MutantGossypium universal stress protein-2 (GUSP-2) gene confers resistance to various abiotic stresses in E. coli BL-21 and CIM-496-Gossypium hirsutum

Gossypium arboreum is considered a rich source of stress-responsive genes and the EST database revealed that most of its genes are uncharacterized. The full-length Gossypium universal stress protein-2 (GUSP-2) gene (510 bp) was cloned in E. coli and Gossypium hirsutum, characterized and point mutated at three positions, 352–354, Lysine to proline (M1-usp-2) & 214–216, aspartic acid to serine (M2-usp-2) & 145–147, Lysine to Threonine (M3-usp-2) to study its role in abiotic stress tolerance. It was found that heterologous expression of one mutant (M1-usp-2) provided enhanced tolerance against salt and osmotic stresses, recombinant cells have higher growth up to 10-5dilution in spot assay as compared to cells expressing W-usp-2 (wild type GUSP-2), M2-usp-2 and M3-usp-2 genes. M1-usp-2 gene transcript profiling exhibited significant expression (8.7 fold) in CIM-496-Gossypium hirsutum transgenic plants and enhance drought tolerance. However, little tolerance against heat and cold stresses in bacterial cells was observed. The results from our study concluded that the activity of GUSP-2 was enhanced in M1-usp-2 but wipe out in M2-usp-2 and M3-usp-2 response remained almost parallel to W-usp-2. Further, it was predicted through in silico analysis that M1-usp-2, W-usp-2 and M3-usp-2 may be directly involved in stress tolerance or function as a signaling molecule to activate the stress adaptive mechanism. However, further investigation will be required to ascertain its role in the adaptive mechanism of stress tolerance.

In Silico analysis. Site directed mutagenesis of GUSP-2 gene. Three point mutations were identified in GUSP-2 gene (510 bp) with computational analysis. The Sequence of GUSP-2 gene was retrieved from NCBI (Accession # EU107767) and 3D model was predicted through SWISS-MODEL expasy tool (https:// swiss model. expasy. org/ .expasy.org/) and evaluated with RAMACHANDRAN plot (http:// mordr ed. bioc. cam. ac. uk/ ~rapper/ rampa ge. php). MOE software (https:// www. chemc omp. com/ Produ cts. htm) was used for the identification of ATP-binding sites in template protein structure which interacted with ATP. For the alignment of sequences of both template (2gm3.A) and GUSP-2-protein CLC-Bio work bench tool was used. During alignment corresponding mismatched amino acids of GUSP-2 were analyzed. The residues which were matched with template were left and the mismatched amino acids in GUSP-2 were changed according to the template and three mutated GUSP-2 protein models were designed and named as M1-usp-2, M2-usp-2, M3-usp-2 accordingly.
Functional validation of wild type and mutated (M1-usp-2, M2-usp-2, M2-usp-2 and W-usp-2) genes in E. coli under various abiotic stress conditions. Spot and liquid culture assays were carried out to ascertain the function  M3-CeS3 and W-CeS constructs and with pET30b vector alone (negative control) were grown overnight in Luria-Bertani (LB) media. Next day overnight culture was 1:100 diluted and allowed to grow to an absorbance (OD600 nm) 0.6 and induced by adding 1 mM IPTG and incubating further at 37 °C up to 12 h under various abiotic stress conditions. For the spot assay 18 the absorbance of culture was adjusted to 0.6 (OD600 nm) by diluting using broth. Different serial dilutions (10 -2 , 10 -3 , 10 -4 , 10 -5 ) of the culture were made, spotted on LB plates supplemented with salt (800 mM NaCl) and PEG (8%). For cold stress, serially diluted culture were spotted on LB plates and placed at 4 °C. Spotted plates were removed from 4 °C after 10d and photos were taken after 12 h of incubation at 37 °C. For heat stress, after 2 h of IPTG induction 1 ml culture (OD600 nm 0.6) was added in 10 ml media and incubated at elevated temperatures (46 °C). Samples were removed after 8 h and spotted on LB plates with serial dilutions (10 -2 , 10 -3 , 10 -4 , 10 -5 ). For liquid culture assay (salt, osmotic and heat stresses) 1 ml culture after 2 h of IPTG induction (OD600 nm 0.6) was added in 10 ml media containing salt (NaCl 800 mM), PEG (8%) and incubated at 37 °C (for heat stress at 48 °C), with shaking (180 rpm). The aliquots were removed after every 2 h up to 12 h and absorbance (OD600 nm) was measured. Abiotic stress (salt, osmotic, heat & cold) tolerance was determined with respect to control cultures (bacterial cells and vector control).
