemented with mannitol, NaCl, and ABA. After stratification at 4 °C for 3 days, the seeds were germinated at 22 °C under 16-h-light/8-h-dark photoperiod. The germination rate was calculated when a white radicle was exposed from most of the seeds, and the cotyledon greening rates was calculated after 11 days, when most of the cotyledons were fully extended and had turned green.
To document the effects of NaCl or ABA on seedling growth, seeds were germinated on MS medium in petri dishes. Six days after germination, seedlings were transferred to petri dishes containing MS medium supplemented with mannitol, NaCl or ABA, and petri dishes were placed vertically. After 14 days, photographs were taken and root lengths were measured.
To determine the effect of NaCl, mannitol or ABA on chlorophyll content, relative conductivity, and malondialdehyde (MDA) content, the germinated and grown seedlings on MS medium for 7 days were transferred to liquid MS medium containing 130 mM NaCl, 200 mM mannitol or 0.75 μM ABA and grown for an additional 7 days prior to analyses.
To study the effect of heterologous MdbZIP26 expression on the phenotype of Arabidopsis plants under drought and salt tolerance, 3-week-old Arabidopsis plants grown under 16-h-light/8-h-dark photoperiod were treated with drought and salt stresses. For the drought treatment, Arabidopsis plants were dried for 7 days to observe the phenotype, then rehydrated for 2 days and the phenotype was observed again. For the salt treatment, Arabidopsis plants were watered thoroughly with 130 mM NaCl and phenotypes were observed after 5 days.

2.9. Analysis of Chlorophyll, Relative Electrolyte Leakage, and MDA Content

Chlorophyll content was determined by 80% acetone according to the method of Hui et al. [44]. Relative conductivity was determined as described by Cao et al. [45]. For determination of malondialdehyde content, seedlings were cut into pieces and mixed uniformly. Then, 0.5 g of seedling tissues was homogenized in 10 mL 10% TCA solution, and the sample was subjected to centrifugation at 4000× g rpm for 10 min. The absorbance of the supernatant at 450, 532, and 600 nm was determined with a UV spectrophotometer (SHIMADZU UV-1700) with 10% TCA as a reference.

2.10. Histochemical Observation of Cell Death and Superoxide Accumulation

Plant cell necrosis was observed with trypan blue staining using the method of Frye and Innes [46]. For H2O2 detection, Arabidopsis thaliana leaves were immersed in 1 mg/mL diaminobenzidine (DAB) solution (DAB was dissolved in pH 7.0 phosphate buffer to 20 mM, and the solution was adjusted to pH 3.8–4.0 with 1 M HCl), and incubated in the dark for 8 h [47]. In addition, for the detection of O2−, Arabidopsis thaliana leaves were immersed in 6 mM Nitrotetrazolium blue chloride (NBT) solution (NBT was dissolved in 100 mM Hepes buffer at pH 7.5) and stained for 2 h under light [48].

2.11. Antioxidant Enzyme Activity

Total protein concentration was determined according to the Bradford method [49]. Superoxide dismutase (SOD) content was measured using a photochemical assay system consisting of methionine, riboflavin, and p-nitro blue tetrazolium as previously reported [50]. Peroxidase (POD) activity was measured according to the guaiacol method and expressed as the change in absorbance/min/g FW [51]. Catalase (CAT) activity was assayed as described by Aebi [52].

2.12. Stomatal Aperture Analysis

Stomatal aperture experiments were based on a previous study [53]. Briefly, 3-week-old plants were maintained in low light (100 μmol m−2 s−1) for 3 h, then leaves were excised and incubated in 4-morpholineethanesulfonic acid (MES) buffer (10 mM MES-Tris, 10 mM KCL, 50 M CaCl2, pH 6.2) for 3 h. Thereafter, ABA (5 μm or 10 μm) was added to the solution, respectively. Stomatal apertures were observed with a microscope (BX53, Olympus, Japan) after 1 h and recorded as the aspect ratio of stomatal.

