Heterologous expression of banana MaDREB16 gene in Arabidopsis increases tolerance to dehydration and combined dehydration plus heat stress through stomatal regulation and root elongation

doi:10.21203/rs.3.rs-4436910/v1

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Heterologous expression of banana MaDREB16 gene in Arabidopsis increases tolerance to dehydration and combined dehydration plus heat stress through stomatal regulation and root elongation

https://doi.org/10.21203/rs.3.rs-4436910/v1

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We report here isolation of a dehydration responsive element binding 2 (DREB2) gene (MaDREB16) from Grand Nain (GN) cultivar of Musa acuminata and its involvement in dehydration and heat stress responses in transgenic Arabidopsis plant. Expression analysis of MaDREB16 in root tissues of two banana varieties showed its significant expression under dehydration and combined dehydration plus heat stress. Our findings demonstrate that soil grown transgenic Arabidopsis plants overexpressing MaDREB16 gene display better rate of survival, aerial dry biomass, seed production, relative water content and content of free proline, whereas it has lower ion leakage and malondialdehyde content under the stress conditions studied. Transgenic Arabidopsis plants also showed significant induction of downstream marker genes under dehydration, heat as well as their combination of stresses. Transgenic Arabidopsis displayed reduced stomatal densities and indices even under unstressed conditions suggesting that the transgene modifies the stomatal characteristics and improved regulation of stomatal aperture in dehydration stress as compared to wild-type Arabidopsis. These alterations may be accountable for the observed improved tolerance against individual and combined stress studied.in transgenic as compared to the wild-type. Interestingly the transgenic Arabidopsis showed improved germination rate and root growth under mannitol and combined mannitol plus heat stresses. Altogether, our findings reveal that the MaDREB16 is an important gene of attention to exploit in transgenic breeding to augment tolerance in not only banana against dehydration and its combination with heat stress but also in other crops.

Banana DREB

Abiotic stress tolerance

Dehydration stress

Heat stress

Combined stress

Transgenic Arabidopsis

Plants in their natural habitat get exposed to assorted environmental fluctuations throughout their existence. Dehydration (drought), salinity, cold and high temperatures (heat), oxidative stress and nutrient deficiency are the prime abiotic stresses responsible for worldwide crop losses, reduction of average yields and quality (reviewed by Zandalinas et al. 2018). Dehydration stress alone has main role in significant drop in crop yields per year than the other stresses (FAO, 2018). Global warming and change in average environmental conditions (climate change) are the main issues responsible for heat stress to plants. Wang et al. (2016) verified that when the heat stress was applied at a later stage of plant growth to a C4 grass, Andropogon gerardii and a C3 plant, Solidago canadensis, photosynthetic rate as also productivity was affected greatly. Under dehydration conditions, decreased stomatal conductance and transpiration may induce heat stress due to increase in leaf temperature, therefore heat and dehydration were reported to be linked (reviewed by Lamaoui et al. 2018). In the recent past, climate change is taking us in the direction of hotter and more dehydrated surroundings (FAO, 2019). One needs to consider the effect of both dehydration and heat together as their collective consequence is greater than measured separately. Impact of dehydration and heat stresses on yield when both stresses were applied individually as well as in combination was studied in barley and in wheat by Jedmowski et al. (2015) and Mahrookashani et al. (2017) respectively. They reported yield losses in both crops however combined dehydration and heat stress resulted in additional loss than the separate stresses.

Banana is a staple and nutritive fruit consumed all over the world and grown in many tropical and sub-tropical countries. Banana plants are sensitive to dehydration stress, so that even short periods of dehydration stress also resulted in considerable loss of its yield and productivity (van Asten et al. 2011). The emerging leaves as well as developing fruits are the first to be affected due to dehydration stress. As dehydration stress continues, plant closes stomata to keep leaves highly hydrated and this closure in early stage impacts the productivity (Turner et al. 2007). Global warming resulting in increased temperature stress is another leading abiotic stress resulting in reduced productivity of banana only next to dehydration (Vidya et al. 2018). Response of banana to dehydration stress depends on the genotype and it was predicted that banana possessing B genome as a genetic constituent will be more dehydration stress tolerant. Using proteomics approach, Vanhove et al. (2012) demonstrated less osmotic stress induced growth reduction in ABB banana (constituting more B genome) than other four bananas possessing AAAh, AAA, AABp, and AAB as genetic constitution. More dehydration stress tolerance in banana possessing B genome was again observed by Chaudhari et al. (2019) who reported detrimental result of combined dehydration and heat stress on growth and production of two banana cultivars namely Grand Nain (AAA genome) and Hill Banana (AAB genome). In their studies GN showed no survival and 100% yield loss upon natural high temperature stress combined with an imposed drought stress for a week whereas HB displayed 80% survival and 45.88% yield loss with respect to well watered plants.