RNA isolation and semi quantitative RT-PCR. Five samples were taken from each stress treatment (heat, osmotic and salt), total RNA isolated (RNA isolation kit, cat # 7020). cDNA was synthesized with oligo (dT) primer by using RevertAid TM H minus first strand cDNA synthesis kit (ThermoFisher, cat #1631). This cDNA was used for fold expression of mutated and wild type GUSP-2 through RT-PCR.
SDS-PAGE analysis. Samples were taken from induced and un-induced cultures for protein confirmation with SDS-PAGE. Briefly, cells were lysed by sonication at 50% level 6-7 pulses for 20 s each. To the lysed cells, 2.5 µl Triton X100 and 2.0 µl β-mercaptoethanol were added and centrifuged at 6000 rpm. The pellet was again dissolved in lysis buffer and incubated at 37 °C for 30 min. In 100 µl of supernatant, 18 µl of 6X SDS loading dye was added and placed in boiling water for 8 min, cool down on ice for 5 min and centrifuged at 14000 rpm for 15 min at 4 °C. The supernatant was analyzed by SDS-PAGE analysis.
Incorporation of two mutations in GUSP-2 for pCAMBIA 1301b. After scrutinizing the mutated GUSP-2 genes for their role under osmotic and salt stresses it was decided M1-usp-2 & M3-usp-2 mutant genes should be verified for osmotic stress tolerance enhancement in cotton. Selected point mutations were incorporated into Tw-GP-2p TA construct for CAMBIA1301 (cat # M1592, Marker Gene Technologies Inc). The confirmed mutated TA clones were named Tm1-GP-2p and Tm3-GP-2p. The size of pCAMBIA-1301 was reduced up to 10.8 kb from 11.8 kb by removing 1 kb fragment of hygromycin by using Xho1 site (Fig. 1A) and it was renamed pCEMBIA-1301b. The mutated and wild type GUSP-2 genes were fused with GFP marker, pGWB5 vector was used for GFP (762 bp) amplification and TA cloning (Fd-SalI-GFP + Rv-BstEII-GFP primer pair).
Transformation of Gossypium hirsutum-CIM-496. BioRad electroporator was used for the transformation of confirmed constructs (pGW-usp-2, pGM1-usp-2, and pGM3-usp-2) into competent cells of Agrobacterium-LBA-4404. 2 µl of each plasmid construct (300 ng) was mixed with 120 µl of competent cells and proceed for electroporation and left at 28 °C incubation with 220 rpm shaking for 72 h. Then, the culture was spread onto the YEP-agar plates containing 50 µg/ml kanamycin. For initial confirmation of positive transformants, colony PCR was performed. The transformants were further confirmed with restriction digestion. LBA 4404-Agrobacterium transformed with recombinant plasmids was used to produce transgenic cotton plants.
Seed sterilization, and Agrobacterium culture treatment. Seeds of CIM-496 G. hirsutum were sterilized by adding few drops of Tween-20 in water by vigorous shaking and washed three times with autoclaved distilled water and were kept in the dark for germination at 30 °C overnight. Shoot cut apex method with some modifications was used for transformation as done by Gould et al. 20 . Total 2000 embryos were used in the transformation of each recombinant plasmid (pW-usp-2, pM1-usp-2& pM3-usp-2). After treatment with Agrobacterium embryos were shifted to MS medium and co-cultivated for 72 h. At this stage, antibiotics were not added to the media. The cultures were kept in growth room at 25 °C ± 2 °C and photoperiod of 16 h light and 8 h dark. The survival percentage of the embryos at different growth stages e.g. after co-cultivation, antibiotic selection, shoot and root formation was observed.
