Isolation of GUSP-2 gene, TA-cloning
Total RNA was extracted from leaves of FDH-171-Gossypium arboreum plants after drought stress of 15d and GUSP-2 gene (Fig. 2) was amplified which showed clone number 3 and 5 generated desired fragments of 510 bp and 3.8 kb size (Fig. S1A,B, Supplementary data) and were proceed for Sanger sequencing (Fig. S2A,B, Supplementary data). TA-clone number 7 was proved correct and named Tw-usp-2.
PCR amplification of GUSP-2 gene.
Site directed mutations of GUSP-2 gene
GUSP-2-protein sequence was aligned with 2gm3.A protein sequence, template protein which was used to predict GUSP-2-protein 3D-Model, to verify the mismatched residues in pocket region. The ATP-binding pocket residues of GUSP-2 were ensured while comparing with 2gm3.A, template protein. Two mismatched and one matched residue (Lysine 60, Aspartic acid 26 and Lysine 3) of GUSP-2 were selected for point mutations and were replaced with Proline, Serine and Threonine respectively by using MOE Bioinformatics tool. Three point mutations were separately created in GUSP-2-protein 3D-Model. (Fig. S3, Supplementary data).
In vivo incorporation of mutations
Three mutations were separately incorporated in Tw-usp-2. Mutagenized plasmids (Mp1, Mp2 & Mp3) were transformed into competent cells of DH5αT1R E. coli (Fig. S4A-C, Supplementary data). Positive transforment number 2, 6 from rMp1, 2 from rMp2 and 5 from rMp3 generated two fragments of 510 bp and 3.8 kb (Fig. S5A-C, Supplementary data) and were further confirmed with Sanger sequencing (Fig. S6A-C, Supplementary data). The confirmed clones were named Tm1-GP-2, Tm2-GP-2 & Tm3-GP-2 respectively. TA constructs of mutants (Tm1-GP-2, Tm2-GP-2 & Tm3-GP-2) were used for cloning pET-30b expression vector and transformation into wild type and mutated E. coli expression strains (Table S7, Supplementary data) for functional validation. Five colonies were picked randomly from each transforments (rpE1, rpE2, rpE3 & rpW) and screened via colony PCR (Fig. S7A,B, Supplementary data). Clone number 2 & 3 of rpE1 (Fig. S8A, Supplementary data), 7, 9 & 11 of rpE2 (Fig. S8B, Supplementary data), 2, 3, 4 & 6 of rpE3 (Fig. S8B, Supplementary data) and 7, 8, 9 & 10 of rpW were positive. Positive transforment number 3 from rpE1, 9 from rpE2, 4 & 6 from rpE3 and 8 & 9 from rpEw generated two fragments of 510 bp and 5.2 kb (Fig. S7A,B, Supplementary data) were confirmed positive and named M1-CeS1, M2-CeS2, M3-CeS2 and W-CeS respectively.
Functinal validation under various abiotic stress conditions
Under salt stress (NaCl 800 mM) mutant-1 (M1-usp-2) enhanced survival rate of E. coli cells as compared to cells transformed with W-usp-2 and M2-usp-2, M3-usp-2 genes (Fig. 3). Moreover, viability of BL-21-uspA mutant cells was found to be reduced than that of wild type BL-21 cells under salt stress. Thus we assayed the sensitivity of all mutant strains for salt stress and found, the cells lacking all USP (ABC) genes were drastically more sensitive than that of BL-21-uspA, BL-21-uspB, BL-21-uspC and wild type BL-21 cells, The comparative sensitivity of BL-21-uspABC mutant cells transformed with M1-CeS1, M2-CeS2, M3-CeS2, W-CeS constructs, BL-21 control cells was shown in Fig. 4a-c. The OD (600 nm) after 12 h of growth was observed at 1.93 with M1-usp-2 mutant gene (Fig. 4a). At the same time OD (600 nm) of M2-usp-2 transformed cells was noted 0.79 for E. coli BL-21-uspABC (Fig. 4b) while OD of W-usp-2 transformed cells were 1.74, which almost parallel to the OD of M3-usp-2 transformed cells 1.45 (Fig. 4c).