2.13. RNA Extraction and Gene Expression Analysis

Total RNA was extracted from apple and Arabidopsis thaliana leaves using the E.Z.N.A. Plant RNA Kit (Omega Bio-Tek, USA, R6827-01) following the manufacturer’s instructions. Reverse transcription was performed using the PrimeScript TM RTase 1st Strand cDNA Synthesis Kit (TaKaRa Biotechnology, Dalian, China). RT-qPCR was carried out with a Step OnePlus Real-Time PCR System (Applied Biosystems, Foster, CA, USA) using a 6-fold dilution of cDNA in sterile water, and SYBR Green Mix (TaKaRa Biotechnology Dalian, China) with the following parameters: 95 °C for 30 s, then 42 cycles of 95 °C for 5 s, and 60 °C for 30 s. The apple ACTIN1 gene (GenBank Acc. No. AY680701) or Arabidopsis thaliana ACTIN1 gene (GenBank Acc. No. AT3G18780) was used as an internal control. The specific primers used for qRT-PCR are shown in Supplementary Table S1. The relative expression levels were analyzed by the normalized expression method of IQ5 software(Version 2.1, Bio-Rad, Richmond, CA, USA).

2.14. Statistical Analysis

All experimental data were based on three independent biological experiments. Data analyses including determination of means and standard errors were conducted using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and SigmaPlot 10.0. Paired t-tests were performed using the SPSS Statistics 17.0 software (IBM China Company Ltd., Beijing, China).

3. Results

3.1. Isolation and Identification of MdbZIP26 from Apple

MdbZIP26 (MDP0000248567) predicted full-length open reading frame is 1296 bp long, encoding a protein of 432 amino acids. MdbZIP26 has typical features of the group a bZIP subfamily, including a basic leucine zipper domain, three amino terminal (C1, C2, and C3) conserved domains, and a carboxyl terminal (C4) conserved structure, which is predicted to be a potential phosphorylation site responsive to ABA-related signal transduction pathways (Supplementary Figure S1). There is a Pfam domain and an HNS domain at the C-terminal 32-65 and 115-151 amino acids, respectively, which are mainly involved in the binding of sequence-specific DNA. A phylogenetic analysis indicated that MdbZIP26 has a close relationship with other ABF/AREB TFs from Arabidopsis thaliana, grapevine, rice, tobacco or tomatoes (Figure 1A).

3.2. The Expression Pattern of MdbZI26 under Abiotic Stress

In general, the expression level of MdbZIP26 changed significantly after each of these treatments. At 1 h after ABA treatment, the expression of MdbZIP26 was higher than the control plants. Expression continued to increase until 24 hpt, then decreased by 48 hpt (Figure 1b), but was still higher than the control plants. For NaCl treatment, MdbZIP26 was significantly higher than the control plants by 1 hpt, and continued to increase to 48 hpt. After drought treatment, expression was higher than the control plants by 48 hpt and remained elevated at least until 128 hpt. After rehydration, the expression level of MdbZIP26 decreased (Figure 1B). These data indicate that MdbZIP26 is regulated by multiple stress-related pathways.
To gain additional insight into the regulation of MdbZIP26, we expressed a Β-GLUCURONIDASE (GUS) marker gene under control of the MdbZIP26 promoter in Arabidopsis and analyzed seedlings by histochemical staining. GUS expression was observed at all of the developmental stages that we assessed. In non-stressed plants, low GUS expression was seen in 2-day-old (germinating) seedlings, as well as in 7- and 14-day-old seedlings (Figure 1a,e,i). After addition of ABA (Figure 1b,f,j), mannitol (Figure 1c,g,k) or NaCl (Figure 1d,h,l), GUS activity was significantly enhanced. In mature plants, weak GUS activity was observed in the leaves (Figure 1m), roots (Figure 1q), and stomata (Figure 1u). In addition, a clear GUS activity was seen in the flower, with the exception of the anthers (Figure 1y), and in the base of siliques (Figure 1z). After treatment with dehydration (Figure 1n,r,v), ABA (Figure 1o,s,w) or NaCl (Figure 1p,t,x) GUS levels were enhanced in the root and leaf microtubule tissues and stomata, respectively.

3.3. Sequence Analysis of the MdbZIP26 Promoter

The 2500 bp region of MdbZIP26 upstream of the start codon was cloned from “Fuji” leaves, and its putative cis-regulatory element was analyzed. The MdbZIP26 promoter sequence and putative cis-acting elements are shown in Supplementary Figure S4. As anticipated, we found that the MdbZIP26 promoter region also contains a TATA- and CAAT-box, both of which participate in basal expression. In addition, we identified potential cis-elements including an ABA response/stress response element ABRE/G-box, drought response element DRE, and salt response element GT1GMSCAM4, etc. (Supplementary Table S2). The presence of these presumed cis-elements suggests that MdbZIP26 may be involved in the response to ABA and osmatic stress caused by drought and high salinity.