Selection on antibiotic medium and hardening of transgenic plants to soil. After three days (72 h) of co-cultivation, plantlets were shifted to selection medium i.e. MS plus kanamycin (100 mg/ml) and cefotaxime (250 μg/ ml). Medium was also supplemented with different growth hormones i.e. Kinetin (1 mg/ml). Control plants were maintained on MS media with the 2.5 mg/L BAP + 0.1% (w/v) AC, rapid shoot growth occurred with kinetin supplemented media.. Plants were subcultured to fresh selection medium after every 10 days till 6-8 weeks and transformation efficiency was calculated. Soil mixture (clay + sand + peat of 1:1:1) was prepared to shift the transformed plants from culture tubes to soil. Plants were kept in a growth room at 30 ºC + 2 °C for 16 h photoperiod in light intensity (250-300 µmol m -2 S −1 ). After 2-4 weeks plants were ready to shift to glass house.
Confirmation of transgenic plants. Transgenic plants were confirmed with PCR, and ELISA. Genomic DNA (GDNA) was isolated from leaves, shoots and roots of plants by following the procedure with modifications described by Dunwell 21 . Isolated GDNA was used for PCR amplification of GUSP-2 gene by using primer pair (Fd-USP-full and Rv-USP-full). Total protein from root, stem and leaves was extracted according to the modified method described by Sambrook et al. 22 . For ELISA anti GFP monoclonal antibody (diluted 1:2500 in 1× PBS) was used to coat the wells of microtitre plate and incubated at 4 °C. Wells were washed with 1X PBS and 5% BSA and 100 μl of supernatant from each sample was added in wells and incubated at 25 °C. Washed with 1× PBS after 2 h of incubation and then secondary antibody (Sigma-Aldrich, USA, diluted 1:10,000) was added in wells and placed on shaker (30 rpm) for 2 h at 25 °C. Then NBT/BCIP substrate (Sigma-Aldrich, USA) was added in wells and left at 25 °C for 30-60 min. When color was developed then 1 N HCl was added to stop the reaction and the protein was quantified by using spectrophotometer at 450 nm .
RT-qPCR expression analysis. Total RNA was isolated from different plant tissues (leaves, roots, stem) of control and drought stressed (15d) transgenic plants and cDNA was synthesized. Then spatial expression of transgene was conducted through RT-qPCR analysis. The expression analysis was carried on ABI 7500 real time PCR (Applied Biosystem, USA) by using SYBER green PCR master mix (Fermentas) with primer pairs (Fd-GAPDH + Rv-GAPDH & Fd-USP full + Rv-USP full). The GAPDH was used as a reference gene for data normalization. The relative gene expression analysis was done by using SDS 3.1 software (https:// www. therm ofish er. com/ us/ en/ home/ techn ical-resou rces/ softw aredo wnloa ds/ appli ed-biosy stems-7500-real-time-pcr-system-for-human-identification.html) provided by ABI on the basis of CT values normalized with GAPDH.  and after drought stress was measured. The initial height of the plants was measured from the soil surface to the apex before withholding water. Final height of the plants was measured at 15d of drought stress treatment. Five normal plants were selected at random and uprooted from each transgenic line and the length of their roots was measured from their tips to the base of hypocotyl and mean root length was expressed in centimeters.

Physiological parameters. Relative water content.
To determine relative water content (RWC), leaves were removed from selected plants as method described by Turner 23 was followed. The (RWC) was determined using the following formula: Measurement of ion leakage (relative membrane permeability). Leaves were removed from selected and were processed for the measurement of ion leakage from cellular membranes via conductivity measurement method.
Detached leaves of drought-stressed and non-stressed plants were washed with deionized water and placed them on filter paper for air drying then placed in 25 ml of deionized water. Initial electrical conductivity (EC 0 ) of deionized water was measured before the leaves to be soaked in it and final electrical conductivity (EC 1 ) was determined after 24 h. Then leaves along with water were autoclaved and after autoclaving electrical conductivity was again measured (EC 2 ). Ion leakage expressed in the form of percentage which was calculated according to the method described by Quisenberry 24 .