Spot assay of E. coli-BL21-uspABC. Transformed BL21-usp-ABC cells with wild type (W-usp-2) and mutant (M1-usp-2, M2-usp-2, M3-usp-2) genes were spotted on LB plates supplemented with salt (800 mM NaCl), PEG (8%). For heat stress, after 2 h of induction (IPTG) 1 ml culture was inoculated in 10 ml media (LB) and incubated at elevated temperatures (46 °C). Samples were removed after 8 h and spotted on respective plates. For cold stress, spotted plates were removed from 4 °C after 10d and photos were taken after incubation at 37 °C.
Comparison of growth under 800 mM (salt stress) in E. coli BL-21-uspABC mutant, expressing W-usp-2, M1-usp-2, M2-usp-2 & M3-usp-2 genes. (a) E. coli BL-21-uspABC mutant cells expressing M1-usp-2 showed high tolerance then that of vector control, bacterial control cells and W-usp-2 gene expressing cells. (b) BL-21-uspABC cells expressing W-usp-2 gene are more tolerant as compared to M2-usp-2 cloned cells of BL-21-uspABC. (c) growth rate of BL-21-uspABC cells transformed with M3-usp-2 gene is almost similar to the growth rate of BL-21-uspABC cells expressing W-usp-2 gene.
Survival rate of bacterial cells under osmotic stress (PEG 8%) was observed stronger as compared to other abiotic stress conditions (heat & salt). Sensitivity of all mutant strains for PEG stress was analyzed and found, the cells lacking all USP (Fig. 3) genes were drastically more sensitive. Here we observed again that mutant-1 (M1-usp-2) has significant effect on cell survival as compared to mutant-2 (M1-usp-2), mutant-3 (M3-usp-2) and wild type (W-usp-2). The OD (600 nm) after 12 h of growth for BL-21-uspABC (M1-usp-2 mutant) was 1.94 (Fig. 5a) and of M2-usp-2 transformed cells was noted 0.99 for BL-21-uspABC (Fig. 5b) while OD of W-usp-2 transformed cells were 1.64 which almost similar to the growth rate of bacterial cells transformed with M3-usp-2 gene 1.65 (Fig. 5c).
Osmotic stress tolerance in E. coli BL-21, (a) E. coli BL-21 BL-21-uspABC expressing M1-usp-2 showed significant tolerance as compared to vector control, cell control and W-usp-2 gene expressing cells, (b) W-usp-2 gene expressing BL-21-uspABC cells are more tolerant as compared to M2-usp-2 cloned cells of E. coli, (c) OD of BL-21-uspABC cells expressing M3-usp-2 gene is almost similar to BL-21-uspABC mutant cells expressing W-usp-2 gene.
Wild type (W-usp-2) and mutant (M1-usp-2) gene confer slight tolerance against heat stress in E. coli (Fig. 6). BL-21-uspABC cells expressing M1-usp-2 gene showed modest tolerance (OD 0.76 after 12 h at 600 nm). Furthermore, E. coli BL-21-usp-ABC (OD 0.73 after 12 h at 600 nm) transformed with W-usp-2 gene showed minute survival rate as compared to control cells. It was also observed, E. coli mutant cells transformed with M3-usp-2 gene showed equivalent survival rate to W-usp-2 expressing cells. However, mutant-2 (M-2usp-2) in cells (OD at 600 nm 0.54 after 12 h) failed to impart considerable resistance as compared to vector control and cell control (Fig. 6a-c).
Comparison of Heat stress tolerance in E. coli BL-21. Both E. coli BL-21-uspABC (a–c) cells expressing W-usp-2, M1-usp-2, M2-usp-2 and M3-usp-2 showed slight tolerance as compared to control. (b) W-usp-2 and M3-usp-2 gene expressing BL-21-uspABC cells are modestly tolerant as compared to M2-usp-2 cloned cells. It remained difficult to claim difference of survival rate among wild type and mutant clones.
Relative expression level of under osmotic stress
M1-usp-2 expression under osmotic stress (8% PEG) in BL-21-uspABC cells was observed at uppermost level (5.7 folds) as compared to M2-usp-2 (1.0 folds), M3-usp-2 (3.8 folds) and W-usp-2 (4.0 folds) (Fig. 7b). However, under heat (46 °C) stress all genes were poorly expressed (Fig. 7c) either because of cell death or genes don’t confer resistance against heat stress. Expression of W-usp-2, M1-usp-2, M2-usp-2 & M3-usp-2 genes were 0.4, 0.6, 0.4 & 0.5fold in E. coli BL-21-uspABC. Under salt stress expression level 4.4, 3.9, 4.1 & 4.2 of W-usp-2, M1-usp-2, M2-usp-2 & M3-usp-2 in E. coli BL-21 (Fig. 7a).