3.4. Subcellular Localization of MdbZIP26

To determine the subcellular localization of the MdbZIP26 protein, we generated MdbZIP26-GFP fusion constructs under the control of the CaMV35S promoter, and expressed these transiently in tobacco leaves. The results showed that the MdbZIP26-GFP fusion protein is localized to the nucleus (Figure 1C). This suggested that MdbZIP26 is a nuclear protein and may thus have a role as a transcription factor in apple.

3.5. Arabidopsis Plants Expressing MdbZIP26 Show Enhanced Tolerance to Osmotic

We over-expressed MdbZIP26 in Arabidopsis, and selected three transgenic lines for further analysis based on their high levels of MdbZIP26 expression (Supplementary Figure S3). After the stress treatment, the seed germination and cotyledon greening rates of transgenic Arabidopsis thaliana were significantly higher than the wild-type (Figure 2A–D). Transgenic Arabidopsis lines have longer root lengths and more lateral roots (Figure 3A), which were beneficial to plant growth under drought stress. Both the root length and the number of lateral roots of 35S:MdbZIP26 lines were greater than the control lines when seedlings were grown on MS medium containing mannitol and NaCl, although both transgenic lines and controls grew more slowly than on MS alone (Figure 3A,B). Next, we evaluated the effects of MdbZIP26 expression on tolerance of drought and salt stress. Under drought conditions, non-transgenic control plants had wilted and turned yellow after 7 days. After 7 days of drought treatment, 35S:MdbZIP26 plants began to wilt, and the wild-type had almost completely wilted and yellowed. At 2 days after rehydration, the transgenic plants basically returned to normal growth, while the wild-type survival rate was only 12.50%, and the survival rate of the transgenic lines was 8 times the rate of the wild-type (Figure 4A,B). After 5 days of salt stress treatment, leaves of non-transgenic plants had turned chlorotic, while there was little chlorosis in transgenic lines (Figure 4A).

3.6. Mdbzip26 Overexpression Increases Arabidopsis Resistance to Drought and High Salinity

To examine the effect of MdbZIP26 expression on drought tolerance in more detail, we analyzed physiological indicators of osmotic stress. Leaves of transgenic lines showed higher (1.5–2.2 times) chlorophyll content than the control plants (Figure 3C). Following the elongation of dehydration to 7 days, the transgenic strains exhibited lighter wilting (Figure 4A) and lighter trypan blue staining (Figure 4C) compared to the control plants, which indicated that overexpression of the MdbZIP26 gene in Arabidopsis thaliana enhanced the resistance to dehydration by reducing the death rate of cells. There was no significant difference in MDA content and relative electrolyte leakage between transgenic and wild-type seedlings grown on MS medium. However, under salt, dehydration, and ABA stress processing, both the MDA contents and the relative electrolyte leakage are measured at a lower level in transgenic plants than WT (Figure 3C), indicating that overexpression of MdbZIP26 in Arabidopsis thaliana can reduce the degree of membrane damage caused by osmotic stress. We studied the ABA sensitivity of guard cells in transgenic Arabidopsis thaliana since guard cells from salt- and drought-tolerant plants are highly sensitive to ABA. There were little differences in stomatal opening between transgenic Arabidopsis thaliana and control plants under ABA-free conditions. In contrast, when different concentrations of ABA were applied to MdbZIP26 overexpressing plants, the stomatal aperture of the transgenic lines was smaller than the control. This phenomenon was clearer under 10 μm concentration of ABA treatment (Figure 5A,B).

3.7. The Strain Overexpressing MdbZIP26 Had Lower ROS Content and Higher Antioxidant Enzyme Activity under Dehydration Stress

We observed the accumulation of two reactive oxygen species (ROS), superoxide radical (O2−), and hydrogen peroxide (H2O2), in transgenic lines and controls, by means of histochemical staining with Nitrotetrazolium blue chloride (NBT) (Figure 4D) and 3,3-diaminobenzidine (DAB) (Figure 6B), respectively. We found that both the transgenic plants and controls had little accumulation of the two reactive oxygen species before stress treatment. However, under dehydration and high salt stress treatment, the controls stained more deeply than the transgenics. In addition, we measured the activity of three antioxidant enzymes, peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), under the abovementioned stresses. The results showed that the activity of these three enzymes in transgenic plants was higher than the control plants (Figure 6A). This result indicated that the MdbZIP26 expression line improved the tolerance to osmotic stress.