Photosynthesis. CO 2 assimilation and transpiration rates were measured in the leaf with a handheld IRGA, gas exchange system (CI-340 Bioscientific Ltd., UK). The net photosynthesis rate by unit of leaf area (Pn, μmol CO 2 m −2 s) was calculated.
Biochemical parameters. Proline content. About 1 g leaf sample was crushed in 10 ml of 3% Sulfosalicylic acid and then the mixture was filtered through Whatman #1 filter paper. 2 ml of filtrate was mixed with 2 ml of acid ninhydrin in glass tubes and incubated for 1 h in boiling water bath. The reaction was terminated after 1 h by providing chilling temperature to the glass tubes. After cooling the reaction mixture, it was vigorously mixed with 4 ml toluene. Layers were formed within the reaction mixture when the glass tubes were allowed to settle at room temperature. The upper layer was separated and the intensity of red color, which indicating the presence of proline, was measured at 520 nm . The concentration of proline was estimated with the help of standard curve, which was drawn by using a known concentration of proline.
Confocal microscopy for GFP fluorescence. Transient expression of GUSP-2 via GFP was determined in representative leaves through confocal microscopy. Transgenic and control leaves were taken, and slides were prepared for confocal imaging. Digital images were taken by confocal laser-scanning microscope (Zeiss LSM 510) equipped with argon laser. Argon laser was used for excitation of GFP at 488 nm and emission wavelength 505 nm was recorded as digital image.

Statistical analysis.
The results were statistically analyzed with software STATISTIX V 10.0 (Tallahassee, USA: https:// stati stix. infor mer. com). By using this statistical software factorial complete randomized design was applied on the data for analysis of variance (ANOVA). LSD (Lest Significant Difference) test was performed to access the significant differences among the transgenic and non-transgenic cotton plant.

Results
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.
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).     ). 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).

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       www.nature.com/scientificreports/ 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.

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).
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  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-M3usp-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-M1usp-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).

Discussions
We characterized cDNA clone of GUSP-2 from Gossypium arboreum that encodes predicted 19.1 kDa protein. This protein has 81% homology with GUSP-1, another protein from cotton. GUSP-2 has two conserved domain, one at N-terminus and other at C-terminus and both are different from each other. This difference may partially explain why this protein has resistance against stress conditions. Interaction of GUSP-2-potein with adenosine monophosphate and the presence of glycosylation, phosphorylation and ATP-binding sites (predicted by in-silico analysis) suggested its involvement in signal transduction. Previously, it was observed that rice OsUSP1 belongs to subfamily of ATP-binding USP, and plant USPs might contain ATP binding domain dimmers [25][26][27] . www.nature.com/scientificreports/ The functinal acitivity of GUSP-2-protein was enhanced by site directed mutations. Three different mutant of GUSP-2 proteins were produced by point mutated the wild type GUSP-2 gene at three different positions 21,28 . The predicted model of GUSP-2 was used as reference to insert three mutations by using MOE tool. By replacing Lysine with proline in mutant-1 (M1-usp-2) its ATP binding capacity was enhanced by 2× and 2nd mutant protein (M2-usp-2) was abnormal with zero ATP-binding capacity, however, CMP capacity of 3rd mutant protein (M3-usp-2) was enhanced by replacing Lysine with Threonine residue. The interaction of 1st and 3rd GUSP-2 mutated proteins with 2gm3. A template was increased by increase in number of the hydrogen bonds, so it was predicted that functional activity of GUSP-2 protein will be enhanced. Previously, it was studied that activity of Mycobacterium-USP-protein was enhanced by enhancing its ATP-binding capacity and it became more virulent [29][30][31][32] .
