Overexpression of AtDREB1 and BcZAT12 Genes Confers Drought Tolerance by Reducing Oxidative Stress in Double Transgenic Tomato (Solanum Lycopersicum Mill.)

Ram Krishna Banaras Hindu University Institute of Environment &amp; Sustainable Development Waquar Akhter Ansari IIVR: Indian Institute of Vegetable Research Durgesh Kumar Jaiswal Banaras Hindu University Institute of Environment &amp; Sustainable Development Ram Prasad Mahatma Gandhi Central University JAY PRAKASH VERMA (  verma_bhu@yahoo.co.in ) Banaras Hindu University Institute of Environment &amp; Sustainable Development https://orcid.org/0000-0002-2643-9623 Major Singh Directorate of Onion and Garlic Research


Introduction
In agriculture system plants generally face water scarcity due to different types of stresses which include drought, cold, and salinity. The water de cit severely affects growth, development, yield and crops quality depends on the duration, degree of water de cit, and crop stage Karkute et al. 2019;Krishna et al. 2021). Throughout the world about 20% of agricultural land is under irrigation; of which 2. Method And Materials

Plant material and drought stress
In the present study, Kashi Vishesh (H86), developed at Indian Institute of Vegetable Research, Varanasi, India using Solanum hirsutum f. glabratum B '6013' as donor parent following backcross pedigree selection method, used for the experiments as performs very well in the Indian sub-continent and show moderate eld resistance to tomato leaf curl virus. The research materials was consisted of a total eight (H86-cv. Kashi Vishesh, non-transgenic (NT); D86-AtDREB1A/CBF3 (Accession no. AF07460) and ZT1-BcZAT12 (Accession no. DQ166621.1) single transgenic (ST) lines and ve (DZ1, DZ2, DZ3, DZ4, and DZ5) double transgenic (DT) lines expressing, AtDREB1A::BcZAT12 genes developed by crossing D86 and ZT1 transgenic lines were taken for the drought stress study. The 25 days old seedlings were transplanted in 7 kg earthen pots having 5:1 ratios of soil and manure which were kept in the transgenic glass house in a completely randomized design (CRD) at ICAR-Indian Institute of Vegetable Research, Varanasi. Transgenic glass houses were equipped with an air cooling system to maintain the standard growth condition of day/night temperature of 25°C/15°C and 50% relative humidity; 16 h/8 h of photoperiod with a light intensity of 350 mmol m − 2 s − 1 ux. For drought stress treatments different days of water de cit (DWD) imposed on 50 d old tomato plants (from seed germination), during late vegetative stage, by withholding 7, 14, or 21 d irrigation. The regularly irrigated plants were kept as control (0 d), which contains up to 80% moisture of total eld capacity while 7, 14, 21 DWD exposed plants pot have nearly 40, 25 and 15% soil moisture contents of eld capacity. After 0, 7, 14 and 21 DWD treatment, from the top fully expanded fourth leaf was collected and stored at -80°C till the completion of different analysis. The experiment was replicated thrice with three tomato plants in each replication. For various biochemical analyses, leaf samples were collected and pooled from three replications of each tomato lines/ events/ variety of each replicate.

Phyto-pigments (chlorophyll and carotenoid)
The leaf samples (200 mg) were homogenized in 80% acetone to extract phyto-pigments like chlorophyll and carotenoids as described by Porra et al. (1989). From the extract, the supernatant was collected and subsequently measured the absorbance at 663, 645 and 480, 510 nm for chlorophyll and carotenoid respectively, with SmartSpec 3000 UV/Visible Spectrophotometer (Bio-Rad, Hercules, California, USA). Arnon (1949) equation was used to compute the concentration of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids. Results were expressed in milligrams per gram of fresh weight of the sample.