Plants have developed elegant regulatory mechanisms involving interactions with several plant hormone signaling pathways during adaptations to unfavourable abiotic stress conditions. Abiotic stresses modify the production and spreading of plant hormones that later facilitate stress responses through hormone and stress responsive transcription factors (TFs) interfaces (Xie et al. 2019). More recently Singh et al. (2023) demonstrated that a WRKY TF gene (WRKY 23) from tomato enhanced salt and osmotic tolerance in transgenic Arabidopsis through interfering with the ethylene and auxin pathways. Amongst these TFs, DREB is a diverse sub-family of TFs (belonging to the AP2/ERF family of TFs) associated with plant responses against environmental stresses (Agarwal et al. 2017). Sakuma et al. (2002) and Nakano et al. (2006) divided Arabidopsis DREB proteins and later Jangale et al. (2019) divided banana DREB proteins into six subgroups, designated as A-1 to A-6. Sub-group A-1 comprises of DREB1-type and sub-group A-2 comprises of DREB2-type TFs and are the most studied TFs in relation to abiotic stresses (Agarwal et al. 2017). Matsukura et al. (2010) in rice, Mizoi et al. (2013) in soybean and Chaudhari et al. (2023) in banana segregated DREB2-type proteins into three subtypes i.e. 1 to 3. Most of the earlier studies in a literature survey demonstrated that tolerance to abiotic stress is supplemented by members of subtype1 of DREB2 genes in plants (Sakuma et al. 2006 a, b; Qin et al. 2007; Matsukura et al. 2010; Mizoi et al. 2013; Li et al. 2014; Sadhukhan et al. 2014; Chen et al. 2016; Wu et al. 2018; Meena et al. 2022). Very few reports on abiotic stress tolerance related to subtype2 members (Lim et al. 2007; Yin et al. 2018) and no report related to subtype3 members of DREB2 genes are available. Regardless of the intricacy of plant responses to dehydration and heat and the partial progress to date attained in reducing the impact of these stresses suggest that there are opportunities to improve yield of plants under these stresses. To help breeders and biotechnologists it is essential to recognize genes and their promoters from different crops that confer tolerance against abiotic stresses that could be used to alter the genetic makeup of crop plants using the marker assisted breeding or genome editing strategies. Identification of such genes from tolerant genotypes, and if their expression in other species confers tolerance, it may be possible to use such information to produce abiotic stress tolerant plants of commercial importance. In this study, a subtype2 candidate of banana DREB2 proteins, MaDREB16 has been chosen for functional analysis in transgenic Arabidopsis. Our study shows remarkable expression of MaDREB16 in root tissues of GN and HB under dehydration, heat as also their combination. Further, overexpression of MaDREB16 in Arabidopsis showed better regulation of dehydration and combined dehydration plus heat stress tolerance in transgenic Arabidopsis in comparison to wild-type plants.

Plant material and growing conditions

Two distinct banana genotypes, stress sensitive i.e. Grand Nain (M. acuminata L. AAA Cavendish Group) and stress tolerant i.e. Hill Banana (M. acuminata L. AAB Group), were used for experimentation. Young banana plantlets of five leaf stages were grown in controlled conditions of relative humidity 85 ± 10% and light intensity 300–350 µmol m^− 2 sec^− 1 for 16 h. The temperature conditions were 30°C during 6 to 10 h, 35°C during 10:00 to 18:00 h and 25°C during 18:00 to 6:00 h ) for a week before imposition of dehydration, heat and combined (dehydration plus heat) stress as described later. To examine the stress tolerance parameters of MaDREB16 gene, Columbia (Col-0) an ecotype of Arabidopsis was used as a model plant for transformation. Growing of Arabidopsis was performed in growth chambers maintained at temperature of 22 ± 1°C, light of 120–150 µmol m^− 2 sec^− 1 for 16 h light/8 h dark cycle and relative humidity of 55 ± 5%. A combination of peat moss and soilrite in equal quantity was used to grow plants in 150 cc plastic pots in bottom trays of mini-greenhouse. To avoid over irrigation and soil drying, around 2 cm of water level was maintained in trays during irrigation. Stress treatments were imposed on three week old Arabidopsis plants grown under controlled environmental conditions.

RNA extraction and gene expression analysis

RNA from banana root was extracted as per Jangale et al. (2019). Arabidopsis leaf RNA was extracted by Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, St. Louis, Missouri, USA). RNA from banana and Arabidopsis was reverse-transcribed using Revertaid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Vilnius, Lithuania, EU). SsoFast EvaGreen Supermix (Bio-Rad, Hercules, California, USA) was used to perform gene expression analysis on CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, California,USA). Triplicate quantitative assays were performed in qRT–PCR by using 10 fold diluted cDNA of each biological replicate. In case of banana, MaEF-1 and MaRPS (Chaudhari et al. 2019) whereas in case of Arabidopsis, Actin8 (Guo et al. 2015) and α-Tubulin (Liu et al. 2015) were utilized as internal control genes. All relevant primers of this study are presented in Table S1. Gene expression was calculated by delta–delta CT method described by Livak and Schmittgen (2001).

Cloning and transformation in Arabidopsis

Overexpression construct was produced by cloning full-length sequence of MaDREB16 gene (Ma07_g16880) in a pENTR^TM/D-TOPO as stated by producer. (Invitrogen, California, USA). Positively cloned plasmids were sequenced from both ends to confirm correct sequence and aligned with original MaDREB16 (Ma07_g16880) sequence (Fig. S1) and cloned into pK2GW7 vector through LR recombination reaction according to the instructions of manufacturer. Schematic representation of MaDREB16 gene in pK2GW7 is given in Fig. S2. By utilizing freeze and thaw technique (Xing et al. 2007) plasmids carrying MaDREB16 were transformed into GV3101 strain of Agrobacterium tumefaciens. Transgenic Arabidopsis plants overexpressing MaDREB16 gene were generated by utilizing the method given by Clough and Bent (1998). Positive plants in T1 generation were selected on the MS agar (Himedia Laboratories Pvt. Ltd., Mumbai, India) medium containing 50 µg/mL kanamycin (Himedia Laboratories Pvt. Ltd., Mumbai, India) (Fig. S3a) and confirmed through PCR. Genomic DNA of recognized plants was isolated from young leaf as per Clarke (2009) and used as template in PCR. In T2 generation, transgenic lines containing single insert were confirmed by analysing 3:1 segregation ratio for the kanamycin resistance using Chi-square (χ²) test. Seed Homozygocity of all the independent lines in T3 generation was confirmed by kanamycin selection (Fig. S3b) and used for the application of abiotic stress treatments.

