Seeds of horsegram (Macrotyloma uniflorum (Lam) Verdc.) cultivar VZM1 and groundnut (Arachis hypogaea L.) cultivar K-6 were procured from Regional Agricultural Station, Rekulakunta and Kadiri, Anantapuram, respectively. Seeds were sown in earthen pots containing soil and farmyard manure in a 3:1 proportion maintained in the departmental botanical garden under natural photoperiod (10–12 h; 27 ± 4 °C). After 30 days post-sowing, drought stress was induced by withholding water to one set of pots, and respective fully watered controls were maintained in another set of pots. Ten days after stress imposition, fully opened fresh leaf samples were collected, pooled, flash frozen in liquid nitrogen, for futher analysis.

Total RNA was isolated from stress-adapted horse gram leaves subjected to drought stress using the Trizol reagent (Invitrogen). The leaf material (100mg) from drought stressed horsegram plants was ground to amorphous powder using liquid nitrogen and added with 1ml of Trizol reagent containing guanidium thiocyanate (Simms et al., 1993). The supernatant was separated after centrifugation and nucleic acids portion was aspired using chloroform. The RNA was precipitated with isopropanol and sodium citrate/NaCl (1:1) solution. The RNA precipitate washed with ethanol, dried and dissolved in sterile diethylpyrocarbonate (DEPC) water and the same was used as a template for cDNA synthesis using the RevertAid reverse transcriptase enzyme (Thermo Scientific, USA).

Individual gene-specific primers were used to isolate individual genes. The PCR setup and annealing temperatures were optimized for all three genes MuMYB96, MuWRKY3, and MuNAC4, individually to get the specific gene amplification in the gradient thermal cycler (Eppendorf, Hamburg). PCR was initiated by a hot start at 94 ˚C for 5 min followed by 30 cycles of 94 ˚C for 1 min, 59.1 ˚C (MuMYB96), 58.2 ˚C (MuWRKY3) and 53.1 ˚C (MuNAC4) for 45 s and 72 ˚C for 1 min with a final extension of 10 min. and the list of primer sets were given in Supplementary Table 1 . The amplification was checked on 0.8% agarose gel. The authenticity of the PCR product was checked by restriction enzyme digestion, confirmed and cloned into a T/A vector (Thermo Scientific, USA) and sequenced.

Each gene was cloned under the specific promoter by conventional restriction digestion and ligation strategy to develop gene expression cassettes. MuMYB96 gene was cloned under rbcs promoter and terminator in the Impact vector (IM1.1) (P_rbcs: MuMYB96:T_rbcs), MuWRKY3 gene was cloned into the pRT100 vector under CaMV2x35S promoter and a polyA tail terminator (P_CaMV2x35S: MuWRKY3:T_polyA) and MuNAC4 gene expression cassette were developed using a pB4NU plasmid vector carrying ubiquitin promoter and nos terminator (P_Ubi: MuNAC4:T_nos) using specific restriction enzymes. Then, these three gene cassettes were sub-cloned into modified gateway entry vectors. The MuMYB96 gene cassette was released by digesting with HindIII and PacI and was ligated to the linearized pGATE L1-L4 entry vector, and the MuWRKY3 gene construct was sub-cloned into pGATE R4-R3 using SphI enzyme. Finally, the MuNAC4 expression cassette was excised from the pB4NU vector and introduced into the pGATE L3-L2 vector between EcoRI and HindIII restriction sites to prepare the entry clones.

The three genes were stacked together in a plant expression binary vector by recombination reaction. Three gateway entry vectors, pGATEL1L4-P_rbcs : MuMYB96:T_rbcs , pGATE R4R3-P_CaMV2x35S:MuWRKY3:T_polyA, and pGATEL3L2-P_Ubi : MuNAC4:T_nos were allowed to a recombination reaction with destination vector, pKM12GW containing a neomycin phosphotransferase (nptII) gene as plant selectable marker in 1:1:1:3 ratio respectively. The reaction was carried out at 25°C overnight in the presence of LR clonase enzyme, and proteinase K was used to terminate the reaction (Vemanna et al., 2013). The resulting recombinant vector was used to transform Agrobacterium tumefaceins.