Superoxide radical (O ·− 2 ) measurement
The superoxide anion (O ·− 2 ) generation rate was quanti ed by following the procedure of Shah et al. (2001). The assay reaction was set with 3 ml nal volume of 100 mM sodium phosphate buffer (pH 7.2) containing 1 mM sodium diethyl dithiocarbamate and 0.25 mM nitro blue tetrazolium (NBT). Approximately 200 mg of fresh leaf sample was powdered with liquid nitrogen in sodium phosphate buffer (100 mM; pH 7.2) containing sodium diethyl dithiocarbamate (1 mM) to prevent the activity of SOD and 0.25 mM NBT to detect O ·− 2 . Further centrifuged at 22,000 x g for 20 min and the supernatant was collected subsequently. In the presence of O ·− 2 , NBT gets oxidized to form a dark blue insoluble precipitate. Absorbance was recorded at 540 nm using a SmartSpec 3000 UV/Visible Spectrophotometer (Bio-Rad, Hercules, California, USA) and O ·− 2 generation was expressed as ∆A 540 min − 1 mg − 1 protein.

Ascorbate peroxidase (APX) activity assay
According to the procedure of Nakano and Asada (1981) ascorbate peroxidase (APX; EC: 1.11.1.11) activity was estimated from 200 mg of fresh leaf sample homogenized in 5 ml of 50 mM potassium phosphate buffer (pH 7.8) having 1 mM EDTA, 1 mM ascorbic acid, 1% PVP; and 1 mM phenylmethylsulfonyl uoride in a pre-chilled mortar and pestle at 4°C. The extract was centrifuged at of 100 mM Tris-HCl buffer (pH 7.8), 25 mM sodium phosphate buffer (pH 7.0), and 100 mM HEPES-HCl buffer (pH 7.6) respectively for GR, DHAR, and MDHAR at 4°C in a pre-chilled mortar and pestle. The enzymatic extract was centrifuged at 22,000 × g for 20 min on 4°C and the supernatant was collected for the estimation of enzymatic activity. For the GR, a reaction mixture of 2 ml was prepared with Tris-HCl buffer (100 mM, pH 7.8), oxidized glutathione (0.5 mM, GSSG), MgCl 2 (3 mM), NADPH (0.2 mM), and 200 µl of enzyme extract at 25°C. By adding NADPH to the mixture at room temperature, the reaction was initiated and the reduction in the absorbance of NADPH for 5 min was monitored at 340 nm with an extinction coe cient of 6.2 mM − 1 cm − 1 using SmartSpec 3000 UV/Visible Spectrophotometer (Bio-Rad, Hercules, California, USA), the GSSG absence ameliorated NADPH oxidation. The speci c activity of the enzyme was expressed as µmol of NADPH oxidized mg − 1 (protein) min − 1 . For DHAR, a 2 ml enzyme assay reaction mixture consisting of 200 µl enzyme extract, 25 mM sodium phosphate buffer (pH 7.0), 2.5 mM GSH, and 0.4 mM DHA. The enzymatic reaction was initiated by adding DHA at ambient temperature. The increase in absorbance due to ascorbate was monitored at 265 nm for 3 min with an extinction coe cient of 14 mM − 1 cm − 1 using SmartSpec 3000 UV/Visible Spectrophotometer (Bio-Rad, Hercules, California, USA) and as µmol of NADPH oxidized mg − 1 (protein) min − 1 , the enzyme-speci c activity was expressed. For non-enzymatic reduction in DHA by GSH, the reaction rate was corrected. The MDHAR activity measured by a reaction mixture of 2 ml was prepared from 200 µl of enzymatic supernatant with 0.15 mM NADP, 2.5 mM AsA and 100 mM HEPES-HCl buffer (pH 7.6). The NADPH oxidation rate detected as an increase in absorbance up to 3 min at 340 nm (extinction coe cient of 6.2 mM − 1 cm − 1 ) with SmartSpec 3000 UV/Visible Spectrophotometer (Bio Rad, Hercules, California, USA) and was expressed as µmol of ascorbate oxidized mg − 1 (protein) min − 1 .

