Introduction

Being sessile in nature, plants have to cope with adverse environmental conditions throughout their life span. Drought and soil salinity are the major abiotic stresses that negatively affect plant growth and development, which account for the poor crop yield worldwide (FAO 2011). Both, drought and salt stress conditions, adversely affect the plant physiology and metabolic processes, by creating cellular metabolic inequity that leads to ROS generation and ultimately results in cellular damage (Singh et al. 2010). Plants tend to bear these abiotic stresses through both, physical and cellular adjustments; and by complex molecular mechanisms in response to these stresses (Knight and Knight 2001). Drought and salinity tolerance are quantitative characters and controlled by multiple stress-responsive genes (Cushman and Bohnert 2000; Hasegawa et al. 2000). The stress-responsive genes involved in stress signal transduction pathways play significant roles in regulation of stress tolerance. Transcription factors (TFs) are regulatory proteins, which regulate expression of a number of genes that underlie the plant’s response to abiotic stresses by interacting with specific cis-acting elements in their promoters (Chen and Zhu 2004; Agarwal et al. 2006). Till now, several TFs families, such as MYC, MYB, bZIP, NAC, AP2/ERF have been identified, that are implicated in various transcriptional regulatory mechanisms in abiotic stress signal transduction pathways (Agarwal et al. 2006; Shinozaki and Yamaguchi-Shinozaki 2007). Among these, plant-specific APETALA2/ethylene response factors (AP2/ERF) transcription factor family plays significant role in plant growth and development, as well as in biotic and abiotic stress response (Sakuma et al. 2002). The dehydration-responsive element (DRE)-binding protein (DREB) transcription factors belong to AP2/ERF family and regulate a number of abiotic stress-related genes by following both, ABA-dependent and ABA-independent pathways (Yamaguchi-Shinozaki and Shinozaki 1994; Liu et al. 1998; Kizis and Pages 2002; Ito et al. 2006). The CBF/DREB proteins recognize DRE/C-repeat (CRT) having common core sequence, A/GCCGAC and modulate the expression of downstream genes to confer abiotic stress tolerance. The genetic engineering through overexpression of TFs can be a promising tool to enhance abiotic stress tolerance in plants (Agarwal and Jha 2010; Hussain et al. 2011). Earlier studies showed that the overexpression of DREB TFs impart drought, salt, heat, and freezing stress tolerance in transgenic plants (Agarwal et al. 2006; Lata and Prasad 2011; Jiang et al. 2014). The overexpression of VrDREB2 in Arabidopsis and EaDREB2 in sugarcane led to improved salinity and drought tolerance (Chen et al. 2016; Augustine et al. 2015). Recently, Zhang et al. (2015a) revealed that overexpression of SsDREB from succulent halophyte, Suaeda salsa in tobacco led to dehydration and salinity stress tolerance. Similarly, the overexpression of PgDREB2a from Pennisetum glaucum (Agarwal et al. 2010), EsDREB2B from Eremosparton songoricum (Li et al. 2014) and SbDREB2A from halophyte Salicornia brachiata (Gupta et al. 2014) improved multiple abiotic stress tolerance in tobacco plants. The ectopic expression of StDREB1 imparts salinity and drought tolerance to the potato plants (Bouaziz et al. 2015). Similarly, OsDREB2a-overexpressing rice plants exhibited tolerance against salt and drought stresses (Mallikarjun et al. 2011).

Apple (Malus x domestica) is a major fruit crop, grown in temperate regions of the world (Potter et al. 2007). The chilling during winter season plays a crucial role in setting and development of apple fruit. At the onset of winters, already developed buds of apple tree undergo dormancy to survive the chilling temperature. These buds remain dormant until they accumulate sufficient chilling during winter and reinitiate growth at the onset of spring (Kumar et al. 2017). This represents a well-developed adaptive strategy of apple tree to survive seasonal changes in temperature in temperate regions (Rivero et al. 2016). There are few reports, where overexpression of apple genes has been shown to confer abiotic stress tolerance in transgenic plants. For example, overexpression of an apple calcineurin B-like protein (CBL)-interacting protein kinase gene, MdSOS2L1in apple and tomato plants conferred salt stress tolerance by enhancing the antioxidant metabolites (Hu et al. 2015). Tomato plants overexpressing a vacuolar-type ATPase, MdVHA-B gene exhibited drought tolerance (Hu et al. 2012). Overexpression of a phytocystatin gene from Malus prunifolia, MpCYS4-enhanced stomatal closure to confer drought stress tolerance in transgenic Arabidopsis and apple (Tan et al. 2017). Overexpression of an apple cytosolic malate dehydrogenase gene, MdcyMDH conferred salt and cold stress tolerance in transgenic apple by modifying the redox state and salicylic acid content (Wang et al. 2016).

Although DREB TFs have been cloned and characterized in several plant species and shown to confer abiotic stress tolerance upon overexpression (Agarwal et al. 2006, 2017; Lata and Prasad 2011), detailed functional characterization of apple DREB TF has not been reported, so far. Thus, to study if apple DREB TF also plays role in abiotic stress tolerance and regulate expression of stress-responsive genes, we cloned MdDREB76 from apple and performed its detailed functional characterization. The results demonstrated that MdDREB76 possess transactivation activity and its overexpression in tobacco plants confers salinity and drought stress tolerance by inducing the expression of stress-responsive genes.

Materials and methods

Plant material and tissue sampling

Plant material during complete phenological cycle of apple (cultivar Royal delicious) was collected from apple orchard located at Palchan (32°18′36″N, 77°10′40E″; altitude 2350 masl) Kullu district, Himachal Pradesh, India throughout the calendar year 2012–2013 (Kumar and Singh 2015). These samples include, 3 stages of apical buds collected within 70 days, 5 stages of spur buds collected within 126 days, 6 stages of floral development, namely, green tip, tight cluster, initial pink, full pink, full flower and petal fall collected within 39 days interval and 6 different stages of fruit development collected within 161 days post anthesis. All the samples were immediately frozen in liquid nitrogen and stored at − 80 °C for further use.

RNA extraction and cDNA synthesis

Total RNA was extracted from 100 mg of plant material as described (Kumar and Singh 2015). The integrity of RNA was confirmed on 1% formaldehyde denaturing agarose gel. The RNA was quantified using a NanoDrop 1000 (Thermo Fisher, USA). The first-strand cDNA synthesis was carried out using RevertAid RNAse H minus cDNA synthesis kit according to the manufacturer’s instructions (Fermentas Life Sciences, USA).