The binary vector expressing pKM12GW-MuMYB96:MuWRKY3:MuNAC4 was transferred into Agrobacterium tumefaciens EHA105 strain by Freeze-thaw method, and colony PCR was carried out to identify positive transformants (Weigel and Glazabrook, 2006). A tissue culture-independent agrobacterium mediated in planta transformation protocol (Rohini and Rao, 2001) was adopted to develop the groundnut transgenics. Two-day-old germinating groundnut (cultivar variety K6) sprouts were pricked at the embryonic site and co-cultivated with agrobacterium culture for 16 hours at 28°C with gentle agitation, followed by rinsing with cefotaxime (500μg/ml) for 2 minutes and later with sterile distilled water. The seedlings were acclimatized in a plant growth chamber on a sterilized soilrite at controlled conditions (28 ± 2°C; 16 hours of light/day; light intensity-400-550 μE/m²/s) RH- 60%). Another set of seeds were transformed with Agrobacterium cells carrying pKM12GW vector without transgenes and were treated as mock plants. After acclimatization, plantlets were transferred to a greenhouse, maintained at 28°C, and allowed to grow under natural photoperiodic conditions till harvest. Putative transgenic groundnut plants were identified by kanamycin screening and PCR analysis using npt-II primers. The seeds from the T₀ generation were germinated on MS agar medium (Himedia, Mumbai, India) supplemented with 200mg/L of kanamycin under controlled environment chambers (Conviron A1000, Canada). The seedlings showing normal shoot and root growth were transferred to sterile soilrite for acclimatization, then transferred to the earthen pots and maintained in the greenhouse. Integration of all three genes in genomic DNA was confirmed by PCR using gene-specific primers. Transgenic plants showing the integration of three genes were considered positive transgenic plants and advanced to the subsequent generations.

The expression of transgenes, MuMYB96, MuWRKY3, and MuNAC4 was analyzed in putative T₃ transgenic groundnut plants and wild type, subjected to drought stress for 10 days. Fully opened leaf samples were used for analysis. Total RNA isolated from leaf samples was treated with a Turbo DNase treatment kit (Thermo Fisher Scientific, USA) as per the manufacturer’s protocol to remove any DNA traces. cDNA was synthesized using Revert Aid M-MuLV Reverse Transcriptase (Thermo Fisher Scientific) as per the manufacturer’s instructions. qRT-PCR mix was comprised of 1× using Power SYBR Green Master Mix (Ambion, USA), 20 ng of cDNA, and 0.2 µM of forward and reverse primers. The housekeeping gene, actin, was used as an internal control in the reaction. The RT-PCR analysis was done on Applied Biosystems Step One Real-Time PCR machine with standard cycling comprising 95°C for 30 s, 40 cycles of 95°C for 1 s, 60°C for 20 s, and a melt curve analysis. Relative quantification was studied using 2−^{Δ Δ} CT method (Livak and Schmittgen, 2001).

In addition to transgenes, a few downstream genes such as KCS6, KCR1, APX3, CSD, LBD16 and DBP were also analyzed using qRT-PCR. The gene sequences of the selected down-stream genes were obtained from genome of Arachis hypogaea (http://peanutbase.org/home) and the same sequences were used to design primers using Primer Express™ Software v3.0.1. Each gene was analyzed in three biological samples, and three reaction replicates were performed for each biological sample. The primers used for PCR analysis were given in Supplementary Table 2 .

Scanning Electron Microscopy (SEM) was employed to investigate the cuticular wax depositions and stomatal structure on the leaf surface of transgenic, mock, and wild type groundnut plants subjected to drought stress. First, the freshly harvested leaf bits of 1cm² were vacuum dried and mounted onto aluminum stubs, followed by gold nanoparticle coating with a fully automated vacuum sputter smart coater (DII-29030SCTR, JOEL, USA). Then, leaf-mounted stubs were transferred to the scanning electron microscope (JOEL JSM-IT500, Japan) to visualize the extent of epicuticular wax depositions on the leaf surface (Lokesh et al., 2019).