The GUS has been replaced in pMV vector with 550 bp SpUSP gene from tomato which exhibited significant tolerance to heat, cold, drought and salt stress 33,34 . G. arboreum is known for its resistance against abiotic and biotic stresses, it has priceless gene pool to improve future cotton cultivar 8,32 . Similarly, two genotypes of cotton (FDH-786 and FDH-171) studied by Hassan et al. 5 under 800 mM NaCl stress and declared both are tolerant to salt stress and can be utilized for the improvement of traits in crops. In a comparative study, CIM-496 G. hirsutum was reported drought susceptible cultivar as compared to FDH-786 G. arboreum 4 . Several barley stress related genes (USP-gens) were reported to express under NaCl stress by Li et al. 35 . SbUSP-gene 783 bp from Salicornia brachiata was expressed in E. coli which conferred to osmotic and salt stress 36 . Previously, three salt responsive genes cloned from S. brachiata showed significant expression under abiotic stresses in host plant. Among them expression of SbMT-2 was maximum under salt (500 mM), heat (45 °C) treatments; however, expression decreased under cold stress 37 . Similarly, expression of SbpAPX gene was dercreased under osmatic stress but highest expression was noted under (NaCl 500 mM) stress 38 . The expression of SbGST gene was increased under different NaCl treatments 37 , however, expression of both SbGST and SbpAPX genes was strongly induced under cold stress 36,39 10 reported elevated expression of GUSP-1 and GUSP-2 genes in leaves of G. arboreum under drought stress. Several other putative stress responsive genes were isolated and charachterized in barely 35 . Similarly, USp gene cloned from solanum pennellii was expressed under salt, heat and osmatic stress treatments. It was observed that USP involved in stress adaptation mechanisms under various abiotic stress conditions at cellular level in plants and these genes belonged to ubiquitous gene family.
In spot assay growth of cells heat or might be mutant-1 and wild type GUSP-2 gene exhibited slight tolerance against heat and cold stresses. However, LEA gene cloned from Pogonatherum paniceum differentially affected bacterial tolerance to cold and heat stress 41 . Similarly, bacteria expressing OsLEA5 enhanced tolerance against various abiotic stress conditions including heat 42 and E. coli transformed with MuNAC4 gene demonstrated tolerance in saline medium but grow poorly at 46 °C 43 . Relative fold expression of M1-usp-2 gene under 8% PEG stress in bacterial cells was observed at uppermost level (5.7 fold) as compared to M2-usp-2 (1.8 fold) and W-usp-2 (4.00 fold) which is almost similar to the expression level of M3-usp-2 gene. However, under heat stress all genes did not expressed significantly either because of cell death or genes don't confer resistance against heat stress. Stress associated proteins (SAPs) are concerned with stress related response of plants 28 and universal stress protein (USP) genes provide tolerance against prolgonged abiotic stress condtions and their relative expression level was observed to be increased.
The transcription rate of M1-usp-2, M3-usp-2 &W-usp-2 under various stresses viz. salt and osmatic was enhanced. M2-usp-2 expression in host organisms was also noticable but this mutated protein stay behind to impart significant tolerance. It means mutatnt-2 protein (dephosphorylated, zero ATP-binding capacity) failed to initiate any metabolic process of phosphorylated containing compound (signal transduction). However, mutant-1 (M1-usp-2) protein was found more active (enhanced phosphorylation or max ATP-binding ability) under salt and osmatic stresses as compared to wild type stress protein. Point mutated GUSP-1 gene from G. arboreum was cloned and expressed in G. hirsutum, was more acitve (having more ATP-binding sites) and its expression was maximun in different parts of plants (root, stem, and leaves) as compared to control GUSP-1 gene under drought stress 5 .