Application of stress treatments to banana and Arabidopsis plants

In case of banana, dehydration stress was imposed by no watering for 10 days. Heat stress was imposed by subjecting plants to 45°C daily up to 5 days for 6 h (from 12:00 to 18:00 h), however watered regularly. Combined, dehydration plus heat stress, was imposed by exposing plants to dehydration (no watering) in addition to heat as mentioned above for 5 days. To analyse expression of MaDREB16 gene in banana, root samples were collected on 0d, 2d, 4d, 6d, 8d and 10d for dehydration whereas on 0d, 1d, 2d, 3d, 4d and 5d for heat and combined stress.

To impose abiotic stresses three independent homozygous transgenic lines of 35S::MaDREB16 were selected randomly. Transgenic and wild-type (Col-0) Arabidopsis were grown in five repeats; each containing 60 plants. Dehydration stress to Arabidopsis was imposed by discontinuing watering from 21 to 35 days of seed sowing; heat stress consist of exposure to extreme temperature of 44 ± 1°C up to 24 h. Combined stress was forced by subjecting 20 days old plants to extreme temperature coupled with discontinuation of water for 24 h. By the end of stress treatments, growth situations were brought to normal for recovery and plants were watered. Leaf samples for dehydration treatment were taken before dehydration (BD), day 7 of dehydration (7 d), day 14 of dehydration (14 d) and on day 5 after dehydration recovery (DR). Samples for heat stress were taken prior to heat stress (BH), 12 h and 24 h during heat stress and on 5th day after recovery from heat (HR). Leaf sampling for combined stress was done as in case of heat i.e. prior to combine dehydration plus heat (BDH), during stress and after recovery (DHR). Leaf samples were stored at − 80°C after instant freezing with liquid nitrogen and later utilized to analyse biochemical and molecular parameters. In all treatments, survival rate (subsequently 5 days from stress rescue) and aerial dry biomass (ABM) besides yield (at seed harvest) were quantified in wild-type and transgenic plants. Since 24 h heat did not show any damage in wild-type and transgenic Arabidopsis plants; heat treatment was prolonged (up to 72 h) and after heat stress recovery, rate of survival (RS), ABM and seed production of top performer line were analysed.

Assessment of stress linked parameters of Arabidopsis plants

At four stages, throughout each abiotic stress treatments, plants were photographed and their leaf samples were collected to study alterations in stress-related physiological and biochemical indicators besides expression of downstream targets of the transgene. For the assessment of abiotic stress tolerance by transgenic Arabidopsis, certain stress linked parameters like RS, relative water content (RWC), ion leakage (IL), malondialdehyde (MDA) and content of free proline were analysed. In Arabidopsis leaves relative water content in fresh leaves was studied as per method described by Butt et al. (2017) and ion leakage was examined as per Jangale et al. (2019). MAD content to monitor lipid peroxidation was assessed by the protocol of Hodges et al. (1999) using thiobarbituric acid (TBA). Procedure of Bates et al. (1973) was used for estimating free proline content.. Based on previous published literature (Table S1) expression of four downstream marker genes namely AtRD29B, AtHsfA3, AtTCH4/AtXTH 22 and AtIAA1 were analyzed through qRT-PCR in Arabidopsis plants subjected to dehydration, heat and their combination of stresses.

Measurement of stomatal density, stomatal index and stomatal aperture

Stomatal density (SD, total number of stomata per unit area) as well as stomatal index (SI, proportion of stomata to all epidermal cells per unit area) of Arabidopsis plants was measured as per protocol defined by Wu and Zhao (2017). Fully developed leaves (fourth/fifth from the top) of four week old plants were detached and colourless nail polish was put on abaxial side of leaves and permitted to dry completely. Dried layer of nail polish was gently peeled off using forceps. Peeled off film was placed on microscopic slide, covered with cover-slip and observed under 40X objective of a microscope (Labomed Digi3 1500, Labomed Inc., Los Angeles, California, USA); pictures were captured with help of DigiPro 4.0 (Labomed Inc., Los Angeles, California, USA). Stomatal aperture index (SAI, width to length ratio of stomatal opening) was determined according to Eisele et al. (2016).

Assay for seed germination and root growth elongation under mannitol, heat and combined mannitol + heat treatment

Mannitol is used as a stress inducing agent in plant growth media mostly to induce osmotic stress. Therefore, effect of mannitol, heat and combined mannitol + heat treatments on seed germination and root growth in transgenic and wild-type plants was evaluated by growing seeds and seedlings on half-strength MS agar media in presence of mannitol, heat and their combination. For testing seed germination sensitivity, osmotic stress was created as per Luo et al. (2022) by utilizing mannitol (Himedia Laboratories Pvt. Ltd., Mumbai, India) of 250 mM concentration; heat stress was applied by subjecting seeds to high temperature (44 ± 1°C) up to 6 h (Zhao et al. 2017); combined mannitol + heat stress was enforced by using 250 mM mannitol and simultaneous exposure of seeds to 44 ± 1°C for 6 h. Approximately 100 seeds of wild-type and transgenic Arabidopsis were seeded on half-strength MS agar media with or without 250 mM mannitol. Plates were kept at 4°C for synchronization of germination for 4 d and subsequently incubated in a growth chamber. After 4 d of incubation, germination was recorded as the radicles produced out from the seed coat. Germination rate (GR) was calculated as, GR= (number of seed germinated/total number of seeds seeded) X 100. To test the result of mannitol, heat as well as combined mannitol + heat on root development, initially the wild-type and transgenic Arabidopsis seeds were planted in half-strength MS medium with agar. After 4 d, 10 seedlings of wild-type and each transgenic lines were relocated to vertical culture plates consisting half-strength MS agar with or without supplementation of 300 mM mannitol (Luo et al. 2022). After overnight incubation at standard growth conditions in a growth chamber, vertical culture plates for heat and combined mannitol + heat treatment were subjected to high temperature up to 6 h. All plates were grown vertically for 7d at standard growth conditions and root length was recorded.