Physiological and biochemical parameters related to cellular level tolerance and WUE were carried out under drought stress in putative transgenic groundnut lines along with mock and wild type plants. The drought stress was imposed on thirty-day-old plants by withholding irrigation for ten days and fully opened leaf samples were collected uniformly from each set of plants. Three biological samples, and three reaction replicates were performed for each physiological and biochemical assay.

Relative water content (RWC) was measured in multigene transgenic plants along with wild type plants under drought stress conditions. First, leaf discs were prepared from matured leaves, and fresh weight was measured. Then the leaf discs were immersed in sterile water for four hours, and the weight was recorded as turgid weight; then, the leaf discs were dried in a hot air oven for 48 h, and the dry weight was determined. Finally, RWC was calculated using the formula (Vemanna et al., 2017).

Chlorophyll pigments were extracted from drought-stressed leaves of multigene transgenics and wild type plants by boiling them in dimethyl sulfoxide (DMSO) at 65°C for 10 min. The extracted chlorophylls were read at 645nm and 663nm using a spectrophotometer (Shimadzu UV 1800, Japan). The total chlorophyll content was estimated according to Hiscox and Israelstam (1979) and expressed as mg/g F.W (Vennapusa et al., 2022).

Epicuticular waxes on the leaf surface were separated and quantified according to the method given by Mamrutha et al. (2010). The waxes were extracted with chloroform from the leaf surface and treated with acidic-potassium dichromate (K₂Cr₂O₇) to give a coloured compound. The ECW content was calculated using a colorimetric method and expressed as µg/g F.W.

Total soluble sugar content was determined following Kiranmai et al. (2018). Water extract of leaf was treated with 5% phenol and 98% sulphuric acid and incubated at room temperature for 1 hr, and the absorbance was measured at 485nm. The TSS content was expressed as µg/g F.W.

Accumulation of proline content in the leaf samples was determined as described by Bates et al. (1973). Leaf extract was prepared in 3% sulphosalicylic acid, heated, treated with acid ninhydrin and acetic acid, and incubated at 100°C for 1hr. The reaction was terminated on ice, and the chromophore was extracted with 4 mL toluene and mixed thoroughly. The toluene phase was separated and measured with a spectrophotometer 540nm using toluene as blank, and the proline content was calculated from the standard curve and expressed as µmol/g F.W (Vemanna et al., 2017).

The extent of cell membrane damage was calculated indirectly by measuring the malondialdehyde content, a product of lipid peroxidation of membrane lipids. The leaf material from drought stressed transgenic, mock and wild type plants was used to estimate thiobarbituric acid, a reactive compound of malondialdehyde, and calculated against a standard MDA graph (Nisarga et al., 2017).

Superoxide ion and hydrogen peroxide contents were quantified in the transgenic groundnut plants, wild type and mock plants exposed to drought stress. Superoxide ions were estimated by a colorimetric method according to Kiranmai et al. (2018) by treating with nitroblue tetrazolium (NBT) solution, and hydrogen peroxide content was measured as described by Junglee et al. (2014) and Nareshkumar et al. (2015).

The efficacy of the anti-oxidant defense system was analyzed by measuring the activity of superoxide dismutase (SOD) (Kiranmai et al., 2018) and ascorbate peroxidase (APX) (Vemanna et al., 2017) in the leaves of multigene transgenic and wild type plants using a colorimetric method.

After harvesting, morphological and yield traits such as shoot length, root length, shoot dry weight, root dry weight, number of pegs, number of pods, and dry weight of pods were measured for transgenic plants along with the wild type and mock plants (Vemanna et al., 2017).

Briefly, multigene transgenic groundnut lines, wild type and mock plants were gently uprooted from the pots. Roots were cleaned using tap water to remove debris and soil particles properly and maximum care was taken to avoid the loss of roots. Number of pegs and pods were recorded. Shoot and root parts were separated and their length was recorded using an ordinary ruler. Then the shoot, root parts and pods were dried at 50°C for 48 hours in a hot air oven and dry weight was recorded using digital scale. Data was recoded in three biological sets with triplicates and the results were shown mean-values per plant.