Incorporation of two mutations in GUSP-2 for plant expression vector
Two mutants (M1-usp2 & M3-usp-2) and wild type genes of GUSP-2 for the verification of osmotic stress tolerance enhancement in CIM-496 G. hirsutum were selected. GUSP-2 was amplified product (Fd-BglII-P + Rv-SalI-P) and TA ligated in pCR2.1 vector. Five clonies (Fig. S9A,B, Supplementary data) were screened via clony PCR, colony number 1, 3, 4 & 5 were found positive. These positive transforments were confirmed with double digestion by using BglII and SalI enzymes (Fig. S10, Supplementary data). Clone number 1, 3 & 5 generated desired fragments of 510 bp and 3.8 kb. Similarly, GFP (762 bp) was TA cloned (Fig. S11, Supplementary data) and confirmed clone was named T-GFp. Lane1 maker 1 kb, Lane2-5 are positive PCR amplification. Positive transforments number 2, 3 & 4 were confirmed with double digestion (SalI and BstEII).
Relative expression level of mutant and wild type genes (M1-usp-2, M2-usp-2, M3-usp-2 & W-usp-2) in E. coli and, data was analyzed by comparative CT method and presented as relative fold gene expression (2−ΔΔCT) with reference rssA gene. The graph indicates the mean ± SD (a) relative expression under NaCl 800 mM stress. Expression of M1-usp-2 was significant in in BL-21-uspABC cells (4.2 fold). While M2-usp-2 W-usp-2, M3-usp-2 genes have slight expression (4.4 & 4.1fold), (b) relative expression under PEG 8% stress. M1-usp-2 relative expression was significant in E. coli BL-21-uspABC cells (5.7 fold). Again M2-usp-2 gene expression was not notably different (4 & 3.8 fold) (c) relative expression under heat stress in E. coli BL-21-uspABC cells had not been observed at notable difference either because the death of cells at high temperature or because the wild type and mutant genes conferred no resistance against heat stress.
The selected mutants M1-usp-2 & M3-usp-2 were incorporated into Tw-GP-2p construct. The positive Mutagenized plasmids (Gp1 &Gp3) were screened via colony PCR (Fig. S10, Supplementary data) and with restriction digestion (Fig. S12A,B, Supplementary data). The transformants were confirmed via Sanger sequencing and named Tm1-GP-2p and Tm3-GP-2p.
Hygromycin fragment (1 kb) was excised from pCAMBIA-1301 and was renamed pCEMBIA-1301b. Mutated and Wild type TA constructs of GUSP-2 (Tm1-GP-2p, Tm3-GP-2p and Tw-GP-2p) and pCEMBIA-1301b vector (5 µl each) and T-GFp were double digested. Then three fragments ligation was conducted for Tm1-GP-2-GFP, Tm3-GP-2-GFP and Tw-GP-2–GFP fusion in pCEMBIA 1301b vector (Fig. S13A-C, Supplementary data). Cloned pCAMBIA-1301b vectors were confirmed through colony PCR and restriction digestion and constructs were named pW-usp-2, pM1-usp-2 and pM3-usp-2 (Fig. S1A-C, Supplementary data) and were transformed in competent cells Agrobacterium tumefaciens-LBA-4404.
Transformation of CIM-496 G. hirsutum
The confirmed pW-usp-2, pM1-usp-2 and pM3-usp-2 clones were used to make transgenic CIM-496 G. hirsutum. After three weeks of growth on selection (kanamycin) medium, transgenic cotton plants were switched to shoot and root induction medium. After one and half month, healthy plants with prominent roots were shifted to pots containing loamy soil (Fig. 8A-C).
(A) Transgenic plants were growing in culture tubes, (B, C) Transgenic plants were shifted to pots.
Transformation efficiency
Total 6000 mature embryos were used in all the transformation experiments. After four weeks of selection on kanamycin (100 mg/ml), 154 plants of pW-usp-2, 132 pM1-usp-2 and 109 plants of pM3-usp-2 were obtained. Only 16 plants of pW-usp-2 out of 88, 11 plants of pM1-usp-2 out of 91 and 9 plants of pM3-usp2 out of 76 were successfully shifted to CEMB-greenhouse. The overall transformation efficiency remains 0.83% (Table 1).