Cloning, subcellular localization and transactivation assay

The 0.972 Kb CDS of MdDREB76 (LOC_MDP0000683814; Supplementary Fig. S1a) was PCR-amplified from pre-synthesized apple cDNA using full-length gene-specific primers and cloned into the pGEM-T Easy vector (Promega, Madison, WI). For transactivation assay, the CDS of MdDREB76 was cloned into the GAL4 DNA-binding domain of the pGBKT7 vector (Clontech Laboratories, Inc.) at NdeI and BamHI restriction sites to produce the plasmid pGBKT7-MdDREB76 (Supplementary Fig. S1b). The recombinant plasmid and pGBKT7 empty vector (negative control) were introduced into yeast strain AH109. The X-gal agarose overlay assay was performed for the PCR-positive clones to check the activation of reporter gene. The transformed AH109 yeast cells were streaked on SD/-Trp and SD/-Trp/-His/-Ade medium plates to observe yeast growth at 30 °C for 3–4 days. The β-galactosidase activity assay was performed with the addition of X-β-gal to the grown colonies. For colony lift filter assay, the transformed yeast colonies were streaked on SD selective agar plates and incubated at 30 °C for 3–4 days. The filter paper (Whatman No. 5) was placed over the master plate containing transformants to be assayed and rubbed gently with the side of forceps to cling the colonies on the filter paper. The filter paper with colonies replica, was dipped in liquid nitrogen for 10 s. and then allowed to thaw at room temperature. After thawing, the filter paper with colonies upside was placed over another filter paper pre-wetted with Z buffer/X-gal solution (for 100 ml; 1.61 g Na2HPO4·7H2O; 0.55 g NaH2PO4·H2O; 0.075 g KCl; 0.025 g MgSO4.7H2O; 1.67 ml 20 mg/ml of X-gal solution; 0.27 ml of β mercaptoethanol) and incubated at room temperature for the appearance of blue color. The intracellular localization of MdDREB76 was predicted in silico by online tools viz., Cell-PLoc, WoLF PSORT and SCL Pred (Mooney et al. 2011; Horton et al. 2007; Chou and Shen 2010). For confirmation of subcellular localization, the CDS of MdDREB76 was cloned in pCAMBIA-1302 as translational fusion with green fluorescent protein (GFP; Supplementary Fig. S1c). The resulting plasmid was transferred into Agrobacterium tumefaciens strain GV3101. The onion inner epidermal peels were transformed with Agrobacterium-harboring MdDREB76:GFP construct and empty vector as described previously (Sun et al. 2007). After co-cultivation with Agrobacterium for 48 h, the peels were immersed in 30% sucrose solution and transferred onto the glass slides. The images were captured using a confocal laser scanning microscope (Zeiss LSM510 Meta Gmbh, Germany). The primers used in this study are listed in Supplementary Table S1.

Tobacco transformation and molecular confirmation of transgenics

For overexpression in tobacco, MdDREB76 gene was cloned in binary vector pCAMBIA-1302 through an intermediate pRT-101 plant expression vector carrying CaMV35S promoter (Supplementary Fig. S1d). The MdDREB76-pCAMBIA construct containing a hygromycin resistance selectable marker regulated by CaMV35S promoter was introduced into tobacco (Nicotiana tabacum cv. Xanthi) plants through Agrobacterium tumefaciens (strain LBA4404)-mediated leaf disc method as described (Singh et al. 2012). The regenerated putative transformants were screened by growing on selection media containing hygromycin (20 µg L−1). The presence of transgene was confirmed by PCR amplification with CaMV35S forward and MdDREB76 reverse primers using genomic DNA as template. The copy number of transgene was determined in transgenic lines using qRT-PCR method as described by Zhang et al. (2015b). The Tobacco NtWBC1 gene (LOC_107826765) was used as endogenous reference gene (Yuan et al. 2007). The ratio of the copy number of the transgene was determined using the equation: ratio = 2Ct reference−Ct transgene. The expression of MdDREB76 gene was analyzed in transgenic lines by qRT-PCR with three biological and three technical replicates. To normalize the variance in cDNA input, elongation factor-1α (accession number AF120093) gene was used as the internal control (Schmidt and Delaney 2010). The transcript abundance was calculated using comparative delta–delta Ct method as described (Livak and Schmittgen 2001).

Stress tolerance of MdDREB76 transgenic tobacco plants

For initial assessment of the stress tolerance capacity of transgenic lines, leaf disc assays were performed with 60 day old T0 plants as described previously (Singh et al. 2012). The 1 cm diameter leaf discs of transgenic lines and WT were kept on 8 ml solution of NaCl (200 mM) or 8 ml solution of mannitol (400 mM) for salinity or osmotic stress tolerance assays, respectively. Leaf discs kept on 8 ml water served as control. The experiment was maintained at 26 ± 1 °C in culture room until the phenotypic changes were observed. The chlorophyll content was measured as described earlier (Arnon 1949). The experiment was carried out with three replicates and repeated three times.

For percent seed germination, the seeds were germinated on MS media supplemented with NaCl (100, 200, 300, and 400 mM) and mannitol (200, 400, and 500 mM). The seeds germinated on MS medium (unstressed condition) were used as control. After 21 days, germination rate of transgenic lines compared to WT was calculated. To study the effect of salt and dehydration on the growth and physiological parameters of transgenics as compared to WT plants, the 8 days old germinated seedlings were transferred to MS media containing NaCl (100 and 200 mM) and mannitol (200 and 400 mM) and grown for 30 days. Plant growth and physiological parameters, including root length, shoot length, fresh weight (FW), dry weight (DW) and chlorophyll content were recorded.

The T3 seedlings (transgenic and WT) grown on MS medium for 21 days were transferred to half-strength MS hydroponic medium. Hydroponically grown plants (15 days) were treated with salinity (200 mM) or osmotic stress (PEG 20%) for 48 h. The tissues were harvested for H2O2 estimation, in vivo localization of free radicals, enzymatic activity and expression analysis of genes encoding enzymes of ROS scavenging pathway.

The H2O2 content from leaves of WT and transgenic plants was measured as described earlier (Shafi et al. 2015). The in situ accumulation of H2O2 and O2− was analyzed through histochemical staining of seedlings with 3,3-diaminobenzidine (DAB) and nitro-blue tetrazolium (NBT), respectively, as described earlier (Shafi et al. 2017). The enzymatic activities of ROS scavenging enzymes (SOD, APX, and CAT) in response to salinity and dehydration stresses were also analyzed. For SOD and CAT activity measurement, the soluble proteins were extracted with 500 mg leaf tissue in extraction buffer (100 mM potassium phosphate buffer, pH 7.8, 1 mM EDTA, 1% (w/v) PVP and 10% glycerol). Whereas, for APX activity, the tissue was homogenized in 100 mM sodium phosphate (pH 7.0) containing 1 mM EDTA and 5 mM ascorbate as described previously by Lee and Lee (2000). The APX activity was determined using reaction mixture harboring 50 mM phosphate buffer (pH 7.0), 0.5 mM ascorbate and 0.2 mM H2O2 by the change in absorbance at 290 nm assuming an absorption coefficient of 2.8 mM−1 cm−1 and results were evaluated in terms of micromole of ascorbate oxidized per minute (Chen and Asada 1989). CAT activity was determined using reaction mixture containing the appropriate extract in 50 mM phosphate buffer (pH 7.0). The reaction was initiated by addition of 10 mM H2O2. The 1 U of CAT activity was defined as micromole H2O2 degraded per minute (Aebi 1974). The SOD activity was analyzed by examining the reduction of nitro blue tetrazolium (NBT) as described previously (Giannopolitis and Ries 1977).

The expression analyses of genes encoding ROS scavenging enzymes, viz., NtSOD, NtAPX, and NtCAT were carried out in stress treated and control transgenics as compared to WT plants using qRT-PCR. First-strand cDNA synthesis was done as described above and the qRT-PCR were performed with three biological and three technical replicates for each sample along with negative control. Elongation factor-1α (accession number AF120093) gene was used as internal control.

To evaluate the transgenic plants for their tolerance to salinity and dehydration stresses at pot level, the WT and transgenic seeds were germinated on MS medium with or without hygromycin selection. The 21 day old T3 plantlets were transferred to pots (containing garden soil) and grown under greenhouse conditions (16 h light/8 h dark; 25 ± 1 °C) for 21 d. Initially, WT and three independent transgenic lines with three plants each were divided in three sets. One set was irrigated (twice a week) with 200 mM NaCl solution for salinity stress. For dehydration stress, irrigation was withheld for second set. Third set was watered biweekly with normal tap water, which served as control. After 30 days of stress treatments, leaf tissues from each plant (control and treated) were harvested to analyze physio-biochemical parameters such as electrolyte leakage, membrane stability index, relative water content, proline content, total soluble sugar, H2O2 content, lipid peroxidation (MDA content) and downstream gene expression analysis compared to control plants. After 90 days of stress treatments, parameters including shoot and root dry weight and total seed yield per plant were also recorded.