All the physiological and biochemical experiments were conducted in three biologically independent experiments, statistical analyses were performed using R version 4.2.0, and ANOVA was performed using the R package agricolae with Fisher’s LSD test to separate means and significance at P ≤ 0.05 (de Mendiburu, 2014; Pandian et al., 2020). Data presented are mean values and standard error ( ± SE).

The three TF genes, MuMYB96, MuWRKY3, and MuNAC4 were amplified from cDNA synthesized from horse gram leaf RNA samples ( Supplementary Figure 1 ) and sequence ( Supplementary Table 3 ). All the three genes, were sub cloned to pRT100 vectors under CaMV35S promoter and polyA terminator at Apa1 and Nco1, Kpn1 and Nco1, and Kpn1 and BamH1 sites, respectively ( Supplementary Figure 1A ). The individual gene cassettes, P_rbcs: MuMYB96:T_rbcs P_CaMV2x35S:MuWRKY3:T_polyA and P_Ubi : MuNAC4:T_nos were stacked into a single multigene construct in a plant binary vector, pKM12GW through the LR clonase reaction using a modified multisite gateway cloning technology (Vemanna et al., 2013). The plant destination vector carrying all the three genes, pKM-MuMYB96:MuWRKY3:MuNAC4 in Agrobacterium ( Figures 1A, B ), was transferred to groundnut seedlings. Putative transgenic groundnut lines were selected on a kanamycin medium. The putative multigene transgenic groundnut plants showed growth on the kanamycin selection medium, whereas the wild type plants failed to germinate or showed stunted growth. Transgenic plants that showed normal growth were acclimatized in the greenhouse, maintained till harvest, and/or advanced to the next generation ( Figure 2 ). Integration of all three transgenes was confirmed in putative transgenic plants by PCR analysis using genomic DNA as a template ( Supplementary Figure 2 ). The multigene transgenic groundnut plants showing kanamycin resistance and gene integration were advanced to the next generation and the transgenic events were shown in Supplementary Figure 3 .

Quantitative real-time expression analysis (qRT-PCR) of transgenes was carried out in transgenic groundnut lines along with wild type plants in the T₃ generation. The transgenic groundnut plants showed enhanced transcript levels compared to wild type under drought stress conditions. For example, MuMYB96 showed a 2.88 to 4.38-fold increase in transcript abundance in multigene transgenic plants over wild type plants, whereas MuWRKY3 transcript levels showed a 3.50 to 3.618-fold increase and 3.15 to 3.46-fold increase in transcript levels of MuNAC4 gene. The overexpression of transcription factors resulted a significant increase in transcript level of downstream genes such as KCS6, and KCR1, APX3 and CSD1 LBD16 and DBP in transgenic plants over wild type plants under drought stress conditions ( Figure 3 ).

In T₃ generation, 30-days-old multigene transgenic groundnut plants, wild type and mock plants were subjected to drought stress by withholding the water for 10-days. Drought stress resulted visible leaf wilting in both multigene transgenic plants, wild type and mock plants under drought stress, however, the symptoms appeared much earlier in wild type and mock plants, with significant phenotypic difference under drought stress. The transgenic plants showed mild wilting symptoms and remained green after ten days of drought stress imposition whereas wild type and mock plants showed severe visible wilting symptoms ( Figure 4A ).

Further, morphological parameters and yield related data was recorded for transgenic lines, wild type and mock plants after harvest and the multigene transgenic groundnut plants showed better growth and increased root length, more number of pegs and pods compared to wild type and mock plants ( Figure 4B ). In general, multigene transgenic lines, wild and mock plants showed significant difference in their growth. Multigene transgenic plants exhibited superior growth than the wild type and mock plants. Growth of lateral roots and overall root length of multigene transgenic groundnut lines significantly increased compared to wild type and mock plants. Consequently, pronounced increase in the root dry weights observed. Total number of pods was more in multigene transgenic lines compared to wild type and mock plants ( Table 1 ).