Confirmation of transgenic plants of CIM-496 G. hirsutum
Total 16, 18, 19 transgenic plants of pW-usp-2, pM1-usp-2 and pM3-usp-2 respectively were confirmed PCR amplification (Fig. 9). The transgenic plants of wild type GUSP-2 was named pT-W-usp-2, similarly, transgenic plants of pM1-usp-2 and pM3-usp-2 was labeled as pT-M1-usp-2 and pT-M3-usp-2 respectively. All transgenic plants were found phenotypically healthy and their growth rate was normal.
PCR amplification of W-usp-2, M1-usp-2&M3-usp-2 in transgenic plants.
Molecular analysis of transgenic cotton plants
Expression analysis revealed that under drought stress condition both mutated and non-mutated GUSP-2 genes were expressed higher in leaves as compared to root and stem. The leaves of drought stressed transgenic (pT-M1-usp-2) plants contained pM1-usp-2 construct showed 7.8 folds expression of M1-usp-2 as compared to (well-watered) control plants. The stem of same plants also showed 2.6 folds expression as compared to control and their roots also express M1-usp-2 at 2.1 folds more than that of control plants. The expression of M3-usp-2 in leaves of transgenic plants (6.2 folds) was almost similar to the expression of W-usp-2 wild type GUSP-2 (5.8 folds). However, roots of pT-M3-usp-2 and pT-W-usp-2 transgenic plants showed 1.4 to 1.5 folds more expression as compared with control. Similarly, stem of both transgenic plants expressed respective transformed genes at 1.7–1.9 folds more than that of controls. Both mutated and wild type GUSP-2 genes were more expressed in leaves of transgenic plants but the expression of M1-usp-2 was relatively higher in pT-M1-usp-2 (Fig. 10).
Spatial Expression of wild type and mutated GUSP-2 genes in transgenic plants under drought stress conditions.
Quantification of protein with ELISA
The protein concentration was found more in drought stressed leaves as compared with roots and stems of all transgenic plants. The concentration mutated protein (M1-usp-2) was slightly more than that of wild type (W-usp-2) and mutant-3 (M3-usp-2) protein. The protein in leaves of drought stressed transgenic cotton plants (pT-W-usp-2, pT-M1-usp-2 & pT-M3-usp-2) was found at 31.7, 37.1 and 28.8 ng/ml respectively. However, protein concentration in leaves of control plants was observed at 16.2 ng/ml. Both wild type and mutated GUSP-2 proteins in roots and stems of transgenic plants was found closer to that of control plant (Fig. 11).
Concentration of wild type and mutated GUSP-2-proteins in different tissues of pT-W-usp-2, pT- M1-usp-2& pT-M3-usp-2 transgenic cotton plants.
Morphological analysis of transgenic plants
Plant height
The initial plant height of control plants before the application of stress treatment was noted as 15.6 cm. Similarly, initial plant height of transgenic plants (pT-W-usp-2) transformed with pW-usp-2 construct was 16.1 cm. The initial heights of transgenic plants (pT-M1 -usp-2 & pT-M3-usp-2) transformed with pM1-usp-2 & pM3-usp-2 constructs was note down as 14.7 cm and 15.3 cm respectively. After 15d of drought stress the height of pT-W-usp-2, pT-M -usp-2 and pT-M3-usp-2 plants were observed as 21.9, 23.4 and 22.7 cm respectively. However, the height of non-transgenic CIM-496 was increased as 17.2 cm after 15d of drought stress. Although, after 15d the final height of control transgenic and control non-transgenic plants were 25.2, 24.3, 24.8 and 24.8 cm for pT-W-usp-2, pT-M1 -usp-2, pT-M3-usp-2 and control plants respectively (Fig. 12).
Comparison of Plant height (cm) of pT-W-usp-2, pT-M1 -usp-2, pT-M3-usp-2 plants under control and drought stress conditions.
Root length
Root length of transgenic plants (pT-W-usp-2, pT-M1 -usp-2 & pT-M3-usp-2) before stress treatment was 6.2, 5.5, 5.7 cm respectively. While, root length of non-transgenic control plants was noted as 6 cm. The transgenic control plants growing under control conditions, root length after 15d was 11.6, 12.8, 12 and 10.9 cm for pT-W-usp-2, pT-M1 -usp-2, pT-M3-usp-2 and control plants respectively (Fig. 13). After 15d, the root length of transgenic plants (pT-W-usp-2, pT-M1 -usp-2 and pT-M3-usp-2) growing under drought stress was observed as 8.7, 10.1 and 9 cm respectively, while root length of non-transgenic plant after 15d of stress was 6.9 cm.