3.8. Overexpression of MdbZIP26 Increased the Expression Levels of Many Drought Response Genes and ABA-Related Genes

To explore the potential molecular regulation of MdbZIP26, the expression levels of various stress response and ABA response genes in transgenic lines and controls were evaluated by qRT-PCR under dehydration and high salinity (Figure 7). Under osmotic stress, the expression level of AtNCED3, a key ABA biosynthesis gene, was higher in transgenic lines than the controls. In addition, we measured changes in expression of the ABA or stress inducible genes AtRD22, AtRAB18, AtRD29A, and AtRD29B. Compared with the expression in the WT, these genes were strongly induced and had higher expression levels in transgenic plants under drought treatments. When plants under drought conditions were rehydrated, the expression level of all four genes showed a downward trend. Of note, they were both slightly lower in transgenic lines than wild-type. On the other hand, the expression levels of two genes AtERD1 and AtDREB2A, which are independent of the ABA pathway, were lower in transgenic lines than WT. Finally, ionic signaling pathways are also involved in the response to salt and drought stress. The ionic aspect of osmotic stress, especially salt stress, is signaled via the SOS pathway. The expression level of the SOS pathway-related gene SOS2 in transgenic lines was also significantly higher than the controls.

4. Discussion

Apple is rich in nutrients, especially polyphenols, flavonoids, and other substances, which have antioxidant properties [54]. However, increasing conditions of drought and salinization of the land result in great challenges to apple production. Therefore, studying the gene’s function, and the expression of stress-related genes in apple, is of great significance for improving resistance to stress and varieties breeding [55].
In recent years, the bZIP family transcription factor has attracted wide attention. The function of bZIP genes has been reported in many plants, and many of these genes are involved in response to drought and salt stress. AREB (ABA responsive element binding protein)/ABFs (ABRE binding factors) belong to a subfamily of bZIP transcription factors. Three ABFs, AREB1, AREB2, and ABF3, are upregulated by ABA and water stress [12]. The AREB1AREB2ABF3 triple mutant showed resistance to ABA and reduced drought tolerance [20]. The bZIP proteins, OsbZIP23 and OsbZIP46 in rice, were also characterized to have a high potential for drought resistance [22,23]. The bZIP transcription factors VlbZIP36 and VlbZIP30 of grape were expressed heterogeneously in Arabidopsis thaliana, which enhanced the drought resistance [27,56]. In tomato, the expression of SlAREB1 and SlAREB2 were induced by drought and salinity in both leaves and roots [57]. In this study, we identified MdbZIP26 in apple, through multiple sequence alignments. In addition, phylogenetic tree analysis showed that it has the highest homology with the ABF subfamily from Arabidopsis thaliana and grape.
By analyzing the open reading frame of MdbZIP26, we found that MdbZIP26 contained a highly conserved bZIP domain, as well as a nuclear localization signal (Figure 1C) and typical DNA-specific sequence binding structure (Supplementary Figure S1). This was similar to the three-dimensional structure of the bZIP highly conserved amino acid sequences and GCN4 bZIP domain in Arabidopsis thaliana. Furthermore, previous studies have shown that many members of the bZIP family have been shown to localize to the nucleus. For example, subcellular localization analysis revealed that MebZIP3, MebZIP5, TabZIP6, StbZIP25, and 84 PbbZIP proteins are located in the nucleus [58,59,60,61]. Consistent with previous studies, we demonstrated that MdbZIP26 was indeed localized in the nucleus. The expression of some bZIP transcription factors depends on the phosphorylation of SnRks to be fully activated [16,62]. Moreover, we found these phosphorylation sites in the MdbZIP26 amino acid sequence.
The overexpression of drought-inducing genes in Arabidopsis thaliana were able to increase ABA sensitivity and thus improved tolerance to drought stress. However, in this study, overexpression of MdbZIP26 did not lead to more sensitivity to ABA in transgenic Arabidopsis thaliana, but increased their tolerance to osmotic stress during and after germination. After stress treatment, the seed germination rate (Figure 2B) and cotyledon greening rate (Figure 2D) of transgenic Arabidopsis thaliana were significantly higher than the wild-type, and transgenic Arabidopsis lines had longer and more lateral roots (Figure 3AB), which were beneficial to plant growth under drought stress. A previous study reported that in deep soils and water layers, plants increased the root length to improve the chance of contact with water and water retention [63,64], which was consistent with this study.