Statistical analysis

Results of relative expression of genes were compared by the Dunnett (two sided) one-way ANOVA test at P < 0.05. Comparisons of stress related physiological, biochemical and molecular indices in transgenic and wild-type Arabidopsis plants were performed with Student-t test at P < 0.01.

Expression of MaDREB16 under dehydration, heat and combined stress

To inspect the probable participation of MaDREB16 in abiotic stress response, we checked expression patterns of MaDREB16 in roots of banana under dehydration, heat and their combination. qRT-PCR expression analysis showed that MaDREB16 expression was higher at initial (d 2) and final (d 8 and d 10) stage in GN, but in HB it showed higher expression on d 4, 6 and 10 of dehydration treatment (Fig. 1). At earlier stages of heat treatment, it showed reduction in its expression in GN with slight increase at a later stage, but showed continuous reduction in HB throughout heat treatment. Under combined dehydration plus heat stress, expression of MaDREB16 was more in GN as compared to HB during the treatment; however it showed an expression peak in HB at d 3 of combined stress. In general, it showed very low expression in heat than dehydration and combined stress both in GN and HB. Manifestation of MaDREB16 in root tissues of two banana genotypes, GN (stress sensitive) and HB (stress tolerant), particularly under dehydration but not under heat stress suggests its important function in dehydration stress response of banana.

Functional evaluation of MaDREB16 gene in transgenic Arabidopsis

Transgenic Arabidopsis thaliana plants overexpressing MaDREB16 gene were produced. In T1 generation 10 of 15 independent lines (those recognised as positive on half-strength MS agar plates) i.e. MaDREB16_L1, L3-6, L8, L9, L12 and L15 showed positive integration of transgene in Arabidopsis thaliana checked by PCR (Fig. S4a). Transgenic line number 1 (L1), L3-6, L8 and L9 in T2 generation were Chi-square significant i.e. possessing single copy of insert (Table S2). All Chi-square significant lines were observed to be homozygous in T3 generation. Expression of MaDREB16 in seven independent lines of the T3 generation and no expression in wild-type confirmed that MaDREB16 was overexpressed in transgenic plants (Fig. S4b). Homozygous transgenic lines (i.e. MaDREB16_L1, L4 and L5) constitutively expressing MaDREB16 in T3 generation were used for evaluation of abiotic stress response. Under normal (without stress) environments, both the transgenic and wild-type plants grew normally however transgenic plants displayed narrow or elongated leaf morphology than wild-type plants (Fig. S4c). Although the transgenic lines under normal conditions (before stress) showed similar constitutive expression of the transgene (Fig. S4b), they showed differences in their tolerance to the stresses as shown later. Arabidopsis At5g18450 is the homologue of MaDREB16 and its expression was also checked as shown in Fig. 2. Both the homologue and the transgene showed increased expression under dehydration and heat but much higher expression under the combined stress. To assess the abiotic stress tolerance function of MaDREB16, transgenic 35S::MaDREB16 Arabidopsis and wild-type plants were put through individual dehydration, heat and their combination of stresses as defined in material and methods.

Evaluation under dehydration stress

When watering was suspended for 7 d, loss in leaf turgidity was observed in wild-type but not in transgenic plants. When watering was withheld for 14 d, leaves in both wild-type and transgenic lines showed temporary wilting, leaf margin drying and loss of greenness. After 5 d of dehydration recovery, 35S::MaDRERB16 plants showed higher RS from 84–95% however wild-type plants displayed RS of 49% only. During recovery, leaves in wild-type, 35S::MaDRERB16_L1 and 35S::MaDRERB16_L4 turned white in color (chlorosis) whereas that of 35S::MaDRERB16_L5 turned green and started expanding (Fig. 3a) within 5 d. Transgenic plants produced more ABM and seeds in the range of 32.06–34.56 mg and 4.70–5.38 mg respectively, but wild-type produced less i.e. 21.87 mg of ABM and 2.89 mg of seeds (Fig. 3b).

Under dehydration stress, water content is estimated as leaf RWC. Transgenic plants displayed gradual reduction of average RWC from 83.98 to 63.65% from before dehydration up to 14 d but in wild-type RWC decreased from 83.84 to 51.45% (Fig. 3c). In subsequent recovery, average RWC in transgenic plants improved to 75.15% however in wild-type it did not improve because of more drying in leaves and went down to 41.61%. Ion leakage values in transgenic and wild-type plants were similar before dehydration (about 25%). Dehydration till 7th d did not affect IL of either transgenic or the wild-type but on 14th d it increased to average 54.47% and 74.52% in transgenic and wild-type respectively. After 5 d of dehydration recovery, average ion leakage in transgenic plants was reduced to 43.98%; in contrast it was further increased to 72.29% in wild-type plants (Fig. 3d).

Proline is a well-known osmolyte that accumulates under abiotic stress in plants. In both wild-type and transgenic plants, less proline content was observed at BD and 7 d of dehydration as compared to 14 d. On 14 d, average proline content increased to 11.32 nmol/g FW in transgenic however in wild-type, proline content improved merely up to 4.05 nmol/g FW (Fig. 3e). Extent of lipid peroxidation under dehydration stress is estimated by calculating malondialdehyde content. Lower MDA content was noted in transgenics than in wild-type plants throughout the dehydration stress treatment. Transgenic plants contained 4.57, 5.97, 31.38 and 24.20 nmol/g FW MDA however wild-type plants contained 4.80, 10.27, 60.03 and 52.14nmol/g FW at BD, 7 d, 14 d and DR respectively (Fig. 3f).