Electrolyte leakage and membrane stability index

The cell membrane injuries due to salinity and dehydration stresses were assessed by estimating the electrolyte leakage using an electrical conductivity meter as described by Lutts et al. (1995). The 1 g fresh weight leaves were cut into small pieces and immersed in 20 ml of deionized water at room temperature. Electrical conductivity (EC1) of bathing solution was recorded after 24 h. The samples were then autoclaved at 121 °C for 20 min to release all electrolytes. The samples were then cooled to room temperature and final electrical conductivity was recorded (EC2). Electrolyte leakage was calculated by formula: EL = EC1/EC2 × 100. The membrane stability index of stress-treated plants was determined as described previously (Hayat et al. 2010). Leaf tissue was used in two sets and washed with Milli-Q water to eradicate surface-adhered electrolytes. The first set of samples was heated at 40 °C for 30 min followed by electrical conductivity (C1) measurement. The electrical conductivity (C2) was measured for second set after boiling at 100 °C for 10 min. The membrane stability index was calculated using the formula: MSI (%) = (1 − C1/C2) × 100.

Measurement of relative water content, proline, and total soluble sugars

The relative water content (RWC) was determined according to Barrs and Weatherley (1962). Leaves were excised and fresh weight (FW) was immediately recorded, then leaves were soaked in distilled water at room temperature for 24 h and their turgid weights (TW) were recorded. The leaves were dried in hot air oven at 65 °C for 24 h and dry weight (DW) was recorded. The RWC was determined using the formula: RWC (%) = (FW − DW)/(TW − DW) × 100.

To evaluate plant physiological response to salinity and dehydration stresses, proline and total soluble sugar contents were measured. The proline content was measured according to the methods of Bates et al. (1973). The 0.5 g (fresh weight) leaves were homogenized with 3% sulfosalicylic acid. The homogenate was centrifuged at 3000 r.p.m. for 10 min. The supernatant was treated with acetic acid and ninhydrin, boiled for 1 h and then absorbance was determined at 520 nm with UV–Visible spectrophotometer. Proline content was expressed as µmol g−1 fw. Whereas, total soluble sugar content was measured using method of Loewus (1952). The 0.5 g fresh weight of leaves was homogenized with 80% ethanol, centrifuged at 3000 r.p.m. for 15 min and the extract was collected. Cold anthrone reagent (3 ml) was added to 0.05 ml of extract and heated at 100 °C for 10 min and absorbance was determined at 630 nm using spectrophotometer. The contents of soluble sugar were expressed as mg g−1 FW.

Lipid peroxidation

The lipid peroxidation was estimated by measuring the concentration of MDA produced by thiobarbituric acid (TBA) in plants as described earlier (Heath and Packer 1968). Leaf tissue (0.5 g) was homogenized in 5% trichloroacetic acid (TCA) and centrifuged at 12,000 r.p.m. for 15 min. 2 ml of 20% TCA was added to the extract and kept at 95 °C for 30 min. Reaction mixture was cooled to room temperature and centrifuged at 7500 r.p.m. for 5 min. Absorbance was determined at 532 nm and 600 nm using spectrophotometer. MDA content was calculated by formula: MDA (n mol ml−1) = (A532 − A600/155,000) × 106.

Expression analysis of abiotic stress-responsive genes

The leaf tissues from stressed and control plants (transgenic and WT) were used for RNA extraction followed by first-strand cDNA synthesis as described above. The expression analysis of 12 selected downstream genes related to abiotic stress signaling and tolerance was carried out through quantitative real-time PCR (qRT-PCR) with three biological and three technical replicates for each sample along with negative control. The L25 ribosomal protein (accession number L18908) gene was used as reference gene for data normalization (Schmidt and Delaney 2010). Relative fold expression was calculated using comparative delta–delta Ct method as described earlier (Livak and Schmittgen 2001).

Statistical analysis

All the experiments were performed with three replicates, each containing three independent biological replicates. The results were expressed as the mean ± SE. The statistical analyses between WT and transgenics were carried out for statistical significance using Student’s t test. The p values of P < 0.05 and P < 0.01were considered as significant and highly significant and marked with * and **, respectively. The results were evaluated individually and in combination by principal component analysis (PCA). Plants subjected to salt and osmotic stresses were considered as observations and various morphological and physio-biochemical parameters were regarded variables. For principal component analysis (PCA), PAST software (Ver. 2.17c) was used to examine the correlation of plant response and the stress condition. The heat maps were generated using the institute for genomic research (TIGR) MeV v4.9.0 software (Eisen et al. 1998).

Results

MdDREB76 acts as transcription factor

The localization of MdDREB76 protein was predicted to be in nucleus using in silico methods. To confirm its localization in vivo, MdDREB76:mGFP plasmid was constructed using pCAMBIA-1302 vector and transient expression analysis was carried out. The appearance of green fluorescence in the nuclei of onion epidermal cells confirmed nuclear localization of MdDREB76 protein, whereas, in control (empty vector) GFP was localized throughout the cell (Fig. 1a). Moreover, the transcription activation activity of MdDREB76 was investigated using yeast one hybrid (Y1H) system. MdDREB76:pGBKT7 plasmid along with empty vector was transformed in yeast strain AH109 and grown on SD-Trp and SD/-Trp/-His/-Ade media. The recombinant plasmid-containing strain could grow on both the media, whereas, the control plasmid-containing strain could grow only on SD-Trp medium. The yeast cells transformed with MdDREB76:pGBKT7 plasmid formed blue colonies on x-gal-containing medium which confirmed its transactivation activity (Fig. 1b, c). These results confirmed that MdDREB76 encodes for a functional transcription factor protein.

Fig. 1
figure 1

Subcellular localization and transactivation assay of MdDREB76 protein. a GFP and MdDREB76:GFP fusion proteins were transiently expressed under the control of the CaMV 35S promoter in onion epidermal cells and observed with a laser scanning confocal microscope. The GFP (empty vector) was used as control. b The fusion proteins of the GAL4 DNA-binding domain and MdDREB76 were expressed in yeast strain AH109. The empty vector pGBKT7 was used as control. The culture solution of the transformed yeast was streaked on an SD plate with or without tryptophan, histidine and adenine. The plates were incubated for 3 days and subjected to β-galactosidase assay. c Colony lift assay was performed by putting the AH109 cells containing empty pGBKT7 or pGBKT7-MdDREB76 vector on to the filter paper with addition of X-β-gal on the paper

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Development of transgenic tobacco plants overexpressing MdDREB76

In total, 18 putative transgenic tobacco lines were regenerated on media containing hygromycin (20 mg l−1). The PCR analysis using CaMV35S forward and MdDREB76 reverse primers indicated the presence of transgene in all the lines (Supplementary Fig. S2a). The expression analysis using quantitative real-time PCR (qRT-PCR) revealed relatively higher transcript levels of MdDREB76 in transgenic lines S1, S2, S3, S4, S8, S10, S11, S14 and S17 among all the PCR-positive lines (Supplementary Fig. S3). The copy number determination of transgene in T0 transgenics using qRT-PCR method confirmed the presence of single copy number in S1, S3, S4, S8, S11, and S14 lines (Supplementary Table S2). The seed germination of confirmed transgenic lines was checked on MS media-containing hygromycin and the better grown lines (S3, S4, and S11) were selected for further characterization. The presence and copy number of transgene were again confirmed in selected T3 transgenic lines by PCR and qRT-PCR methods, respectively (Supplementary Fig. S2b, Supplementary Table S3).