Comparison of Root Length (cm) of pT-W-usp-2, pT-M1 -usp-2, pT-M3-usp-2 plants under control and drought stress conditions.
Physiological analysis of transgenic plants
Relative water content
Relative water content (RWC) of transgenic cotton plants (pT-W-usp-2, pT-M1 -usp-2 & pT-M3-usp-2) after 15d of stress treatment was 40.7, 43.8, 41.3% respectively while, RWC of control plants under same stress conditions was noted as 30.6 cm (Fig. 14). However, RWC of transgenic and non-transgenic control plants growing under control condition was 47.6, 48.3, 52.7 and 52.1% respectively for pT-W-usp-2, pT-M1 -usp-2, pT-M3-usp-2 and non-transgenic control plants. RWC of transgenic plants (pT-M3-usp-2, pT-W-usp-2) contained mutated, pM3-usp-2, and non-mutated, pW-usp-2, constructs respectively was remained almost same (41.3% ≈ 40.7%) after 15d of drought stress but remained higher than that of control plants (30.6%). Transgenic plants, pT-M1-usp-2, performed well in terms of RWC under drought stress treatment (43.8%) as compared to 3rd mutated, non-mutated and control plants. Comparison between the leaf fresh weight with leaf turgor weight and dry weight revealed that the leaf relative water content was decreased under drought stress treatment more rapidly in non-transgenic plants then that of transgenic plants especially pT-M1-usp-2.
RWC of pT-W-usp-2, pT-M1 -usp-2, pT-M3-usp-2 plants under control and drought stress conditions.
Photosynthetic activity
We observed 10.6 µmol m−2 s−1 maximum photosynthesis rate for transgenic plants contained mutated (pM1-usp-2) GUSP-2 gene under drought stress of 15d. However, photosynthetic activity of same transgenic plants was observed as 13 µmol m−2 s−1 under control conditions. Photosynthesis of regularly watered transgenic plants containing mutated construct (pM3-usp-2) and non-mutated construct (pW-usp-2) was noted as 11.7, 12.3 µmolm−2 s−1 respectively and under drought stress treatment their photosynthetic rate was decreased to 9.7, 9.4µmolm-2 s−1 but remain almost equal. The photosynthesis activity was decreased to 6.8 from 12.8 µmolm−2 s−1 under control to stressed conditions (Fig. 15). With the application of drought stress treatment, photosynthetic activity was observed to be decreased in non-transgenic control plants as compared to transgenic plants. However, among transgenic plants pT-M1-usp-2 (10.6 µmolm−2 s−1) rated at higher level as compared to pT-W-usp-2 and pT-M3-usp-2 (9.4 µmolm−2 s−1≈9.7 µmolm−2 s−1) plants.
Photosynthetic rate of pT-W-usp-2, pT-M1-usp-2, pT-M3-usp-2 plants under control and drought stress conditions.
Biochemical analysis of transgenic plants
Proline content
The concentration of proline was found higher in transgenic plants, which were subjected to drought stress conditions as compared to the plants under control conditions. Minimum proline contents were observed as 25.2 mg/g under control condition for transgenic plant containing mutated construct (pM1-usp-2) and after 15d of stress the contents were noted as 43.6 mg/g for the same plant. Proline content of regularly watered transgenic plants containing mutated construct, pM3-usp-2, and non-mutated construct, pW-usp-2, were noted as 27.5, 26.4 mg/g respectively, however, it was increased up to 40.2, 40.8 mg/g after 15d of drought stress. For controls non-transgenic plants, it was 26.6 mg/g under regular watering conditions and it was decreased to 20.86 mg/g under drought stress treatment. With the application of drought stress treatment, proline content was increased in transgenic plants but decreased in non-transgenic plants (Fig. 16). When comparing among transgenic plants, proline content from leaves of pT-M1-usp-2 transgenic plants containing pM1-usp-2 mutated construct was highest (51.5 mg/g) as compared to pT-M3-usp-2plants (40.2 mg/g), pT-W-usp-2 plants (40.86 mg/g). The proline contents from pT-W-usp-2 plants pT-M3-usp-2 plants were observed almost same (40.86 mg/g ≈ 40.26 mg/g).