Assessment of stomatal density, stomatal index and stomatal aperture in Arabidopsis plants

Stomatal characters like SD, SI and SAI are important factors determining leaf RWC in plants. RWC depleted faster in wild-type than in transgenic plants. Out of three transgenic lines, MaDREB16_L5 that had shown the best tolerance against dehydration was taken to study stomatal characters. At normal growth conditions, both SD and SI were observed to be reduced by 29% and 33% respectively in 35S::MaDRERB16_L5 as compared to the wild-type (Fig. 4a and b). Dehydration treatment resulted in substantial reduction of SAI in 35S::MaDRERB16_L5 in contrast to wild-type plants. It was observed to be significantly lower in 35S::MaDRERB16_L5 plants throughout the dehydration treatment. It was 14.8%, 31% and 25% lower prior to dehydration, upon 7 d and upon 14 d of dehydration respectively in 35S::MaDRERB16_L5 plants in contrast to wild-type plants. After dehydration recovery, SAI was reinstated in overexpressing plants however in the wild-type plants it displayed further reduction after 14 d (Fig. 4c and d).

Heat stress evaluation

Wild-type and transgenic plants were not affected by heat stress of 12 h, but under 24 h heat stress only wild-type plants lost their leaf color. In wild-type plants the upper half of leaves turned white after heat recovery (Fig. 5a). However, during recovery both types of plants showed 100% survival, almost similar ABM and seed production (Fig. 5b). Average RWC after 24 h heat stress was reduced from 84.63 to 61.52% in transgenic plants whereas in wild-type it decreased from 85.61 to 52.12% and after recovery from heat both returned to their original levels (Fig. 5c). Transgenic plants showed slower increase in ion leakage under heat stress for 24 h (28.62–53.25%) in comparison to wild-type plants (29.06–69.19%). On subsequent heat recovery, IL in transgenic plants returned to their original level however in wild-type Arabidopsis IL failed to return to its initial level (Fig. 5d). Heat treatment did not display remarkable influence on proline and MDA content in both types of plants unlike during dehydration treatment (Fig. 5e and f). 35S::MaDRERB16_L1 showed higher RWC and proline accumulation but less IL and MDA content as compared to 35S::MaDRERB16_L4 and 35S::MaDRERB16_L5 under heat stress. However, all the transgenics were better than wild-type although there was no statistical difference amongst them.

Evaluation during prolonged heat treatment

Heat stress of 24 h had no adverse effects on RS, ABM, and seed production of both types of plants, therefore heat stress was prolonged to 72 h. For this purpose the transgenic line (L1) that had shown the best tolerance (among L1, L4 and L5) against heat was chosen. The RS, ABM and seed production (from 48 to 72 h) of the transgenic plants were estimated after 5 d of recovery. RS at all-time points was statistically similar throughout prolonged heat treatment in both types of Arabidopsis plants (Fig. 6a). Upon 60 h of heat stress 35S::DREB16_L1 produced 94.47 mg ABM while wild-type produced only 72.57 mg of ABM. However on 72 h of heat treatment, both 35S::DREB16_L1 as well as wild-type Arabidopsis plants produced nearly similar amount of ABM (Fig. 6b). 35S::DREB16_L1 plants produced 33% significantly lower amount of seeds upon 54 h of heat stress than wild-type plants (Fig. 6c). Heat stress in both 35S::DREB16_L1 and wild-type plants failed to produce seeds after 72 hours.

Evaluation under combined dehydration plus heat stress

Combined dehydration plus heat stress did not show any adverse result up to 12 h however after 24 h, wild-type plants lost their leaf color and their leaves became dry. Transgenic Arabidopsis plants did not show any visible adverse effect during combined stress (Fig. 7a). After recovery from combined dehydration plus heat stress, transgenic plants displayed more RS (83 to 87%) whereas wild-type plants displayed only 72% of RS. Transgenic plants produced more ABM and seeds in the range of 75.76–83.03 mg and 15.25–18.08 mg respectively however wild-types generated 35.15 mg of ABM and 7.35 mg of seeds (Fig. 7b). RWC in transgenic plants was reduced to a lesser amount (86.53 to 66.67%) than wild-type in which RWC was reduced from 87.36 to 41.91%. Both transgenic and wild-type genotypes showed slight improvement in RWC after recovery (Fig. 7c). In wild-type, combined dehydration plus heat stress resulted greater increase of ion leakage (22.89 to 80.51%) however in transgenic plants its increase was only 22.63 to 35.61% (Fig. 7d). Proline content was not affected prominently under combination of stresses in either genotype but it showed prominent effect on MDA content in case of wild-type plants. MDA content was raised from 3.93 to 23.95nmol/g FW in wild-type plants, under combined stress treatment of 24 h however average MDA content was increased to a considerably lesser amount in transgenic plants i.e. 3.47 to 13.57nmol/g FW (Fig. 7e and f). Under combined stress, 35S::MaDRERB16_L4 showed higher relative water and proline content in addition to RS, ABM and seed production but less ion leakage and MDA content as compared to L1 and L5 of 35S::MaDREB16.

Seed germination and root growth elongation under mannitol, heat and combined mannitol + heat treatment

Seed germination analysis showed that, wild-type and transgenic Arabidopsis seeds exhibited indistinguishable seed germination in control. But, with 250 mM mannitol, the germination percentage in wild-type was 17.12% while in overexpressed lines it was from 37.84 to 44.87% (Fig. 8a). Under heat stress, the germination in wild-type was 5.52%, whereas in the overexpressed lines it was remarkably high, reaching 27.07 to 40.94%. Under combined mannitol + heat treatment, wild-type seeds failed to germinate, however seeds of the overexpressed lines showed 13.67 to 27.47% germination.