Superoxide dismutase (SOD) activity assay
According to the procedure as suggested by Nahakpam and Shah (2011) superoxide dismutase (SOD, EC 1.15.1.1) activity was determined from 200 mg of fresh leaf tissue samples crushed in a pre-chilled mortar and pestle with 5 ml potassium phosphate buffer (100 mM; pH 7.8), containing EDTA (0.1 mM), Triton X-100 (0.1% v/v), and polyvinyl pyrrolidone (PVP) (2% w/v). The enzymatic extract was then centrifuged in 4°C for 15 min at 22000 × g; and the resultant supernatant was dialyzed with the help of cellophane membrane tube against cold extraction buffer, sodium carbonate-bicarbonate buffer (50 mM; pH 9.8) for 4 hours. For the enzyme assay a 3 ml reaction mixture was prepared using 100 µl of enzyme extract and EDTA (0.1 mM), to which epinephrine was added nally. The formation of adrenochrome during next 5 min was monitored at 470 nm with a SmartSpec 3000 UV/Visible Spectrophotometer (Bio-Rad, Hercules, California, USA). The amount of enzyme required to stimulate 50 percent epinephrine oxidation under experimental conditions are described as one unit of SOD activity.

Guaiacol peroxidase (POD) activity assay
Guaiacol peroxidase (EC 1.11.1.7) activity was estimated according to the method suggested by Shah et al. (2001). The fresh leaf sample (200 mg) was crushed with 5 ml sodium phosphate buffer (60 mM; pH 7.0) at 4°C using a pre-chilled mortar and pestle. The supernatant collected after centrifuging the homogenates at 22,000 × g for 15 min, and used for enzymatic preparation. A reaction mixture of 2 ml set up consisting of 40 mM sodium phosphate buffer (pH 6.0), 9 mM guaiacol, 2 mM H 2 O 2 , and 50 µl enzyme extract. The absorbance of the reaction mixture was recorded at 470 nm using SmartSpec 3000 UV/Visible Spectrophotometer (Bio Rad, Hercules, California, USA) with 26.6 mM − 1 cm − 1 extinction coe cient for 5 min and expressed as µmol of H 2 O 2 reduced mg − 1 (protein) min − 1 .
2.9. Glutathione assay Owens and Belcher (1965) described procedure was used for glutathione assay; in which a fresh leaf (200 mg) sample was powdered in mortar and pestle with liquid nitrogen and mixed with 5 ml of 5% (w/v) meta-phosphoric acid at 4°C. The extract was centrifuged at 22,000 × g for 20 min at 4°C and the supernatant collected for assaying both ascorbate and glutathione. Total glutathione and reduced glutathione (GSH) were assayed following the procedure. The reduced glutathione was oxidized by 5,5dithio-bisnitrobenzoicacid (DTNB), and the reaction between GR and NADPH, the oxidized glutathione (GSSG) was reduced to GSH. A reaction mixture of 2 ml prepared by using 100 mM potassium phosphate buffer (pH 8.0), consisting of 50 µl of the extract, 30 µl of 4% (w/v) DTNB, and 1 mM EDTA. The reaction mixtures were incubated at room temperature for 4 min then the absorbance was recorded at 412 nm using SmartSpec 3000 UV/Visible Spectrophotometer (Bio Rad, Hercules, California, USA). Total glutathione was also assayed by adding 0.25 mM NADPH and 0.5 units of glutathione reductase to the same reaction mixture. The concentration of GSH and total glutathione was measured with a standard reference curve of 1-5 µg.
2.10. Ascorbate (AsA) assay Ascorbate (AsA) DHA, total AsA, and reduced AsA were estimated according to Law et al. (1983) procedure. Total AsA was estimated using Dithiothreitol (DTT) by reducing DHA to AsA. Fresh leaf tissue (200 mg) the sample was crushed in 5% of metaphosphoric acid consisting of 100µl of 10 mM DTT and 500 µl of 150 mM sodium phosphate buffer (pH 7.5). The reaction mixture was incubated for 10 min at room temperature in dark, after incubation 100 µl of 0.5% (w/v) N-ethylmaleimide was added along with 200 µl FeCl 3 (3% w/v), 400 µl 2, 20-bipyridyl (4% w/v, in 70% v/v ethanol) and 400 µl orthophosphoric acid (44% v/v) followed by incubation for 30 min at 40°C in dark. The absorbance was recorded at 525 nm with SmartSpec 3000 UV/Visible Spectrophotometer (Bio Rad, Hercules, California, USA), the total AsA content was analyzed using a standard AsA reference curve. With the substitution of 100 µl of DTT with 100 µl of distilled water, the reduced AsA was measured in the similar method as described in the earlier procedure. The DHA was analyzed as a difference between the reduced AsA and total AsA and expressed as mg g − 1 FW.