The aerobic metabolism of plant growth and development is often accompanied by the production of ROS, which is in a dynamic equilibrium under normal conditions. However, when the plants are subjected to drought or salt stress, the balance is broken, and the rapid accumulation of active oxygen induces membrane lipid peroxidation, producing MDA, damaging the cell membrane and the cell leakage of contents, which eventually leads to cell death [65,66,67]. Therefore, plant resistance is indirectly inferred by the accumulation of active oxygen, MDA content, and electrical conductivity [47,68,69]. In our study, histochemical staining results were consistent with phenotypic observations. Transgenic lines were found to exhibit lower levels of programmed cell death and ROS accumulation compared to non-transgenic control plants. Meanwhile, the relative electrolyte leakage and MDA content of the transgenic lines were also significantly lower than the wild-type under drought and salt stress. On the other hand, substances in plants, such as protective enzymes and chlorophyll content, can alleviate the damage caused by osmotic stress. Interestingly, in this study, it was found that the activity of antioxidant enzymes (SOD, CAT, POD) in transgenic lines was significantly higher than the wild-type. Water shortage can inhibit photosynthesis, as well as affect the content of chlorophyll [70,71]. Leaves of transgenic lines showed higher chlorophyll content than the control plants. In addition, stomatal closure is a regulatory mechanism in response to stress in plants, which can increase resistance to stress by reducing transpiration. Our observation was that stomatal aperture in wild-type plants was markedly larger than MdbZIP26 expression, and that transgenic lines accumulated more callose compared to wild-type following stress treatments. In summary, the overexpression of MdbZIP26 gene in Arabidopsis thaliana can enhance the resistance of plants to abiotic stress.
As a transcription factor, MdbZIP26 is likely involved in the regulation of downstream stress-response genes. When plants suffer from drought and salt stress, genes associated with ABA and stress are indeed induced in transgenic Arabidopsis thaliana. Usually, some cross-communication genes are referred to both drought and salt stress. The results showed that the expression level of ABA-dependent, abiotic stress-response genes (AtRD29A, AtRD29B, AtRD22, AtRAB18) were significantly higher in transgenic lines than controls. In addition, an increase in the expression level of AtNECD3, a key enzyme gene for ABA synthesis, was detected in the transgenic plants [72]. After abiotic stress, the ABA content in plants was increased, which was beneficial to the response of plants to drought stress. We speculated that MdbZIP26 may affect the synthesis of ABA and play a role in the upstream of ABA biosynthesis pathway. The SOS pathway is an important defense pathway for plants to tolerate Na+ stress [12]. Through the sequential activation of SOS1-SOS2-SOS3, excessive Na+ in the cell is discharged to the outside, maintaining the intracellular K+/Na+ balance, and regulating the adaptability of plants to salt stress. In the SOS pathway, the activity and expression of SOS2 are regulated by calcium in response to SOS3-SOS2 protein kinase, maintaining ion balance in the cytoplasm and increasing salt stress resistance [12], and its expression level is higher in transgenic plants. The expression levels of AtDREB2A and AtERD1 were slightly higher in the transgenic plants than the wild-type, and there was no significant difference, indicating that the overexpression of MdbZIP26 had little effect on the ABA-independent signaling pathway.

5. Conclusions

In summary, MdbZIP26 localizes to the nucleus. Under drought and high salt treatment, the expression of MdbZIP26 in apple seedlings was significantly different from the control. In response to drought and high salt stress, overexpression of MdbZIP26 in Arabidopsis thaliana enhanced its antioxidant enzyme activity, increased its root length and chlorophyll content, decreased its ROS and malondialdehyde content, and affected the expression of genes related to ABA signaling pathway. Therefore, the present study provides preliminary information on the role of MdbZIP26 in drought and high salt resistance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12112624/s1, Figure S1: Multiple amino acid sequence alignment; Figure S2: The predicted functional domain of the MdbZIP26 protein; Figure S3: Screening of transgenic lines; Figure S4: The promoter nucleotide sequence analysis of the MdbZIP26. Table S1: Primer sequences used in this study; Table S2: Cis-acting regulatory elements analysis of the MdbZIP26 promoter sequence.