The root growth elongation by the osmotic stress of 7 days in both genotypes showed no root growth in wild-type (17.6 mm only) while the transgenic lines achieved significantly longer root growth (21.3–24.4 mm). In the heat stress, the wild-types had root growth of 27.53 mm whereas transgenic lines showed the root growth in the range of 28.61 to 29.52 mm, though it was not significantly different than wild-type. Under combined mannitol + heat treatment, root growth in the wild-type was 8.41 mm while the overexpressed lines had significantly longer roots i.e. 15.84–17.55 mm (Fig. 8b and c). These results suggest the possible role of MaDREB16 in germination of transgenic Arabidopsis seeds under mannitol, heat and combined mannitol + heat stresses. Additionally, it seems to have a role in root elongation of transgenic Arabidopsis under mannitol and combined mannitol + heat stresses, but not in heat alone.

Expression of marker genes in transgenic plants under stress conditions

Better germination rate, root growth and stress tolerance was observed against dehydration, heat and combined dehydration plus heat stress in 35S::MaDREB16 plants than wild-type. For the confirmation of these consequences the expression of four stress-responsive marker genes (AtRD29B, AtHsfA3, AtTCH4 and AtIAA1) as downstream targets were studied in both genotypes under these stresses.

Downstream target AtRD29B was expressed significantly higher at 12 h in heat but not in dehydration and combined stress in transgenic plants in contrast to wild-type plants. AtHsfA3 expression was more in transgenic lines than wild-type in all types of stress treatments; its expression was higher under combined stress in transgenic plants in contrast to individual dehydration and heat stress. Expression of AtTCH4 was significantly more in transgenic line under dehydration and after dehydration recovery as compared to wild-type. However, during heat and combined stress AtTCH4 expression was lower in transgenic plants as compare to wild-type. (Fig. 9). AtIAA1 showed significantly higher expression in all stress treatments in transgenics in contrast to wild-type plants as was observed for AtHsfA3.

DREB2 type proteins among DREB sub-family are major components that mediate abiotic stress reaction in plants responding to a range of stressors e.g. drought, cold, salt, heat, nutrient deficiency, etc. DREB2 TFs are well-known for the induction of various abiotic stress related downstream genes in response to abiotic stresses in several plants and such information has been reviewed earlier (Lata and Prasad, 2011). Involvement of DREB2 type protein (DREB2A) in dehydration and heat tolerance of Arabidopsis using transgenic approach in Arabidopsis was reported initially by Sakuma et al. (2006a, b). Later abiotic stress tolerance by DREB2 genes from several plant species including maize (Qin et al. 2007), rice (Matsukura et al. 2010), soybean (Mizoi et al. 2013), cowpea (Sadhukhan et al. 2014), lily (Wu et al. 2018) and pearl millet (Meena et al. 2022) have been confirmed in transgenic Arabidopsis. In this study, we wanted to find out if DREB2 homolog (MaDREB16) identified from banana plant that was shown to confer tolerance to dehydration, heat and their combination will provide distinctive insights into DREB2 function and gene control by over expressing it in Arabidopsis.

In our earlier studies MaDREB16 gene showed prominent expression in leaves of GN and HB plants in dehydration and combined dehydration plus heat stress; however in heat it displayed very little expression in GN and somewhat reduced expression in HB (Jangale et al. 2019). In this study, MaDREB16 in root tissues of GN and HB displayed similar prominent expression (as seen in leaves) under dehydration and combined dehydration plus heat stress. However under heat stress alone it showed low expression in roots of GN and gradual reduction in HB (Fig. 1). Analysis of cis-acting regulatory elements carried out in the promoter region of MaDREB16 by Jangale et al. (2019) showed existence of six ABRE (abscisic acid responsive element) and one each of CE3 (ABA and VP1 binding elements) and MBS (MYB binding site) for drought sensing but no HSE (heat shock element) for heat sensing. The expression and promoter region analysis of MaDREB16 gene pointed out its strong dehydration and combined dehydration plus heat stress responsive function, but just a little or no responsiveness to heat in banana. Similarly, Liao et al. (2017) in an expression analysis of a DREB gene (MsDREB6.2) in leaf and root tissues of apple when subjected to drought, heat, cold and salt treatments observed its significant expression against drought and salt stresses, but slight expression under heat and cold treatments; concluding strong responsiveness of MsDREB6.2 against drought and salt in apple. Previous studies on drought and heat tolerance have shown that drought and heat tolerance is related to better RWC, improved proline content, less membrane damage and lower oxidative stress (Li et al. 2014; Butt et al. 2017; Liao at al., 2017). Accumulated proline under dehydration stress functions as a compatible osmolyte arising from activation of its biosynthesis as also decreased inactivation of its degradation (Yoshiba et al. 1997). Reverse of this occurs when the drought stress is released and plant returns to its normal growth.

The constitutive expression of MaDREB16 in this study displayed enhanced tolerance to individual dehydration and combined dehydration plus heat stress (but not to individual heat stress) in transgenic Arabidopsis by showing improved RS, ABM, and seed production as compared to wild-type plants (Figs. 3 and 7). This might be because of improved relative water and proline content as also decreased membrane damage and lipid peroxidation in transgenic Arabidopsis. Similar enhanced RS and dehydration stress tolerance was reported by Liao et al. (2017) in 35S::MsDREB6.2 Arabidopsis plants as compare to wild-type. This was attributed to the substantial increase in content of relative water and proline as also creation of smaller amount of ROS and MDA than wild-type plants. Butt et al. (2017) noted significantly more content of proline and better drought resistance in Arabidopsis overexpressing a cotton MYB85 gene as compare to wild-type. These observations indicate increased tolerance to drought due to internal proline content. Li et al. (2014) observed remarkably more proline and less MDA content in transgenic tobacco after overexpression of EsDREB2B gene and noted improved tolerance against cold, osmotic, heat and salt stresses in transgenic tobacco than wild-type.