Overexpression of MdDREB76 improves plant growth under salt and osmotic stress

Leaf disc senescence assay was carried out to screen the T0 transgenic lines for salinity and osmotic stress tolerance. The results revealed that the WT leaf discs undergo severe necrosis under salt and osmotic stress conditions as compared to the transgenics (Fig. 2a). The chlorophyll content was reduced by 86% in WT, whereas, the reduction was 28, 44 and 45% in S3, S4, and S11 transgenic lines, respectively, under salinity stress. Similarly, under osmotic stress, 67% reduction was observed in chlorophyll content in WT, while the transgenic lines S3, S4, and S11 suffered chlorophyll reduction only by 10, 31, and 22%, respectively (Fig. 2b). The higher chlorophyll retention capacity of transgenic lines than WT indicated their improved tolerance against salinity and osmotic stress by overexpression of MdDREB76.

Fig. 2
figure 2

Leaf disc senescence assay and estimation of total chlorophyll. a The leaves of transgenic and WT plants were treated under NaCl (200 mM) and mannitol (400 mM) stress. Leaf discs treated with water were considered as control. Left panel shows leaf discs at 0 days, right panel shows leaf discs after 10 days of incubation. b Estimation of total chlorophyll content of transgenic lines (S3, S4, and S11) and WT plants under control, NaCl (200 mM) and mannitol (400 mM)-containing media. The bars represent mean values ± SE and values with double asterisk are significant at **P < 0.01

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The germination and growth responses of WT and transgenic lines (T3) were studied under salinity and osmotic stress conditions. Under control conditions, the germination percentage of both, WT and transgenic lines was same. Whereas, the percentage germination and growth of transgenics (S3, S4, and S11) were significantly (p < 0.05) higher than WT plants under salinity stress conditions (Fig. 3a–f). The percent seed germination of WT was about 46 and 25%, whereas, that of the transgenic lines was 58–69 and 40–56% under 100 mM and 200 mM NaCl stress, respectively (Fig. 3g). The seeds of transgenic lines S3 and S4 showed reduced germination up to 28 and 18%, while, the line S11 and WT seeds showed severely inhibited seed germination up to 5.3 and 2.6% at 300 mM NaCl (Fig. 3e). At 400 mM NaCl, neither transgenic nor the WT seeds germinated even after 20 days (Fig. 3f). The growth parameters viz., root length, shoot length, fresh weight, dry weight and chlorophyll content of transgenic and WT seedlings were observed after growing under salinity stress (100 and 200 mM NaCl) for 30 days and found to be significantly (p < 0.05) higher in transgenic lines as compared to WT plants (Fig. 3h-l). The root length of WT seedlings was reduced by 25 and 87%, whereas, transgenic lines exhibited only 3–4% and 28–39% reduction in root length under 100 and 200 mM NaCl stress, respectively, as compared to the control seedlings (Fig. 3h). Correspondingly, WT seedlings exhibited reduction in shoot length by 14 and 42%, whereas, a range of 7–14% and 25–32% reduction in shoot length was observed in transgenic lines under 100 and 200 mM NaCl stress, respectively, as compared to seedlings grown under control conditions (Fig. 3i). The fresh weight of WT seedlings was reduced by 36 and 82%, whereas, 20–33% and 57–67% reduction was observed in transgenic lines grown on 100 and 200 mM NaCl-containing media, respectively (Fig. 3j). The dry weight of WT was also decreased by 25 and 79%, while, the transgenic lines exhibited low rate of reduction, i.e., 6–11% and 56–66% reduction in dry weight under 100 and 200 mM NaCl conditions, respectively (Fig. 3k). For assessment of damage caused by salinity stress, chlorophyll (Chl) content of leaves was measured. Both transgenic and WT plants showed almost similar Chl content under control conditions. However, under 100 and 200 mM NaCl, reduced Chl content was observed in transgenics and WT seedlings as compared to seedlings grown under control conditions; however, the reduction was less in transgenic seedlings (19–37 and 52–58%) as compared to WT (47 and 76%) seedlings (Fig. 3l).

Fig. 3
figure 3

Seed germination and growth parameters of WT and transgenic lines under salt stress. Seeds from WT and transgenic lines (S3, S4, and S11) were germinated on a 0 mM NaCl; b 50 mM NaCl; c 100 mM NaCl; d 200 mM NaCl; e 300 mM NaCl; f 400 mM NaCl. The bar diagrams represent mean of three replicates for g germination rate; h root length; i shoot length; j fresh weight; k dry weight; l total chlorophyll content of seedlings. The vertical bars represent ± SE for three replicates. The single or double asterisk over bars represent significant difference at *P < 0.05 and **P < 0.01, respectively

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Under osmotic stress conditions, transgenics (S3, S4, and S11) exhibited significantly (p < 0.05) higher germination percentage than WT (Fig. 4a–d). The WT seed germination was 40 and 10%, whereas, transgenic lines had 53–65% and 20–30% seed germination under 200 and 400 mM mannitol stress, respectively (Fig. 4e). At 500 mM mannitol, both transgenics and WT seeds could not germinate even after 20 days (Fig. 4d). The growth parameters viz., root length, shoot length, fresh weight, dry weight and chlorophyll content of transgenics and WT seedlings were observed after growing under osmotic stress (200 and 400 mM mannitol) for 30 days and found to be significantly (p < 0.05) higher in transgenic lines as compared to WT. The WT seedlings showed reduction in root length by 45 and 88%, however, the root length was reduced by 24–27 and 67–75% in transgenic lines, under 200 and 400 mM mannitol stress, respectively, as compared to the seedlings grown on control media (Fig. 4f). The shoot length of transgenic seedlings was reduced by 4–14 and 29–33%, while, a significant decrease by 20 and 50% was observed in WT seedlings grown on media containing 200 and 400 mM mannitol, respectively (Fig. 4g). WT plants exhibited reduction in fresh weight by 39 and 82%, while the fresh weight of transgenic lines showed reduction by 23–29 and 61–66% under 200 and 400 mM mannitol stress, respectively (Fig. 4h). Likewise, reduction in dry weight of WT by 25 and 73% was observed, while the dry weight of transgenic lines was declined by 4–14.8 and 56–65% under 200 and 400 mM mannitol, respectively, as compared to seedlings grown on control media (Fig. 4i). The chlorophyll content was found to be decreased in WT and transgenic lines under 200 and 400 mM mannitol, although, the reduction was less in transgenic lines (22–34% and 49–53%) than the WT (50 and 83%), as compared to control seedlings (Fig. 4j).