Comparison of Proline content from pT-W-usp-2, pT-M1-usp-2, pT-M3-usp-2 plants under control and drought stress conditions.
Confocal microscopy for localization of GUSP-2 with GFP
Wild type GUSP-2 gene (W-usp-2) was observed localized with GFP fluorescence in the leaves of pT-W-usp-2 transgenic plants by using confocal microscopy. With localization of green fluorescence, it was found that W-usp-2, non-mutated form of GUSP-2, was expressed in guard cells of stomata. Green fluorescence was not seen in the leaves of control plants, but red fluorescence of chloroplast was observed (Fig. 17A). Figure 17B, demonstrated the green fluorescent image of pT-W-usp-2 leaves in guard cells of stomata, Fig. 17C is merged image of both showed green fluorescence of GFP and red fluorescence of chlorophyll. Close observation of confocal images of control and transgenic leaves also revealed that the guard cells were shrinked in which GUSP-2 gene was expressed and stomata was seen closed. The guard cells with no GUSP-2 expression were seen turgid and stomata were remained open. It means GUSP-2 gene is expressing in guard cells of transgenic leaves and playing role in functioning of stomata under drought stress.
Confocal microscopy for localization of GUSP-2 in leaves of transgenic plants, (A) control plant, (B) transgenic plant with GFP fluorescence (C) transgenic plant with GFP fluorescence and chloroplast fluorescence.
Statistical analysis
Analysis of variance (ANOVA) for E. coli transformed with recombinant vectors containing W-usp-2, M1-usp-2, M2-usp-2, M3-usp-2 genes showed significant differences under salt and osmotic stresses for mutant genes, vector control, wild type and cell control at P < 0.05 but no significant difference was observed under heat stress treatment. Comparative study among mutant genes showed no significant difference between W-usp-2 and M3-usp-2 P < 0.05 under all stress conditions (Supplementary Table S1). Similarly, analysis for morphological, physiological, biochemical and molecular data of transgenic plants (pT-M1usp-2, pT-M3-usp-2, pT-W-usp-2) transformed with mutated (pM1-usp-2, pM2-usp-2) and non-mutated (pW-usp-2) constructs and non-transgenic plants showed significant differences among mutated and non-mutated genotypes under drought stress treatment. However, comparison among mutant genes, revealed no significant difference between pT-W-usp-2 and pT-M3-usp-2 at P < 0.05 (Supplementary Table S2). Under control conditions, 25.2 cm plant (shoot) height was recorded for transgenic plants, while under drought stress conditions the mutant gene M1-usp-2 (23.4 cm) showed higher expression in the form of long (higher) plant height. Significant difference was observed among mutant genes, stress and the interactions between gene × stress for morphological traits including shoot length (plant height) and root length (Supplementary Table S3). Major difference was found between pT-M1-usp-2 (10.1, 24.4 cm, 0.43) and pT-M3-usp-2 (9, 22.7 cm, 0.39) at P < 0.05 for root length and shot length but no significant difference was recorded between pT-W-usp-2 (8.7, 21.9 cm, 0.38) and pT-M3-usp-2 (9, 22.7 cm, 0.39).
Significant difference was also reported among mutant genes, stress and the interactions between gene × stress for physiological traits including relative water content, photosynthetic rate, (Supplementary Table S4). It was revealed from results that the relative water content (43.8%) was found higher under drought stress condition for pT-M1-usp-2 as compared to other transgenic plants and non-transgenic (control) plants. Similarly, higher photosynthetic rate and lower transpiration rate and stomatal conductance were found for pT-M1-usp-2 transgenic plants. It was observed significant difference among mutant genes, stress and the interactions between gene × stress for proline content P < 0.05 (Supplementary Table S5). The mean performance of genes under water stress condition indicated that mutant gene M1-usp-2 initiated the formation of higher proline to improve drought stress tolerance and normal growth of transgenic plants. Similarly, significant difference was also recorded among mutant genes, stress and the interactions between gene × stress for ELISA analysis at P < 0.05 (Supplementary Table S6). It was found that M1-usp-2-protein was expressed at higher level (36.885 mg/g) in drought stressed leaves of pT-M1-usp-2 as compared to the expression of 3rd mutant protein (M3-usp-2) in pT-M3-usp-2 (28.570 mg/g) and wild type protein (W-usp-2) in pT-W-usp-2 (31.630 mg/g).