Total fruit number and fruit weight
The total number of fruit per plant counted and fruit yield per plant recorded by weighing total fruit and expressed in gram per plant.

Gene expression analysis
Antioxidative enzymes gene namely APX, CAT, DHAR, GR, MHDAR, POD, and SOD were selected and their sequences were retrieved from the NCBI database (www.ncbi.nlm.nih.gov) to study their expression analysis. qRT-PCR primer designing Primer3 v. 0.9 software (Rozen and Skaletsky 1998) was used (Table 1). RNA was isolated from top young leaves by utilizing TRI reagent (Ambion, CA, USA) according to manufacturer protocol. cDNA strand was synthesized using 1 µg RNA in a 20 µl of reaction volume using Bio-Rad cDNA synthesis kit (Bio-Rad, Hercules, USA), and for RT-qPCR IQ SYBR Green Supermix (Bio-Rad) was utilized, the reaction was set in thermal cycler (iQ5, Bio-Rad), actin gene was used as an internal control. The sequences of actin and other gene-speci c primer are tabulated in Table 1. For PCR reaction 5 µl of cDNA was taken and PCR was programmed as initial denaturation at 95°C for 1 min, then at 95°C for 45 s denaturation, at 56-60°C for 45 s annealing, 72°C for 45 s extension, repeated for 36 cycles and a nal extension of 72°C for 5 min. The relative gene expression was detected by using 2 −ΔΔCT method (Livak and Schmittgen 2001).

Superoxide radical (O ·− 2 )
Superoxide anions increase with increase in DWD in NT, ST and DT plants, after 7 DWD the higher fold change noted in H86 while it was lowest in DZ5. While after 14 and 21 DWD maximum increases recorded in H86, while it was minimum in the case of DZ2 (Table 4).  (Fig. 1c). The activity of SOD enhanced with each water de cit treatment viz; 7, 14 and 21 DWD in each except in H86 and D86 in which after 21 DWD it gets reduced. Highest SOD activity after 7 DWD noted in DZ5 while it was lowest in H86. After 14 DWD signi cant increase in SOD compared to control noted in ZT1 (3.02 fold), while it was signi cantly lower in H86 (2.05 fold). Similarly after 21 DWD highest increase in SOD activity compared to control noted in ZT1 (3.22 fold) however it was decreased in H86 (0.889 fold) (Fig. 1d) (1.39 fold compared to control), while it was minimum in H86 (0.64 fold compared to control) (Fig. 2a). Continuous enhancement in MDHAR recorded with rise in DWD, with maximum value after 21 DWD.

Guaicol peroxidase activity
Guaicol peroxidase activity was highest in DZ5 and minimum in H86 under controlled conditions. It gets reduced in H86 and D86, while increases in ZT1, DZ1, DZ2, DZ3, DZ4 and DZ5 after 7 DWD. Again it increases in NT, ST, and DT after 14 DWD, the increase compared to control was highest in DZ3 (2.07 fold) whereas lowest for H86 (1.41fold). After 21 DWD it reduces in all, the maximum reduction compared to control was recorded in H86 (1.16) and minimum in DZ2 (1.98) (Fig. 2c).

Ascorbate
Under control condition the value of dehydroascorbate was highest in DZ2 (0.693 mg g − 1 DW) while it was lowest in H86 (0.277 mg g − 1 DW). After 7 DWD the dehydroascorbate increases however again decreases after 14 DWD and the values were lower than the values under 7 DWD, however, still, it was higher than the value under control conditions. At the end of maximum 21 DWD, the value in fold change was highest in DZ4 (3.40) and lowest in H86 (1.20) as compared to control (  (Table 5). Compared to control total ascorbate increases highest by 3.47 fold in DZ2 after 7 DWD. However it gets reduces after 14 DWD, and the highest fold value as compared to control was 3.56 (Table 6). After maximum DWD of 21 days, results in increase of total ascorbate compared to control, with a maximum increase of 4.29 fold (DZ4) and a minimum of 2.19 fold (H86).