Fig. 4
figure 4

Seed germination and growth parameters of WT and transgenic lines under osmotic stress. Seeds from WT and transgenic lines (S3, S4, and S11) were germinated on a 0 mm mannitol; b 200 mM mannitol; c 400 mM mannitol; d 500 mM mannitol. The bar diagrams represent mean of three replicates for e germination rate; f root length; g shoot length; h fresh weight; i dry weight; j total chlorophyll content of seedlings under osmotic stress. The vertical bars represent ± SE for three replicates. The single or double asterisk over bars represent significant difference at *P < 0.05 and **P < 0.01, respectively

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Overexpression of MdDREB76 alleviates ROS buildup under salt and osmotic stress

The transgenic (T3) and WT plants were treated with salinity (200 mM) and osmotic stress (PEG 20%) for 48 h under hydroponic system and in situ localization of ROS (H2O2 and O2−) accumulation and H2O2 content estimation was carried out to investigate the degree of oxidative damage caused by stress. During control conditions, the leaves of transgenic lines and WT plants were stained equally. However, intense NBT and DAB staining in WT leaves compared to transgenics was observed, which revealed the higher levels of free radicals and H2O2 accumulation, respectively, in WT plants than transgenic lines when exposed to salinity and osmotic stresses (Fig. 5a, b). Similarly, the H2O2 contents in transgenic and WT were found to be comparable under control conditions. Whereas, significantly (p < 0.01) lower accumulation of H2O2 were observed in transgenic lines as compared to WT, under both the stresses (Fig. 5c). However, the WT plants subjected to osmotic stress, accumulated higher levels of H2O2 than under salinity stress. Among transgenic plants, S11 line accumulated higher H2O2 content, but lesser than WT under osmotic stress condition, while S3 line exhibited least accumulation of H2O2 under both the stress conditions. These results indicated the major involvement of ROS scavengers in stress tolerance exhibited by transgenic lines. To get further insight, the enzymatic activity of ROS scavenging enzymes (SOD, APX and CAT) and expression analysis of their respective genes was performed in transgenic and WT plants under salinity and osmotic stresses (Fig. 6a–f). The activity of SOD, APX, and CAT were similar in all the plants under control conditions, whereas, during salinity and osmotic stress conditions, their enzymatic activities were found to be significantly (p < 0.01) higher in transgenic lines as compared to WT plants. Under salinity stress, the enzymatic activities of SOD and APX were higher in S3 line (Fig. 6a–c), however, the CAT activity was higher in S11 among all the transgenic lines (Fig. 6b). Under osmotic stress, the activity of these enzymes was higher in S3 among all the transgenic lines.

Fig. 5
figure 5

In situ localization of ROS (H2O2 and O2−). a 3,3′-Diaminobenzidine (DAB) staining was used to detect H2O2, b Nitroblue tertazolium (NBT) staining was used to detect O2− in transgenics (S3, S4, and S11) and WT leaves. c The vertical bars in graph represent mean values ± SE over three replicates for H2O2 content in transgenic and WT leaves and values with double asterisk represents significant difference at **P < 0.01

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Fig. 6
figure 6

Enzymatic activity of ROS scavenging enzymes in WT and transgenics and expression analysis of antioxidant system-related genes in transgenic lines (S3, S4, and S11) seedlings under control, NaCl and PEG treatments. The graphs represent activity of ROS scavenging enzymes a SOD (superoxide dismutase); b CAT (catalase); c APX (ascorbate peroxidase) in transgenics (S3, S4, and S11) and WT plants exposed to salinity and osmotic stress. d Transcript level of NtSOD in transgenic lines; e expression of NtCAT in transgenic lines; f expression level of NtAPX in transgenic lines. Data represent mean values ± SE of three replicates. The bars with single or double asterisk represent significant difference at *P < 0.05 and **P < 0.01, respectively

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In addition, the relative expression of genes encoding superoxide dismutase (NtSOD), catalase (NtCAT) and ascorbate peroxidase (NtAPX) were analyzed in transgenic plants as compared to WT under both the stresses (200 mM NaCl and PEG 20%) and control conditions. It was observed that the transcript levels of these genes were significantly (p < 0.01) higher in transgenic plants under stress as compared to control conditions. Under salinity stress, the transcript level of NtSOD was increased up to eight, seven and threefold in S3, S4, and S11 lines, respectively, as compared to WT plants. Similarly, NtSOD transcript level was increased by 8, 6, and 2.4 fold in S3, S4, and S11 lines, respectively, under osmotic stress condition (Fig. 6d). The expression level of NtCAT transcript was significantly increased by eight, seven and fourfold in S3, S4, and S11 lines under salinity stress, respectively. While, under osmotic stress, the NtCAT transcript was elevated by approximately twofold in all transgenic lines as compared to WT (Fig. 6e). In addition, NaCl treatment elevated the expression level of NtAPX by fourfold in S3, threefold in S4 and S11 lines each, with respect to WT plants. Similarly, PEG treatment also elevated NtAPX transcript level in S3 and S4 lines by fivefold, whereas, twofold increase was observed in S11 line (Fig. 6f). However, the transcript level of these genes was similar in all the lines under control conditions. The elevated expression of these genes in transgenic lines revealed the involvement of MdDREB76 in transcriptional regulation of genes encoding reactive oxygen species scavenging enzymes.

MdDREB76 overexpression improves physiological status of transgenic plants under salinity and dehydration stress

The T3 transgenic plants grown in garden soil were subjected to salinity (200 mM NaCl) and drought (by withholding water) stress along with respective lines in control conditions. The physiological status of the stress treated plants was studied by analyzing physio-biochemical parameters such as electrolyte leakage, membrane stability index, relative water content, proline content, total soluble sugar, H2O2 content and lipid peroxidation (MDA content) as compared to WT plants after 30 days of treatment (Fig. 7). Under control conditions, all these parameters were almost comparable in transgenic and WT plants. However, under stress conditions, the transgenic lines showed significantly (p < 0.01) higher relative water content (RWC) than WT plants. The RWC in WT plants was 41 and 36%, whereas, transgenic lines exhibited 54–59% and 53–57% RWC under salinity and drought stress, respectively (Fig. 7a). Similarly, membrane stability index (MSI) in transgenic plants was significantly (p < 0.05) higher as compared to WT plants. The transgenic plants exhibit MSI level of 48–54% and 41–52%, whereas, MSI in WT plants was reduced to 33 and 30% under salinity and drought stress, respectively (Fig. 7b). The MSI of S3 line was highest among all the transgenic lines under both the stress conditions. Significantly (p < 0.05) lower electrolyte leakage (EL) was recorded in transgenic lines as compared to WT plants. The EL in WT plants was elevated above 80% under stress (salinity and drought), but a very low increase was observed in transgenic lines (Fig. 7c). The extent of damage caused by salinity and dehydration stresses was also examined through accumulation of MDA and H2O2 in WT and transgenic plants. Under both the stresses, transgenic and WT plants accumulated higher MDA content as compared to control conditions. However, within the treatments, accumulation of MDA was significantly (p < 0.01) lower in transgenic plants than the WT plants (Fig. 7d). Among transgenic plants, MDA accumulation was least in S3 line under drought stress (Fig. 7d). The analysis of H2O2 content showed significantly (p < 0.05) less accumulation in transgenic lines than WT plants under drought and salinity stresses. However, H2O2 accumulation within the stressed plants was higher than the control plants (Fig. 7e). The less MDA and H2O2 accumulation indicates reduced oxidative damage in transgenic plants as compared to WT plants under both, drought and salinity stresses. Moreover, the levels of osmoprotectants viz., proline and total soluble sugars (TSS) were studied in transgenic and WT plants. Under control conditions, the proline content in transgenic lines was comparable with WT plants. However, the transgenic lines subjected to salinity, accumulated significantly (p < 0.05) higher proline than WT plants. Similarly, transgenic lines under drought stress accumulated significantly (p < 0.01) higher proline content than WT plants (Fig. 7f). The transgenic lines exhibited 1.4–1.7 fold and 1.6–twofold increased proline accumulation under salinity and dehydration stress, respectively, as compared to control plants. While, WT plants showed about 0.8 and 0.7% increase in proline content under salt and dehydration stress, respectively. Likewise, the TSS content was almost similar in all the plants under control conditions. However, under stress conditions, the TSS content was significantly (p < 0.01) higher in transgenic lines as compared to WT plants (Fig. 7g). The transgenic lines exhibited 2.3–2.4 fold and 2.4–3.2 fold elevated sugar accumulation under salinity and dehydration stress, respectively, as compared to control plants (Fig. 7g). All these results revealed the better physiological status of transgenic lines as compared to WT plants under salinity and drought stresses.