In plants, conservation of membrane stability and integrity under drought is an important constituent to measure drought tolerance and measured in terms of ion leakage (Whitlow et al. 1992). In this study, the cell membrane of 35S::MaDREB16 Arabidopsis plants was significantly stable and less leaky than wild-type plants when subjected to dehydration, heat and combination of their stresses. Level of MDA, an important indicator of destructive effects of reactive oxygen species (ROS) in plants under stress conditions (Sharma et al. 2012) was lower in transgenic plants in the present study under dehydration and its combination with heat as compared to wild-type but not under individual heat ; demonstrating little osmotic and oxidative impairment to transgenic plants.

Plants under dehydration and heat stress face a contrasting situation with respect to transpiration. Under combined dehydration and heat stress, regulation of stomata is a challenging situation for plants as they have to close their stomata under dehydration (to halt loss of water) and open their stomata under heat (to keep up leaf temperature) (Mittler and Blumwald, 2010). We had observed (Jangale et al. 2019) that the heat and drought tolerant HB genotype possessed lower stomatal density in comparison to the susceptible GN genotype of banana. Reduced stomatal density causes minimum transpiration through the stomata and results in improved tolerance of dehydration stress in plants. Luo et al. (2013) reported reduced stomatal density and water loss in transgenic Arabidopsis overexpressing a WRKY type TF gene of Glycine soja(GsWRKY20) and conferring enhanced drought tolerance in transgenic than wild-type plants. Better drought tolerance as a result of reduced stomatal aperture, stomatal density (by 32%), as well as transpiration in transgenic apple after overexpression of MsDREB6.2 gene than control plants was observed by Liao et al. (2017). Increased water use efficiency because of reduced stomatal densities as well as stomatal indices in transgenic Arabidopsis overexpressing wheat EPIDERMINAL PATTERING FACTOR (EPF) genes (TaEPF1 and/or TaEFP2) than wild-type was reported by Dunn et al. (2019). In the present study, stomatal densities and stomatal indices in transgenic plants overexpressing MaDREB16 were remarkably reduced in contrast to wild-type Arabidopsis plants. Reduced stomatal indices indicate that the amount of total stomata generated in proportion to pavement cells get reduced due to constitutive expression of MaDREB16 gene in the Arabidopsis background. The enhanced tolerance against dehydration and combined stress in transgenic 35S::MaDREB16 Arabidopsis plants may be because of precise regulation of stomata to stop water loss, upkeep of leaf temperature in addition to regulation of gaseous exchange under drought and its combination with heat stress in this study. It is not clear how the transgene controls stomatal frequencies/indices and this possibly will be a motivating aspect of research. Variations in stomatal density by different stress responsive TFs such as GsWRKY20, MsDREB6.2 and MaDREB16 in transgenic Arabidopsis suggests that stomatal density may be under the control of several TFs that could undergo expression changes against abiotic stresses. For the estimation of thermo-tolerance, Zhao et al. (2017) subjected wild-type Arabidopsis plants (grown up to 4-week in plastic cup) to 45°C up to 72 h and noticed neither heat stress signs nor mortality up to 12 h. In this study, transgenic 35S::MaDREB16 and wild-type Arabidopsis plants exhibited no mortality on exposure to 45°C up to 24 h and no thermo tolerance was observed in both genotypes when exposed to 45°C for 72 h (Fig. 6).

In this study, some plate assays in addition to pot assays, were carried out to examine the role of transgene in progression of seed germination as well as root growth elongation in mannitol (osmotic), heat and their combinatory stress conditions. Water movement into the seeds determines the seed germination rate. To circumvent low water potential under dehydration stress condition, plants try to maintain equilibrium through turgor regulation to secure continual growth of plant and existence (Verslues et al. 2006). Seeds of transgenic plants overexpressing MaDREB16 showed higher germination rate under all treatments than the wild-type. This suggests that MaDREB16 may provide abiotic stress tolerance during seed germination as well (Fig. 8). Previously, Luo et al. (2022) and Song et al. (2017) reported improved germination rate and root growth under mannitol (250 mM) and heat (45°C) in seeds of transgenic Arabidopsis after overexpression of ZmSNAC13 and AtCYS5 genes respectively than wild-type plants. In this study, 35S::MaDREB16 Arabidopsis plants, showed longer roots under mannitol and combined mannitol + heat treatment but no difference against heat treatment than wild-type, signifying that MaDREB16 could have an important role in root development under mannitol and combined mannitol + heat stress however not in individual heat stress.

Abiotic stress tolerance function of two homologous downstream genes i.e. AtRD29A and AtRD29B in Arabidopsis was proved earlier by Msanne et al. (2011). In this study we detected very low expression of AtRD29A (data not shown) but AtRD29B did show some expression (Fig. 9). However the transgene did not seem to induce higher expression under any of the stresses studied suggesting that the transgene does not confer tolerance through the expression of these two genes. Another downstream gene studied was the expression of AtHsfA3. Larkindale and Vierling (2008) had shown that induction of this gene is important in thermo tolerant plant. Wu et al. (2018) observed induced expression of this gene after exposure of plants to heat. The presence ofMaDREB16 in the transgenic Arabidopsis showed higher expression of AtHsfA3 under dehydration, heat and very high under the combined stress than wild-type plants. Though the expression of this gene was several folds higher under heat stress in wild-type, it was further accentuated in the transgenic. Thus it appears that MaDREB16 may confer resistance under high temperature through the increased expression of AtHsfA3.