Glutathione
Under control condition highest glutathione recorded in DZ5 (0.666 µg g-1 FW), however, it was lowest in case of H86 (0.312 µg g-1 FW). After 7 DWD it increases maximum by 1.47 fold in DZ3 compared to value under control condition, similarly after 14 DWD maximum 2.02 fold increase compared to control recorded in DZ2 and after 21 DWD highest 1.49 fold increase compared to control recorded for ZT1 (Table 7). The total GSH increases with an increase in DWD in NT, ST and DT, except in H86 after 21 DWD which reduces slightly compared to the value after 14 DWD. As in glutathione, the highest GSG recorded in DZ5 under control, 7, 14, and 21 DWD condition with respective values of 0.857 µg g-1 FW, 1.52 µg g-1 FW, 2.05 µg g-1 FW, and 2.57 µg g-1 FW, however it was lowest in H86 under the similar condition with respective values 0.40 µg g-1 FW, 0.542 µg g-1 FW, 0.884 µg g-1 FW and 0.855 µg g-1 FW (Table 7). Total glutathione increases after 7, 14 and 21 DWD in NT, ST and DT compared to control, maximum total glutathione recorded in DZ5 under control, and under all the drought treatments viz; 7, 14 and 21 DWD, with respective values 1.60 µg g-1 FW, 2.62 µg g-1 FW, 4.07 µg g-1 FW and 5.21 µg g-1 FW, while it was least in H86 under the similar conditions with respective values 0.67 µg g-1 FW, 0.85 µg g-1 FW, 1.11 µg g-1 FW, and 1.49 µg g-1 FW (Table 8).   The results are mean ± SE of triplicate measurements. Means followed by the same letter along same row are not signi cantly different (P ≤ 0.05), according to Duncan's multiple range test.

Relative gene expression
Relative expression of seven different antioxidant enzyme genes was measured under control, 7, 14 and 21 DWD through RT-PCR. Respective increase in APX gene expression measured after 7, 14, and 21 DWD, and it was observed that a continuous increase takes place in, NT, ST, and DT plants; however, it was higher in DT lines. After 7 DWD it was maximum for DZ5 with 1.98 fold increase compared to control plants (Fig. 3a). Further, CAT expression was measured under the similar condition and it was observed that the CAT expression increases maximum by 1.68, 1.92 and 2.38 as compared to control under 7, 14, and 21 DWD respectively for DZ5, DZ2, and DZ5, however, it was minimum for H86 under similar condition (Fig. 3b). GR expression increases maximum by 2.22 fold in DZ1 under 7 DWD than control plants. However, under 14 DWD it followed the trend DZ1 > DZ5 > DZ3 > ZT1 > DZ2 > D86 > DZ4. GR expression was highest after 21 DWD as compared control plants, with a maximum fold increase of 3.45 fold in DZ5 (Fig. 3c). The expression of SOD increases under 7, 14 and 21 DWD compared to control plants, under 7 DWD the expression level of SOD rises by 2.12 fold as compared to the respective value under control condition. Similarly, under 14 and 21 DWD the DZ5 and DZ3 noted maximum with respective fold increases in SOD expression of 4.52 and 4.93 than control plants (Fig. 3d). The rise in the expression level of DHAR and MDHAR genes were also recorded after 7, 14, and 21 DWD. After 7 DWD the DHAR expression increases highest by 1.77 fold DZ1 as compared to control plant. Similarly, it was highest 2.12 fold under 14 DWD and 2.89 fold in DZ4 under 21 DWD, for the same plants, compared to control condition (Fig. 4a). MDHAR gene expression was highest in DT line DZ4 with a respective fold increase of 3.13 compared to control, under 7 DWD. After 14 DWD it was highest in DZ5 with respective fold increase of 3.47. Similarly, it increases maximum by 3.62 fold for DZ4, and DZ5, as compared to the fold change under control conditions after 21 DWD (Fig. 4b). As in the case of APX, CAT, GR, SOD, DHAR, and MDHAR gene expression, the level of POD expression also rise under water-de cit condition. After 7 DWD POD expression level was highest in DT line DZ4 followed by DZ5 and DZ2 with respective fold change rise 2.45, 2.43 and 2.33. Similarly, after 14 and 21 DWD, it was noticed maximum in DZ5 with respective value 2.44 and 3.82, followed by DZ3 with respective fold change, 2.33 and 3.67 as compared to control (Fig. 4c).