Fig. 7
figure 7

Physiological and biochemical parameters of WT and transgenic lines under salt and drought stress. The physiological response of transgenic plants was observed by estimating a relative water content (RWC%); b membrane stability index (%); c electrolyte leakage (%) from leaves of transgenic (S3, S4, and S11) and WT plants under control, salinity and drought stress. The biochemical analysis included estimation of d MDA content; e H2O2 content; f proline content; g total soluble sugar content from leaves of transgenic (S3, S4, and S11) lines and WT plants under control, salinity and drought stress. The bars represent mean vallues ± SE of three replicates. Single or double asterisk represent significant difference at *P < 0.05 and **P < 0.01

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MdDREB76 regulates stress-responsive gene expression under salt and dehydration stress

To study if MdDREB76 regulates expression of stress-responsive genes in transgenic tobacco, the transcript profiling of 12 known genes related to ROS detoxification (NtAPX and NtCAT), signaling components (NtPLC3 and NtCMK1), stress-responsive proteins (NtERD10B, NtERD10D and NtLEA5), heat-shock proteins (NtHSP26 and NtHSP70), transcription factor (NtERF5), lipid transfer protein (NtLTP1) and Tyramine—hydroxycinnamoyl transferase (THT1) was carried out in transgenic lines as compared to WT plants. The expression analysis showed significant (p < 0.05) upregulation of these genes in transgenic plants under salt and drought stresses as compared to plants under control conditions. The expression analysis showed upregulation of NtAPX in transgenics by 10 and 11 fold in response to salinity and dehydration stresses, respectively (Fig. 8a). Expression of NtCAT was reported to be upregulated by maximum 3.4 fold in S11 line and 9.6 fold in S4 line under salt and drought stress, respectively (Fig. 8b). The expression of Ca2+/Calmodulin-dependant Kinase (NtCMK1) in transgenic lines was induced by a maximum of 4.4 and 4.7 fold compared to WT under salt and dehydration stresses, respectively (Fig. 8c). While, Phospholipase C3 (NtPLC3) was found to be induced by 6 and 3.5 fold in transgenics, under salt and dehydration stress, respectively (Fig. 8d). The upregulation of dehydrins (NtERD10B, NtERD10D and NtLEA5) was observed in transgenic lines under both the stress conditions. In transgenic lines, NtERD10B showed a maximum 2.4 and 6.3-fold induction, whereas, NtERD10D showed 3.5 and 2.3-fold upregulation under salinity and drought stresses, respectively (Fig. 8e, f). The expression of NtLEA5 was also found to be upregulated in transgenics by a maximum of 5.7 and 3.7 fold under salinity and dehydration stress, respectively (Fig. 8g). The expression of NtHSP26 and NtHSP70 was increased in transgenic lines by nine and sixfold, respectively, under drought stress (Fig. 8h, i). Whereas, under salt stress, NtHSP26 was induced in transgenics by 4.8 fold (Fig. 8h). The expression of transcription factor NtERF5 was observed to be upregulated by 3.8 fold in transgenics under drought stress, but its expression remained unaltered under salt stress (Fig. 8j). The transcript levels of NtLTP1 and NtTHT1 genes were elevated in transgenic lines by a maximum of 4.3 and 5.2 fold, respectively, in response to salt stress. However, under dehydration stress, 4.9 fold induction of NtLTP1 was detected in transgenics, while, expression of THT1 remained unaltered (Fig. 8k, l). The elevated expression of most of these genes in the transgenic lines compared to WT plants indicated that MdDREB76 positively regulates expression of stress-associated genes, thereby conferring improved stress tolerance to transgenic tobacco plants.

Fig. 8
figure 8

Gene expression profiling of stress-responsive genes. The relative fold expression of aNtAPX; bNtCAT; cNtCMK1; dNtPLC3; eNtERD10-B; fNtERD10-D; gNtLEA5; hNtHSP26; iNtHSP70-3; jNtERF5; kNtLTP1; lTHT1 genes in transgenics as compared to WT was analyzed through qRT-PCR under control, salinity and drought stress The bars represent mean values ± SE for three replicates

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Overexpression of MdDREB76 provide salt and drought endurance

The tolerance of transgenic plants towards salinity (200 mM NaCl) and drought stress was evaluated until completion of their life cycle. The morphology and growth pattern of all the plants, including WT and transgenic lines were comparable under control conditions. However, growth of WT plants was severely retarded under salinity stress, while growth of transgenic lines was less affected (Fig. 9a). The WT plants suffered 44% reduction in height than the control plants (Fig. 9b), and exhibited arrested flowering, chlorosis, wilting and ultimately did not complete their life cycle under salinity stress. Whereas, height of transgenic line (S11) was reduced only by 12% under salinity stress (Fig. 9b). The yield parameters, such as number of inflorescence, flowers, pods and pod weight were comparable in all the plants under control conditions (Fig. 9b). After maturity of plants, we have also recorded shoot and root dry weight of transgenic and WT plants. The transgenic plants retained significantly (p < 0.01) higher dry weight of shoot and root than WT plants under salinity stress (Fig. 9c,d). Compared to control plants, the shoot dry weight of WT was reduced by 76%, whereas in transgenic lines S3, S4, and S11 it was reduced by 30, 28 and 33%, respectively, under salinity stress (Fig. 9c). Similarly, the root dry weight of WT plants was reduced by 70% under salinity stress, while, it was reduced by 18, 20 and 24% in S3, S4, and S11 transgenic lines, respectively, compared to that of controls plants (Fig. 9d).

Fig. 9
figure 9

Relative salt tolerance of WT and transgenic (S3, S4, and S11) plants at mature plant level. a The WT and three representative transgenic lines (S3, S4, and S11) were grown under control conditions for 30 days followed by continued presence of 200 mM NaCl, till maturity. The graphs show; b height of plants, number of leaves, number of days to flower, number of flowers, number of pods, average weight of pods and seed weight per pod; c shoot dry weight; d root dry weight of WT and transgenic lines. The error bars represent mean values ± SE over three replicates

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Morphologically, it was clearly observed that the growth of WT plants was severely affected, while the transgenic lines grew well under drought stress conditions (Fig. 10a). The height of transgenic lines S3, S4, and S11 was reduced by 18, 20, and 27% than the control plants, respectively, under drought stress (Fig. 10b). Although, the height of transgenic plants was inhibited under drought stress, but showed healthy growth, flowered and produced viable seeds. In contrast, the WT plants exhibited reduction in height by 78% followed by wilting, arrested flowering and ultimately did not complete their life cycle under drought stress. The growth parameters, such as number of inflorescence, flowers, pods and pod weight of transgenic lines were recorded and were comparable in all the plants under control conditions (Fig. 10b). After maturity, the shoot and root dry weight of transgenic and WT plants were recorded. Under drought stress, the transgenic plants retained significantly (p < 0.01) more dry weight of shoot and root than WT plants (Fig. 10c, d). The shoot dry weight of WT was observed to be reduced by 84%, while it was reduced by 34, 33, and 32% in transgenic lines S3, S4, and S11, respectively (Fig. 10c). The root dry weight was reduced by 77% in WT plants, whereas, the reduction by 26, 22 and 35% was observed in transgenic lines S3, S4, and S11, respectively, as compared to their control counterparts (Fig. 10d).