Lim et al. (2007) in transgenic Arabidopsis lines overexpressing DREB2C gene, observed elevated expression of heat inducible downstream genes under temperature stress; AtTCH4 was amongst them that was further induced under mild temperature in these transgenic plants. In the present study, Arabidopsis overexpressing MaDREB16 did show expression of AtTCH4 under normal temperature and dehydration however under increased temperature, either alone or in combination with dehydration, its expression was, if anything, suppressed. The control lines did show enhanced expression of this gene under heat either individually or in combined dehydration plus heat. This suggests that MaDREB16 does not confer thermo-tolerance through the change in the expression of AtTCH4. The hormones/auxins regulate several processes in plants. There are auxin sensitive repressors that are significant in conferring stress tolerance in plants (Shani et al. 2017). The expression of one such gene AtIAA1 was studied to find out whether the MaDREB16 also affects IAA related genes. Interestingly the transgenic Arabidopsis plants showed very high expression of AtIAA1 under combined stress but negligible under dehydration and much higher as compared to the wild-type in heat stress. This suggests that MaDREB16 might be interfering with the induction of IAA associated genes resulting in conferring tolerance. This aspect needs further investigation to elucidate how this increased expression assists in tolerance to combined stress.

These studies associated with the expression of downstream marker genes suggest that overexpression of MaDREB16 may have enhanced their abiotic stress tolerance by regulating the expression of downstream targets in transgenic Arabidopsis plants. Zhao et al. (2013) had observed increased expression levels of downstream genes (RD29A, RD29B, LEA7 and HsfA3) under abiotic stress conditions in the transgenic plants overexpressing MsDREB2C gene as compared to wild-type Arabidopsis plants, resulting in improved tolerance in transgenic to abiotic stresses. Significant expression of downstream targets of DREB genes conferring drought, heat and combined drought + heat stress tolerance in transgenic Arabidopsis plants separately overexpressing MaDREB20 and MaDREB20.CA genes were reported by Chaudhari et al. (2023). Lack of appropriate procedure for developing transgenics in banana did not allow us to study the function of MaDREB 16 gene in banana. However the use of Arabidopsis has provided some leads as to how increased expression of MaDREB16 gene in tolerant banana genotype may be helping tolerance to stresses. From the existence of only dehydration sensing elements in the promoter of MaDREB16 it is clear that it could primarily confer tolerance in response to drought. This is also very evident from the present studies on transgenic Arabidopsis. It appears that MaDREB16 may be providing some tolerance to high temperature indirectly through its interference with such genes as IAA repressors.

Based on the physiological, biochemical and molecular analysis we suggest that significant tolerance in transgenic Arabidopsis overexpressing MaDREB16 against dehydration and combined dehydration plus heat is due to a combined effect of reduced water loss, reduced membrane damage and lower MDA levels and more proline content in addition to improved root growth and up-regulated transcripts of downstream stress-responsive genes. Presence of MaDREB16 appears to regulate the frequency and stomatal indices that provide the transgenic with regulatory control over stomatal function. From our results, we propose that those genotypes of banana that show higher expression of MaDREB16 under normal growing conditions are most likely to provide enhanced dehydration and combined dehydration plus heat stress tolerance. Thus these studies provide a new candidate gene for the selection of banana genotypes from the variants available either from natural population or generated through mutation or other breeding procedures including gene editing strategies.

ABM

aerial dry biomass

cDNA

complementary DNA

Grand Nain

germination rate

Hill banana

ion leakage

MDA

malondialdehyde

Murashige and Skoog

PCR

polymerase chain reaction

qRT-PCR

quantitative real-time PCR

RWC

relative water content

SAI

stomatal aperture index

stomatal density

stomatal index

rate of survival.

Declaration of competing interest

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

Author contributions

P.V.S. and B.K. designed the research. R.S.C. and B. L. J. carry out the experiments. R.S.C., B. L. J., B.K. and P.V.S. wrote the manuscript. All authors read and approved the manuscript.

Acknowledgements

We are deeply thankful to Padmashri Dr. B. H. Jain, founder architect of Jain Irrigation Systems Limited (JISL) for motivation and encouragement to initiate this project and for providing all necessary facilities. We are also thankful to the chairman and management of JISL for all financial support.

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Heterologous expression of banana MaDREB16 gene in Arabidopsis increases tolerance to dehydration and combined dehydration plus heat stress through stomatal regulation and root elongation

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Abstract

Figures

Introduction

Materials and methods

Plant material and growing conditions

RNA extraction and gene expression analysis

Cloning and transformation in Arabidopsis

Application of stress treatments to banana and Arabidopsis plants

Assessment of stress linked parameters of Arabidopsis plants

Measurement of stomatal density, stomatal index and stomatal aperture

Assay for seed germination and root growth elongation under mannitol, heat and combined mannitol + heat treatment

Statistical analysis

Results

Evaluation under dehydration stress

Assessment of stomatal density, stomatal index and stomatal aperture in Arabidopsis plants

Heat stress evaluation

Evaluation during prolonged heat treatment

Evaluation under combined dehydration plus heat stress

Seed germination and root growth elongation under mannitol, heat and combined mannitol + heat treatment

Expression of marker genes in transgenic plants under stress conditions

Discussion

Conclusion

Abbreviations

Declarations

Declaration of competing interest

Author contributions

Acknowledgements

References

Supplementary Files

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