Variation in the epicuticular wax accumulation was observed between the multigene transgenic lines and wild type and mock plants. The transgenic plants showed a significant increase in the deposition of cuticular wax crystals over the wild type and mock samples. Leaf sample of TL2-2-2 transgenic line showed condensed cuticular crystals resulting in plaque-like deposits ( Figure 5 ). The surface of the transgenic leaves (TL19-1-3, TL 40-2-4 and TL 41-1-3) exhibited dense wax crystals accumulation, whereas the mock and wild type plants have sparse wax accumulation In addition to the wax deposition, we observed variations in the stomatal number between multigene transgenic lines and wild type and mock plants. Wild type plants showed more number of stomata, whereas the transgenic plants showed less number of stomata ( Figure 6 ).

The relative water content was significantly less in wild type compared to multigene transgenic lines under drought stress. The transgenic groundnut plants showed a range of 40.27 to 66.89% of relative water content. In contrast, wild type and mock plants showed 32.39% and 34.88%, respectively, demonstrating superior water retention capacity of transgenic plants than wild type plants under water stress conditions ( Figure 7A ). Stress effect was more pronounced in wild type as evidenced by reduction in total chlorophyll content. The transgenic groundnut plants showed significantly higher chlorophyll content (0.26 to 0.42mg/g. FW) compared to wild-type (0.18mg/g. FW) and mock plants (0.218 mg/g. FW) ( Figure 7B ).

The multigene transgenic groundnut plants showed significantly higher epicuticular wax content ranging from 12.42 to 16.51µg/g F.W under drought stress conditions compared to wild type and mock plants (6.54 and 7.08µg/g F.W respectively), which is 2 to 2.5 folds lower than that of the transgenic groundnut plants ( Figure 7C ).

The transgenic groundnut plants showed significantly higher levels of total soluble sugars ranging from 760-997µg/g F.W under stress conditions. In comparison with transgenic plants, wild type and mock plants showed relatively lower levels of TSS, ranging 321.71 and 409.49µg/g F.W respectively ( Figure 8A ). The transgenic lines showed lower proline content ranging from 72-131µg/g F.W than wild type and mock plants which showed 184 and 179µg/g F.W, respectively ( Figure 8B ). There was significant decrease in proline content in multigene transgenic lines compared to wild type and mock plants under drought stress. The lower proline content could be due to better RWC and maintenance of high turgor potential which perhaps not sufficient enough to induce high proline content in multigene transgenic groundnut plants than the wild type and mock plants. Malondialdehyde, the end product of membrane lipid peroxidation, was quantified to assess the extent of oxidative damage caused by imposed drought stress. The multigene transgenic groundnut plants showed significantly lower levels of MDA (310.68-432.28nmol/g F.W) content than the wild type (627.85nmol/g F.W) and mock plants (491.57nmol/g F.W) ( Figure 8C ).

The wild type and mock plants showed a significant increase in superoxide production under drought stress conditions compared to transgenic plants. A two to four fold decrease in superoxide production was observed in multigene transgenic groundnut plants compared to wild type and mock plants ( Figure 9A ). Similarly, H₂O₂ production significantly increased in wild type and mock plants with 4.14μmol/g. F.W and 3.41μmol/g F.W of H₂O₂, respectively. The multigene transgenic plants showed a 2-3 fold lower levels of H₂O₂ production ( Figure 9B ).

The ROS was counter-attacked by antioxidative defense enzymes, such as SOD, and APX were measured in multigene transgenic groundnut plants, wild type, and mock plants under drought stress conditions. Results indicated significantly higher levels of SOD activity with a 2 to 2.2-fold increase in transgenic groundnut plants compared to wild type and mock plants ( Figure 9C ). In addition, the multigene transgenic plants exhibited APX activity with a range of 0.63-1.04µmol/mg protein/min, which is 3-5 folds higher than that of the wild type (0.18µmol/mg protein/min) and mock plants (0.24µmol/mg protein/min) ( Figure 9D ).