Discussion
Plant water interaction plays a signi cant role during initiation and modulation of the antioxidant protective system against water de cit stress. Expanding proofs are available which recommends the oxidative stress which might be the major plants harming factor presented under distressing conditions (Rai et al. 2012b). To increase resistance against drought stress in tomato plants developed DT plants over-expressing two transcription factors: AtDREB1A and BcZAT12. Longer exposer to drought stress causes ROS over-generation, which disrupts the metabolism of plants, prompts oxidative harm to the cellular components (Alexieva et al. 2001), and has signi cant involvement in decreased yield e ciency under stress conditions. APX, CAT, GR, SOD, DHAR, MDHAR, and POD are the crucial enzymatic antioxidants which perform a critical role in plants protection against severe damages generated due to oxidative stresses. Many reports suggested elevated antioxidant activities, in abiotic stress tolerance genes expressing transgenic plants Krishna et al. 2021). Earlier reports presented the impacts on enzymatic and non-enzymatic antioxidant system under drought stress in DREB1A and BcZAT transgenic plants (Rai et al. 2012a;Rai et al. 2013b), although, there combined impact was yet to be explored. Hence, the current experiment was conducted to explore the variation in transgenic tomato plants antioxidant mechanisms carrying AtDREB1A and BcZAT12 transcription factors jointly. The joint overexpression of AtDREB1A and BcZAT12 transcription factors observed effective and accepted strategy for genetic engineering of plant developed to increase tolerance against drought stress (Rai et al. 2012a;Rai et al. 2013b). Additionally, the gene-stacking strategy paves the way to make transgenic plants capable to grow and survive better under abiotic stress conditions (Kudo et al. 2017), by the following (a) ionic and osmotic maintenance through homeostasis, (b) growth and cell division control, and (c) cellular repair through detoxi cation (Saijo et al. 2000). The study showed that, AtDREB1A and BcZAT12 system might function mediated through protection of ionic and osmotic homeostasis. Increased expression of AtDREB1A and BcZAT12 genes enhance the quantity of GR, which most likely contribute in the detoxi cation of ROS, as AtDREB1A and BcZAT12 pathway genes control GSH homeostasis, supporting survival and growth of plants even under the number of abiotic oxidative stresses in distinct species (Rai et al. 2013b;Rai et al. 2102a). The two transcription factors additive overexpression is accepted to be an e cient approach for plant genetic engineering intended to elevate the tolerance against drought stress and improve plant growth.
An expanded drought stress resistance of tomato plants over-expressing AtDREB1A and BcZAT12 separately and regulation of AtDREB1A and BcZAT12 expression were accounted by Rai et al. (2012b, b).
In the present study tomato plants over-expressing both AtDREB1A and BcZAT12 jointly were compared for their drought tolerance and various biochemical parameters with respect to ST and NT plants.
Expression levels of various antioxidants genes were elevated in transgenic plants compared to NT plants, although, appeared higher in DT plants with respect to the single and NT plants, which showed their additive effects.
In the present study the enhanced O. − 2 level recorded with rising days of drought stress in NT, ST, and DT lines emerge to be linked with time and severity of stress. The generation of O. −2 demonstrated to reliant on the level of stress, duration, plant age, and species (Rai et al. 2012a). Diminished superoxide anion generation is reminiscent of enhanced cell ROS homeostasis in DT tomato lines contrasted with ST and NT. In the current study, the drought stress treated AtDREB1A and BcZAT12 DT tomato plants showed signi cantly elevated CAT and SOD activity in comparison to their ST and NT corresponding plants, more speci cally the DT lines DZ3 and DZ5. Under stress condition SOD mediates the removal of superoxide radicals; it evacuates O 2 by catalyzing its dismutation. Earlier under drought stress critical increment in SOD activity has been seen in a number of muskmelon genotypes with better drought tolerance capability (Ansari et al. 2017;Ansari et al. 2018;Ansari et al. 2019), and ST tomato plants over-expressing BcZAT12 (Rai et al. 2012a) and AtDREB1A (Rai et al. 2013a), additionally drought tolerant transgenic peanut (Bhatnagar et al. 2009), chrysanthemum (Hong et al. 2006), and Lolium