Author Contributions

Conceptualization, H.G.; methodology, H.G.; formal analysis, Y.W. (Ye Wan), Y.W. (Yaqiong Wang), and F.W.; investigation, Y.W. (Ye Wan), Y.W. (Yaqiong Wang), F.W., S.F., L.Z., and X.W.; writing—original draft preparation, Y.W. (Ye Wan) and Y.W. (Yaqiong Wang); writing—review and editing, Y.W. (Ye Wan) and F.W.; funding acquisition, H.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Chinese National Apple Industry Technology System (CARS-APPLE).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data supporting the findings of this study are available within the article and its Supplementary Materials.

Acknowledgments

The authors thank all editors and reviewers for their comments on this manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic analysis of VlbZIP30 and expression analysis of MdbZIP26. (A) The phylogenetic tree represents MdbZIP26 (black circle) and other bZIP amino acid sequences (SlAREB1(AFA37978.1), OsABI5 (XP_015612550.1), AtABF1 (AEE32464.1), AtABF2 (NP_001185157.1), AtABF3 (NP_849490.2), and AtABF4 (NP_566629.1). (B) Expression profiles of MdbZIP26 in apple following abscisic acid (ABA), NaCl, and dehydration treatments. Data represent the mean values ± SE from three independent experiments. Asterisks indicate statistical significance (* 0.01 < p < 0.05, ** p < 0.01, Student’s t-test) between the treated and untreated control plants. (C) Nuclear localization of MdbZIP26 protein of transiently transformed tobacco leaves: Fluorescent images of GFP, bright light images, and merged images. (D) Patterns of MdbZIP26 promoter-driven GUS (β-glucosidase) expression in Arabidopsis thaliana at different growth stages. Mature embryos cultivated on Murashige-Skoog (MS) agar medium (a) or MS agar medium supplemented with 0.5 μM ABA (b), 200 mM mannitol (c) or 130 mM NaCl (d) for 2 days. Scale bar = 1 mm. Seven-day-old seedlings cultivated on MS agar medium (e) or MS agar medium supplemented with 0.5 μM ABA (f), 200 mM mannitol (g) or 130 mM NaCl (h) for 7 days. Scale bar = 1 mm. Fourteen-day-old seedlings transferred from MS medium plates into MS agar medium (i) or MS agar medium supplemented with 100 μM ABA (j), 200 mM mannitol (k) or 130 NaCl (l) for 7 days. Scale bar = 2 mm. Leaf of 3-week-old plant (m). Leaf of 3-week-old plant after treatments of dehydration (n), 100 μm ABA (o) or 200 mM NaCl (p) for 24 h, respectively. Root of 3-week-old plant (q). Root of 3-week-old plant after treatments of dehydration (r), 100 μm ABA (s) or 200 mM NaCl (t) for 24 h, respectively. Guard cells of 3-week-old plant (u). Scale bar = 2 mm. Guard cells of 3-week-old plant after treatments of dehydration (v), 100 μm ABA (w) or 200 mM NaCl (x) for 24 h, respectively. Scale bar = 100 μm. Flower (y). Scale bar = 2 mm. Silique (z). Scale bar = 2 mm.
Figure 1. Phylogenetic analysis of VlbZIP30 and expression analysis of MdbZIP26. (A) The phylogenetic tree represents MdbZIP26 (black circle) and other bZIP amino acid sequences (SlAREB1(AFA37978.1), OsABI5 (XP_015612550.1), AtABF1 (AEE32464.1), AtABF2 (NP_001185157.1), AtABF3 (NP_849490.2), and AtABF4 (NP_566629.1). (B) Expression profiles of MdbZIP26 in apple following abscisic acid (ABA), NaCl, and dehydration treatments. Data represent the mean values ± SE from three independent experiments. Asterisks indicate statistical significance (* 0.01 < p < 0.05, ** p < 0.01, Student’s t-test) between the treated and untreated control plants. (C) Nuclear localization of MdbZIP26 protein of transiently transformed tobacco leaves: Fluorescent images of GFP, bright light images, and merged images. (D) Patterns of MdbZIP26 promoter-driven GUS (β-glucosidase) expression in Arabidopsis thaliana at different growth stages. Mature embryos cultivated on Murashige-Skoog (MS) agar medium (a) or MS agar medium supplemented with 0.5 μM ABA (b), 200 mM mannitol (c) or 130 mM NaCl (d) for 2 days. Scale bar = 1 mm. Seven-day-old seedlings cultivated on MS agar medium (e) or MS agar medium supplemented with 0.5 μM ABA (f), 200 mM mannitol (g) or 130 mM NaCl (h) for 7 days. Scale bar = 1 mm. Fourteen-day-old seedlings transferred from MS medium plates into MS agar medium (i) or MS agar medium supplemented with 100 μM ABA (j), 200 mM mannitol (k) or 130 NaCl (l) for 7 days. Scale bar = 2 mm. Leaf of 3-week-old plant (m). Leaf of 3-week-old plant after treatments of dehydration (n), 100 μm ABA (o) or 200 mM NaCl (p) for 24 h, respectively. Root of 3-week-old plant (q). Root of 3-week-old plant after treatments of dehydration (r), 100 μm ABA (s) or 200 mM NaCl (t) for 24 h, respectively. Guard cells of 3-week-old plant (u). Scale bar = 2 mm. Guard cells of 3-week-old plant after treatments of dehydration (v), 100 μm ABA (w) or 200 mM NaCl (x) for 24 h, respectively. Scale bar = 100 μm. Flower (y). Scale bar = 2 mm. Silique (z). Scale bar = 2 mm.