When transgenic plants containing mutated and non-mutated GUSP-2 genes were compared with each other, pTM1-usp-2 was found more stress tolerant on the basis of their morphological characteristics. Reduction in stem length in Soya bean plants was reported by Specht et al. 44 . Similarly, 25% reduction in plant height was observed in water stressed citrus seedlings by Wu and Xia 45 . Significant reduction in the stem length of potato plant was also reported under drought stress. The height of other plant species like Abelmoschus esculentus 46 , Vigna unguiculata 47 and Petrosolinum crispum 48 were reported to be reduced under drought stress. Ferreira et al. 49 reported that progressive decline in root to shoot length ratio is because of drought stress in plants especially in G. hirsutum. As drought stress directly affects leaves, so, measurement of relative water content (RWC) and photosynthetic rate were included in this study to measure the degree of drought stress tolerance in transgenic CIM-496 G. hirsutum plants. Ferreira et al. 49 reported that progressive decline in RWC is because of drought stress in plants especially in Gossypium hirsutum. Assaad and Signer 50 found positive relationship between RWC and leaf water content, however, when the stress is disappeared, RWC progressively recovered within 48 h. High temperature and drought stress alters the structure of membrane proteins which enhanced the permeability of membrane and increased the loss of electrolyte 51 . The increased solute leakage has been used as indirect measure of drought stress effects. It has been studied by Chen et al. 52 in potato and tomato, in cotton by Maqbool et al. 53 54 , in cotton by Ashraf et al. 55 , in sorghum by Marcum 56 , in cowpea by Ismail and Hall 57 and in barley by Waheed and Shabir 58 . Proline is considered as a compatible solute as well as osmo-protectant, it protects the plant tissues by producing stress responsive protein 59 . Kumar and Reddy 60 revealed that, when water potential becomes the amount of osmolytes which are imperative for osmoregulation, allows additional water from environment. This helps in minimizing the immediate effect of drought stress. Similarly, Unyayar and Keles 61 , while studying the characteristics of Helianthus annus under drought condition observed a strong correlation between proline content and water deficiency. Proline also play role to stabilize membranes and cellular proteins in the presence of high level of osmoticum 62 . The results of current study are in accordance with the findings of Krasichkova et al. 63 and Mohammadian and Moghaddam 64 who reported that proline and sugar contents were increased under drought stress in drought tolerant plants. Elevated proline level in wheat plant reported under drought stress by Vereyken et al. 65 . Therefore, elevated concentration of USPs in cotton might help in maintaining the proline synthesis which play role to stabilize membranes and cellular proteins.
Real Time expression analysis revealed that wild type and mutated forms of GUSP-2 genes were expressed in all parts of plants, but maximum expression was found in leaves. Comparative analysis showed that expression of M1-usp-2 was maximum (7.8 folds) in leaves as compared to M3-usp-2 (6.2 folds) and W-usp-2 (5.8 folds). Udawat et al. 37 reported increase in the transcription of SbUSP-gene under abiotic stress treatments. In chimeric proteins, GFP can fuse at C or N-terminus enable the transcription of GFP under same regulatory sequence as for target gene [66][67][68] . The GFP was cloned in fusion with mutated and wild type GUSP-2 in pCAMBIA-1301b by replacing GUS with GFP. Likewise, GFP has been used to describe the localization of RAP2. Zinc finger protein-1 and antiphagocytic protein-1 with GFP from rice to identify their cellular localization in leaves of tobacco and tomato 69 .The leaves of CIM-496 G. hirsutum transgenic plants demonstrated the elevated level of GUSP-2-GFP transgene which was localized into the guard cells of leaves through confocal microscopy. In this study, the expression of GUSP-2-protein is localized in guard cells, which is contrary to the findings of Loukehaich et al. 33 as they reported cellular localization of SpUSP in nucleus of stomata and cell membrane of tomato leaves.

Conclusion
GUSP-2 gene from Gossypium arboreum exhibited salt and osmatic tolerance in E. coli BL-21-uspABC mutant strains. The mutant-1 (M1-usp-2) form of this gene was more active and encoded mutated universal stress protein of adenine nucleotide alpha hydrolases superfamily, which conferred more tolerance aginst salt and osmatic stresses, thus could be utilized as an important genetic resource for abiotic stress tolerance. The mutant-1 (M1-usp-2), mutant-3 (M3-usp-2) and wild type (W-usp-2) ATP bound proteins may function as a signaling molecule to activate stress inducible pathway or my be directly involved in salt and osmatic stress tolerance mechanisms. Stress dependent activation and deactivation are of immense importance for biotechnology applications. Further studies will focus on some more suitable side directed mutations to enhance its activity and transformation in plant to analyse its biochemical mode of action.