Fig. 10
figure 10

Relative drought tolerance of WT and transgenic (S3, S4, and S11) plants at mature plant level. a The WT and three representative transgenic lines (S3, S4, and S11) were grown under control conditions for 30 days followed by withheld water irrigation till maturity. The graphs show; b height of plants, number of leaves, number of days to flower, number of flowers, number of pods, average weight of pods and seed weight per pod; c shoot dry weight; d root dry weight of WT and transgenic lines. The error bars represent mean values ± SE over three replicates

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Principal component analysis differentiates plant response under salt and drought stress

The data of morphological and physio-biochemical parameters in transgenic lines (S3, S4, and S11) and WT plants were analyzed with principal component analysis (PCA) to see the correlation of plant response to salinity and drought stress. Maximum of 97.18 and 95.44% variation was observed by morphological and physio-biochemical responses of plants, respectively, under stress conditions (Supplementary Fig. 4a, b). However, the combined PCA accounts for 95.13% variation under both the stresses (Supplementary Fig. 4c). Moreover, the biplot analysis explained 95.13% variation because of the differential response as evidenced by heat map (Fig. 11a, b). All plants (WT and transgenics) exhibited almost similar response under control conditions, therefore, arranged in same cluster in each analysis (Fig. 11a). The WT plants respond similarly to salinity and drought stresses and clustered together in same axes in every PCA analysis (Supplementary Fig. 4a–c). Likewise, in biplot analysis, the transgenic lines were arranged at same axes and divulged similar response to cope with salinity and drought stresses (Fig. 11a). Among transgenics, a significant correlation of S3 line in response to salinity and drought stresses was observed in integrated PCA, which was supported by both morphological and physio-biochemical plots individually. Therefore, principal component analysis revealed a statistical variation among morphological and physio-biochemical responses of plants (WT and transgenics) under control and stress conditions.

Fig. 11
figure 11

Multivariate data analysis of transgenic lines. Combined comparative a bi-plot-based principal component analysis (PCA) with first two principal components, and b The heat map showing the differential response of transgenic lines (S3, S4, and S11) and WT plants under control, salinity and osmotic stress conditions

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Discussion

Soil salinity and drought are the two major threats to agriculture and have damaging consequences on growth, development and yield of plants. To combat these stresses, plants have evolved complex mechanisms to respond and adapt to these stresses at molecular and cellular levels. Therefore, attention has been paid to identify candidate genes and their functional characterization for developing salinity and drought-tolerant plants. Drought and salinity are quantitative characters that can be controlled by expression of multiple stress-responsive genes at a time (Cushman and Bohnert 2000; Hasegawa et al. 2000). Therefore, rather than engineering single gene, overexpression of a gene that regulates the transcription of several downstream genes can be a promising approach in the development of abiotic stress-tolerant transgenic plants. Being regulatory proteins, TFs act as master regulators of several downstream genes essential for protection against abiotic stress and are better candidates for developing drought-and salt-tolerant transgenic plants.

In the present study, we have identified a salt- and drought-responsive gene, MdDREB76, from apple (Malus x domestica) and performed its functional characterization. Intracellular localization revealed the presence of MdDREB76 protein in nucleus, which supported the in silico analysis. Consistent with these results, the transcription activation activity of MdDREB76 confirmed by the x-gal assay, revealed its function as transcription factor. Likewise, the subcellular localization and transactivation studies revealed that LcDREB2 gene from Leymus chinensis (Peng et al. 2013) and EsDREB2B from Eremosparton songoricum (Li et al. 2014) act as transcription factors. The SbSDR-1 gene cloned from Salicornia brachiata was shown to function as a transcription factor and confer tolerance to salinity and drought stress in transgenic tobacco plants (Singh et al. 2016).

The transgenic plants overexpressing MdDREB76 showed normal phenotype. However, initial leaf disc senescence assay revealed that the transgenic lines retained more chlorophyll than WT under 200 mM NaCl and 400 mM mannitol. The higher chlorophyll retention capacity by transgenic lines over the WT signified their tolerance to salinity and osmotic stress. The chlorophyll degradation in plant leaves under stress conditions has been reported earlier (Santos 2004). While, higher chlorophyll retention capacity has been correlated with improved stress tolerance in transgenic tobacco plants overexpressing OsCBSX4 and SbUSP genes, previously (Singh et al. 2012; Udawat et al. 2016).

On the basis of seed germination assay, three homozygous transgenic tobacco lines S3, S4, and S11, having single gene integration and higher expression of transgene were selected to understand the role of MdDREB76 gene in salinity and drought tolerance. Under stress conditions, MdDREB76 overexpressing seedling showed better growth in terms of root length, fresh weight and dry weight as compared to WT. Similarly, the overexpression of other DREB TFs in tobacco such as PgDREB2A (Agarwal et al. 2010), OsDREB2A (Mallikarjuna et al. 2011) and SbDREB2A (Gupta et al. 2014) conferred enhanced abiotic stress tolerance over WT at seedling level.

Reactive oxygen species (ROS) are produced during the normal metabolism of O2 in chloroplast and mitochondria, but their production is enhanced dramatically under stress conditions. ROS levels also act as the cellular markers of abiotic stresses (Biehler and Fock 1996). Generally, during abiotic stress conditions viz. salinity, drought, freezing, heat, and UV, the level of ROS increases which lead to redox imbalance (Choudhury et al. 2013). In the present study, the leaves of WT plants showed intense NBT and DAB staining under salt and osmotic stress conditions, indicating the high accumulation of free radicals and H2O2, respectively (Fig. 5a, b). Whereas, in MdDREB76 overexpressing lines, reduced accumulation of H2O2 and free radicals was observed as compared to WT under stress conditions. Previously, SbDREB2A overexpressing transgenic lines also showed reduced accumulation of H2O2 than the WT (Gupta et al. 2014). It has been reported that during abiotic stresses, the activity of ROS scavenging enzymes, viz., SOD, APX, and CAT gets elevated in different plants (Shafi et al. 2015, 2017). In this study, the enzymatic activity of SOD, APX, and CAT was found to be elevated in both, WT and transgenic lines, but the transgenic plants exhibit relatively higher activity than WT plants under salinity and drought stress conditions (Fig. 6). The enhanced activity of ROS scavenging enzymes in transgenic lines may be attributed to reduced ROS accumulation and their better adaptation under stress conditions. Similarly, the enhanced activity of SOD and CAT was observed in tobacco plants overexpressing SpAQP1 gene which conferred salt tolerance to transgenics (Chang et al. 2016). The increased expression of genes encoding antioxidant enzymes was also reported in transgenic tobacco plants overexpressing SbUSP gene (Udawat et al. 2016). The overexpression of MdDREB76 in tobacco upregulated the transcript levels of genes encoding ROS scavenging enzymes which enhance the plant tolerance in response to salinity and drought stress. Similar results were reported in rice overexpressing SUB1A, where transcript level of genes encoding ROS scavenging enzymes was induced (Fukao et al. 2011). Likewise, the elevated transcript levels of NtSOD, NtAPX, and NtCAT genes were also reported in plants overexpressing different genes, that imparted salinity and drought stress tolerance (Wang et al. 2015; Udawat et al. 2016). The higher expression level of these genes may be responsible for increased activity of the ROS scavenging enzymes, which in turn account for the lower accumulation of free radicals and H2O2 in transgenic lines, that exhibit improved salinity and drought tolerance.