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Figure 2. Phenotypes of wild-type (WT) and M-overexpressing (OE) transgenic lines at the seed germination and the greening cotyledon stage following osmatic stress and abscisic acid (ABA) treatments. (A) Seed germination from transgenic lines and WT seedlings growing on MS medium with or without 130 mM NaCl, 200 mM mannitol or 0.75 μΜ ABA for 7 days. (B) Germination rates of transgenic lines and WT seedlings grown on MS medium with or without 130 mM NaCl, 200 mM mannitol or 0.75 μM ABA for 7 days. (C) Phenotypes of the greening cotyledon from transgenic lines and WT seedlings growing on MS medium with or without 120 mM NaCl, 200 mM mannitol or 0.5 μΜ ABA for 11 days. (D) Cotyledon greening rates of transgenic lines and WT plants growing on MS medium with or without 120 mM NaCl, 200 mM mannitol or 0.5 μΜ ABA for 11 days. Asterisks represent statistical significance (* 0.01 < p < 0.05, ** p < 0.01, Student’s t-test).
Figure 2. Phenotypes of wild-type (WT) and M-overexpressing (OE) transgenic lines at the seed germination and the greening cotyledon stage following osmatic stress and abscisic acid (ABA) treatments. (A) Seed germination from transgenic lines and WT seedlings growing on MS medium with or without 130 mM NaCl, 200 mM mannitol or 0.75 μΜ ABA for 7 days. (B) Germination rates of transgenic lines and WT seedlings grown on MS medium with or without 130 mM NaCl, 200 mM mannitol or 0.75 μM ABA for 7 days. (C) Phenotypes of the greening cotyledon from transgenic lines and WT seedlings growing on MS medium with or without 120 mM NaCl, 200 mM mannitol or 0.5 μΜ ABA for 11 days. (D) Cotyledon greening rates of transgenic lines and WT plants growing on MS medium with or without 120 mM NaCl, 200 mM mannitol or 0.5 μΜ ABA for 11 days. Asterisks represent statistical significance (* 0.01 < p < 0.05, ** p < 0.01, Student’s t-test).
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Figure 3. Effects on root length and related physiological indexes from MdbZIP26 transgenic and wild-type seedlings under osmatic stress and ABA treatment. (A) Root length and lateral root count of transgenic lines and wild-type seedlings. (B) Phenotypes of root growth of transgenic lines and WT plants growing on MS medium with or without 130 mM NaCl, 200 mM mannitol or 0.75 μΜ ABA. (C) Physiological changes (including chlorophyll content, relative electrolyte leakage, MDA content) associated with osmotic stress response in MdbZIP26 transgenic lines and WT seedlings. Asterisks represent statistical significance (* 0.01 < p < 0.05, ** p < 0.01, Student’s t-test).
Figure 3. Effects on root length and related physiological indexes from MdbZIP26 transgenic and wild-type seedlings under osmatic stress and ABA treatment. (A) Root length and lateral root count of transgenic lines and wild-type seedlings. (B) Phenotypes of root growth of transgenic lines and WT plants growing on MS medium with or without 130 mM NaCl, 200 mM mannitol or 0.75 μΜ ABA. (C) Physiological changes (including chlorophyll content, relative electrolyte leakage, MDA content) associated with osmotic stress response in MdbZIP26 transgenic lines and WT seedlings. Asterisks represent statistical significance (* 0.01 < p < 0.05, ** p < 0.01, Student’s t-test).
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Figure 4. Effects on MdbZIP26 transgenic lines and wild-type seedlings under osmotic stresses. (A) Representative phenotypes of 4-week-old transgenic and WT lines during normal conditions (a), salt stress (b), deprived of water condition for 7 days (c), and re-watered for 2 days (d). (B) Survival rates 2 days after re-watering. (C) Histochemical staining with trypan blue to observe the accumulation of dead cells in transgenic and WT lines. (D) Histochemical staining with diaminobenzidine (DAB) was performed to observe reactive oxygen in transgenic and WT lines. Asterisks represent statistical significance (* 0.01 < p < 0.05, Student’s t-test).