perenne plants (Li et al. 2011) over-expressing AtDREB1A. In cherry tomato enhanced activity of CAT was reported particularly drought exposed tolerant varieties (Bhatnagar et al. 2009), and ST plants over-expressing AtDREB1A and BcZAT12, individually. Enhanced expression of CAT gene in transgenic tomato plants in reaction to heterologous over-expression of AtCBF1 has been reported (Gill et al. 2013). Similar to CAT gene expression, under drought stress catalase the activity was also higher in constitutively over-expressing DT plants of tomato comparatively with the ST and NT plants. In the current experiment, in addition to enhanced APX expression, increased APX activity was also recorded in DT and ST tomato plants exposed to different water de cit condition, however, it was higher in DT lines compared to ST and NT plants. When exposed to drought stress condition enhanced the expression of APX has been reported in many plants, viz., tomato (Rodriguez et al. 2010), muskmelon (Ansari et al. 2017), and APX enhanced activity in ST AtDREB1A (Rai et al. 2013a) and BcZAT12 (Rai et al. 2012a) separately over-expressing. In the present study, drought treated DT tomato lines showed signi cantly increased gene expression and activities compared to ST and NT plants, if see in DT lines DZ5 showed the highest gene expression and enzyme activities. An increased expression of GR was recorded in drought stress treated muskmelon (Ansari et al. 2017), and GR enzyme activity in rice (Sharma and Dubey 2005) and ST tomato (Rai et al. 2012b;Rai et al. 2013a) plants. In the current experiments, increased activities of DHAR up to 14 days and MDHAR up to 21 days of drought stress were observed in the DT tomato lines in comparison to counterpart ST and NT plants. Many reports are available which showed DHAR and MDHAR over-expressing transgenic plants exhibit a higher AsA level (Yin et al. 2010). The elevated MDHAR activity is responsible for drought tolerance in ST tomato (Rai et al. 2012a;Rai et al. 2013a) and cherry tomato (Rodriguez et al. 2010). As reported earlier AtDREB1A (Rai et al. 2012b) over-expressing transgenic tomato plants POD activity was random as it decreases initially then increases and again decreases, instead of this in BcZAT12 (Rai et al. 2012a) transgenic increase noted up to 14 days of water de cit then reduction observed after 21 days of water de cits, in the case of our DT lines the results were similar to BcZAT12, but the level of POD was higher, this might be due to the additive effects of both genes (Kudo et al. 2017). These enzymatic changes bolster the suggestion that AtDREB1A and BcZAT12 over-expressing DT tomato plants eases stress actuated oxidative damages and adjust promptly to the drought stress condition. Observations supplements the knowledge about the proper adjustment of oxidative stress in ST plants over-expressing AtDREB1A and BcZAT12 gene, which is mostly controlled through the enzymes like SOD, CAT, APX, GR and POD (Rai et al. 2012a;Rai et al. 2013b). It responds by neutralizing ROS to limit the harm, having the capability for electrons donation in various non-enzymatic and enzymatic responses, and furthermore goes about as an inherent substrate for numerous plant peroxidases (Gill et al. 2013). It is well recognized that amount of AsA enhances under drought conditions in plants, however, the buffering limit produced by AsA adds to resistance against the stress generated (Rai et al. 2012a). For instance, droughtresistant tobacco, poplar, and cherry tomato genotypes showed an increased leaf AsA and notwithstanding this AtDREB1A and BcZAT12 (Rai et al. 2012a;Rai et al. 13a) ST plants additionally indicated a higher measure of AsA which give plants improved resilience to oxidative stress (Rodriguez et al. 2010). The AsA redox framework comprises of MDHA, DHA and L-ascorbic acid. An elevated proportion of reduced to oxidized AsA is required to combat cells ROS (Rodriguez et al. 2010). In this context, transgenic (DT) tomato plants enlisted increased AsA contrasted with the ST and NT plants, and seem to have a more prominent ability to diminish ROS. In plants Glutathione is considered as important metabolite, moreover, its reduced structure GSH accepted as a signi cant part for intra-cellular defense against ROS-generated oxidative stress. It additionally assumes a signi cant role in the development and advancement of plants, including differentiation, death, and senescence of the cell, also regulation of enzymes (Rai et al. 2012a). Under drought stress, DT tomato lines displayed higher