The measurement of physio-biochemical parameters such as electrolyte leakage, lipid peroxidation, membrane stability index, relative water content, proline and total soluble sugar content can reveal physiological conditions of plants under stress. The MDA is released due to the peroxidation of polyunsaturated fatty acids present in biomembranes and accumulated in higher concentration under salt and drought stresses (Gossett et al. 1994; Zhang and Kirkham 1994). The MdDREB76 overexpressing tobacco plants showed reduced accumulation of MDA, indicating the less oxidative damage than WT plants under salt and drought stresses. Similar results were reported in StDREB1 overexpressing potato lines (Bouaziz et al. 2015) and BdDREB2 overexpressing tobacco lines (Zhang et al. 2014) that exhibited salinity and drought stress tolerance. The electrolyte leakage reflects the extent of damage to membrane integrity in plants under abiotic stresses (Bewley 1979). The MdDREB76 overexpressing transgenic lines exhibited less electrolyte leakage than WT and therefore suffered less damage under salt and drought stress. Similarly, the overexpression of GmDREB1 in alfalfa (Jin et al. 2010) and AtDREB1A in rice (Ravikumar et al. 2014) lead to reduced electrolyte leakage in transgenic lines.

The increased level of membrane stability index, relative water content, proline and total soluble sugar contents in transgenic lines as compared to WT plants, indicate their better physiological state under salinity and drought stress conditions than WT plants. Evaluation of membrane dysfunction due to abiotic stresses can be assessed by membrane stability index, which was higher in MdDREB76 overexpressing tobacco lines than the WT plants. The improved membrane stability index can be correlated with lower accumulation of MDA and less electrolyte leakage in transgenic lines than WT plants. RWC measures the water retention capacity of plants and is generally used as a physiological parameter for dehydration and salinity stress tolerance. The higher RWC was reported in MdDREB76 overexpressing transgenic lines than the WT plants, which was in agreement with the previous reports (Bouaziz et al. 2013; Ravikumar et al. 2014). The osmolyte accumulation is responsible for maintaining enzyme activity and membrane stability under osmotic stress (Du et al. 2014; Singh et al. 2015). Accumulation of proline and total soluble sugars (TSS) under salt and drought stresses was reported previously (Ito et al. 2006; Ravikumar et al. 2014; Zhang et al. 2015a). Similarly, the present study also showed higher accumulation of both, proline and TSS in transgenic lines as compared to WT plants (Fig. 7). This may be attributed to higher water retention capacity and lower level of ion leakage due to improved membrane integrity in transgenic plants under stress conditions.

The DREB/CBF transcription factors are known to regulate expression of genes responsible for multiple abiotic stress tolerance in plants (Mizoi et al. 2012). The present study showed upregulation of reactive oxygen species detoxification-related genes under salinity and drought stress conditions. Similar results were observed in transgenic tobacco overexpressing wheat TaASR1, that increases the transcript level of NtCAT and NtSOD genes which conferred osmotic stress tolerance to the plants (Hu et al. 2013). The higher expression level of these genes may account for the lower accumulation of ROS in transgenic lines, which may be responsible for improved salinity and drought tolerance in transgenic plants. During dehydration conditions, the dehydrins protect the cellular components from congelation (Hanin et al. 2011; Zhu 2002). The transcript levels of NtERD10B and NtLEA5 (dehydrins) were found to be elevated in MdDREB76 transgenics under dehydration and salt stress, respectively. Similarly, upregulation of NtERD10B was also observed in PgDREB2A overexpressing (Agarwal et al. 2010) and SbDREB2A overexpressing tobacco transgenics (Gupta et al. 2014) under osmotic and salt stress, respectively. Likewise, the upregulation of NtLEA5 transcript was also reported in transgenic tobacco plants overexpressing TaASR1 (Hu et al. 2013) and TaWRKY44 (Wang et al. 2015) transcription factors from wheat. The upregulation of dehydrin genes may be responsible for higher levels of proline and total soluble sugars accumulation, which establish osmotic homeostasis and protect the cells from osmotic stress during salt and drought conditions. The upregulation of signaling components viz., Phospholipase C3 (NtPLC3) and Ca2+/Calmodulin-dependant Kinase (NtCMK1) may stimulate the stress-responsive signaling pathways that elicit other components of cell signaling in response to salinity and drought stresses. The heat-shock proteins (HSP) act as molecular chaperons and regulate protein folding, assembly and transport that protect the cells from abiotic stresses (Whitley et al. 1999). Ectopic expression of ZmDREB2A in Arabidopsis elevated the transcript level of LEA and heat-shock proteins and impart tolerance to drought and heat stresses (Qin et al. 2007). Likewise, in the present study, upregulation of NtHSP26 and NtHSP70 genes in response to drought stress was observed. The MdDREB76 transgenic lines showed upregulation of a transcription factor NtERF5 under drought stress. The phenylpropanoid pathway is known to be activated in response to environmental stresses in plants. The hydroxyl cinnamic acids derived from phenypropanoid pathway, conjugate with sugars and organic acids to form amides. The N-(hydroxycinnamoyl)-tyramines synthesis is catalyzed by hydroxycinnamoyl-CoA: tyramine hydroxycinnamoyl transferase (THT1) and has been previously studied in relation to environmental stress, biotic and abiotic elicitor in various plants (Farmer et al. 1999; Ishihara et al. 2000). In this study, we reported the upregulation of THT1 in transgenic tobacco lines under salinity stress, which was in accordance with the previous studies (Agarwal et al. 2010). The NtLTP1 encodes for the enzyme involved in membrane biogenesis and phospholipid transport (Kader 1996). In this study, induction of NtLTP1 gene in transgenic lines was reported under salt and dehydration stress. Similarly, the upregulation of NtLTP1 was also reported in transgenic tobacco plants overexpressing TaASR1 from wheat under drought stress (Hu et al. 2013). Several reports showed the induction of NtLTP1 in response to abiotic stresses (Torres-Schumann et al. 1992; Kalifa et al. 2004; Wang et al. 2015). These observations clearly suggest that MdDREB76 acts as key regulator of genes involved in ROS detoxification, transcription factors, abiotic stress signaling that are important for imparting salt and dehydration stress tolerance to transgenic tobacco plants.

The assessment of transgenic plants for their tolerance to salinity and drought stresses was carried out throughout their lifecycle. The transgenic plants grew healthier, exhibited flowering, produced viable seeds and completed their lifecycle. Whereas, WT plants showed stunted growth, wilting, chlorosis, and were unable to complete their life cycle under salinity and drought stress. Therefore, it can be concluded that overexpression of MdDREB76 activates the transcription factors, abiotic stress signaling, and ROS detoxification machinery that conferred tolerance to the transgenic lines under salinity and drought stresses.

Conclusions

In summary, we functionally characterized a gene from Malus x domestica (MdDREB76) by its overexpression, in tobacco. Our results revealed that MdDREB76 protein exhibit transactivation activity and localized in the nucleus. Moreover, the overexpression of MdDREB76 imparts salt and drought endurance in transgenic tobacco plants. The improved stress tolerance of transgenic plants as compared to WT plants was supported by morphological and physio-biochemical parameters such as higher MSI, RWC, and proline content, which was linked with lower H2O2 and MDA content under salt and drought stresses. Furthermore, MdDREB76 functions as transcriptional regulator of stress-responsive genes that imparted salinity and drought stress tolerance without significant yield penalty in transgenic lines. These results enhance our understanding of the role played by MdDREB76 transcription factor in responses to salt and drought stresses. Therefore, MdDREB76 can be utilized for the genetic engineering of crop plants to improve their tolerance towards salinity and drought stress.

Author contribution statement

AKS conceived and designed the study. VS and PG conducted the experiments. SK guided and supported the research, VS drafted the manuscript. VS and AKS analyzed the data and finalized the manuscript.