Figure 4. Effects on MdbZIP26 transgenic lines and wild-type seedlings under osmotic stresses. (A) Representative phenotypes of 4-week-old transgenic and WT lines during normal conditions (a), salt stress (b), deprived of water condition for 7 days (c), and re-watered for 2 days (d). (B) Survival rates 2 days after re-watering. (C) Histochemical staining with trypan blue to observe the accumulation of dead cells in transgenic and WT lines. (D) Histochemical staining with diaminobenzidine (DAB) was performed to observe reactive oxygen in transgenic and WT lines. Asterisks represent statistical significance (* 0.01 < p < 0.05, Student’s t-test).
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Figure 5. Stomatal aperture in response to ABA treatment in MdbZIP26 transgenic lines and WT plants. (A) Representative images of stomatal apertures in response to different concentrations of exogenous ABA. (B) Stomatal aperture width to length ratios following treatment with different concentrations of exogenous ABA. Asterisks represent statistical significance (** p < 0.01, Student’s t-test).
Figure 5. Stomatal aperture in response to ABA treatment in MdbZIP26 transgenic lines and WT plants. (A) Representative images of stomatal apertures in response to different concentrations of exogenous ABA. (B) Stomatal aperture width to length ratios following treatment with different concentrations of exogenous ABA. Asterisks represent statistical significance (** p < 0.01, Student’s t-test).
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Figure 6. ROS levels and oxidative enzyme activity assays of MdbZIP26 transgenic lines and WT plants under normal and osmotic stress conditions. (A) Activity of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in the leaves of transgenic and WT plants during normal growth and osmotic stress conditions. (B) Staining with DAB of detached leaves from transgenic and WT plants subjected to abiotic stress or normal control. Asterisks represent statistical significance (** p < 0.01, Student’s t-test).
Figure 6. ROS levels and oxidative enzyme activity assays of MdbZIP26 transgenic lines and WT plants under normal and osmotic stress conditions. (A) Activity of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in the leaves of transgenic and WT plants during normal growth and osmotic stress conditions. (B) Staining with DAB of detached leaves from transgenic and WT plants subjected to abiotic stress or normal control. Asterisks represent statistical significance (** p < 0.01, Student’s t-test).
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Figure 7. Stress-related genes expression levels in WT and MdbZIP26-expressing transgenic plants under drought (A) and salt stress (B). Asterisks represent statistical significance (* 0.01 < p < 0.05, ** p < 0.01, Student’s t-test).
Figure 7. Stress-related genes expression levels in WT and MdbZIP26-expressing transgenic plants under drought (A) and salt stress (B). Asterisks represent statistical significance (* 0.01 < p < 0.05, ** p < 0.01, Student’s t-test).
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Wan, Y.; Wang, Y.; Wang, F.; Feng, S.; Zhang, L.; Wang, X.; Gao, H. Heterologous Expression of the Apple MdbZIP26 Gene in Arabidopsis thaliana Improves Resistance to High Salinity and Drought Stress. Agronomy 2022, 12, 2624. https://doi.org/10.3390/agronomy12112624

AMA Style

Wan Y, Wang Y, Wang F, Feng S, Zhang L, Wang X, Gao H. Heterologous Expression of the Apple MdbZIP26 Gene in Arabidopsis thaliana Improves Resistance to High Salinity and Drought Stress. Agronomy. 2022; 12(11):2624. https://doi.org/10.3390/agronomy12112624

Chicago/Turabian Style

Wan, Ye, Yaqiong Wang, Fan Wang, Shuaishuai Feng, Li Zhang, Xiping Wang, and Hua Gao. 2022. "Heterologous Expression of the Apple MdbZIP26 Gene in Arabidopsis thaliana Improves Resistance to High Salinity and Drought Stress" Agronomy 12, no. 11: 2624. https://doi.org/10.3390/agronomy12112624

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