glutathione content contrasted with the ST and NT plants. GSH assumes a key function in the antioxidative protection pathway by recovering another potential water dissolvable antioxidant like AsA, by means of the AsA-GSH cycle (Gill and Tuteja 2010). The GSH and GSSG equilibrium are the vital requirements in continuing the cellular redox state. Continuation of an enhanced reduced to an oxidized ratio of AsA and glutathione is critical for the appropriate diminishing of the ROS in cells (Choudhury et al. 2017;Miller et al. 2018). An expansion in the AsA and glutathione levels in maize and wheat plants exposed to drought stress has been accounted Nayyar and Gupta 2006). In the current experiment, drought stress treated DT tomato lines exhibited distinctly enhanced activities of these enzymes than their ST and NT counterparts. In short, the results of the present study showed that AtDREB1A and BcZAT12 additive overexpression brought about a more prominent actuation of the segments of Halliwell Asada cycle under the conditions of drought stress in DT lines (Fig. 5). The amount of AsA, reduced AsA, glutathione, and GSH in the DT tomato lines demonstrated a positive interaction with ROS level. Accordingly, it might be reasoned that the upgraded oxidative stress resilience under drought stress condition was related to the additive expression of DT lines, which expanded the activities of these enzymes. These backings the conclusion that the enhanced antioxidant levels in ST added to bring down the ROS and a superior osmotic alteration prompting improved drought stress resistance (Rai et al. 2012a). In the current investigation, the activities of the important antioxidant enzymes, were signi cantly higher in DT tomato lines, overall the DT tomato lines exposed to drought stress exhibited reduced oxidative stress comparatively the ST and NT plants. Under drought stress conditions in DT lines, the amount of enzymatic and non-enzymatic antioxidants was more prominent. This demonstrates about the DT tomato lines increased resistance against drought. The enhanced expression of genes linked with antioxidants may be an outcome of the additive expression of AtDREB1A and BcZAT12 genes under drought stress presenting the DT tomato lines increasingly valuable for zones affected with drought stress. Drought mediated diminution of leaf pigments is accepted as an indicator of oxidative stress which associates with the degradation of chlorophyll, photo-oxidation of pigment, or retardation in chlorophyll synthesis (Rai et al. 2012a). Similarly in the current experiments, the photosynthesis pigments degradation observed, however, it was lower in DT compared to the ST and NT plants. which might be attributed to delayed physiological processes (Cabello et al. 2009;Ibrahim et al. 2014;Dasgan et al. 2018). On the contrary, the present study revealed that double transgenic tomato lines recorded higher fruit yield as compared single transgenic (AtDREB1A or BcZAT12 or NT) under different DWD. This increase in fruit yield may be attributed to normal physiological processes under water de cit conditions. Some earlier studies reported that quality parameters such as hardness, TSS, ascorbic acid, acidity, and colour get enhanced in tomato under water de cit condition. Furthermore, reduced fruit size and low dilution from decreased water levels may result in reduced water transport within the plant system but not the photo-assimilates (Zegbe et al. 2006); thereby the improvement in quality parameters.
Under drought stress conditions, double transgenic tomato lines from the present study showed increased fruit size.

Conclusion
In conclusion, in the present study DT tomato lines showed an enhanced antioxidant enzyme activity under drought stress condition. Present results suggest that AtDREB1A and BcZAT12 stacked DT tomato line more speci cally DZ3, DZ4, and DZ5 are increased tolerant to drought stress and able to survive due to increased antioxidant activity, suggesting their signi cance for enhancing the fruit yield in the droughtaffected area. The ndings of the present study demonstrate that genetic engineering of the plant by employing approaches like gene-stacking might be effective for transgenic plants generation which had better tolerance against drought stress. Additionally, co-overexpression of AtDREB1A and BcZAT12 increases the transgenic lines performance as depicted by various enzymatic and non-enzymatic antioxidants (Fig. 6), total chlorophyll content, and genes linked with various antioxidative enzymes, and phenotypic traits.