OsRuvBL1a DNA Helicase Boost Salinity and Drought Tolerance in Transgenic Indica Rice Raised by In-Planta Transformation

RuvBL, is a member of SF6 superfamily of helicases and is conserved among the various model systems. Recently rice homolog of RuvBL has been biochemically characterized for its ATPase and DNA helicase activities, however its involvement in stress is not been studied yet. This study reports the detailed functional characterization of RuvBL homolog of Oryza sativa, under abiotic stress through transgenic approach. An improved Agrobacterium-mediated in-planta transformation method was developed in indica rice to generate the transgenic lines and study was focused on optimization of factors to achieve maximum transformation eciency. Overexpressing OsRuvBL1a transgenics showed enhanced tolerance under in vivo salinity stress as compared to WT plants. The physiological and biochemical analysis of the OsRuvBL1a transgenic lines showed better performance under salinity and drought stresses. Several stress responsive interacting partners of OsRuvBL1a were identied using Y2H method. Working mechanism for boosting the stress tolerance by OsRuvBL1a has been proposed in this study. This integration of OsRuvBL1a gene in rice genome using in-planta transformation method helped us to achieve the abiotic stress tolerant smart crop. This study is the rst direct evidence to show the novel function of RuvBL in boosting abiotic stress tolerance in plants.

RuvB is a highly conserved protein involved in various cellular functions such as cell cycle regulation (Ahmad and Tuteja 2012), mitotic assembly (Morrison and Shen 2009;Sigala et al. 2005), transcriptional regulation of gene expression (Jonsson et al. 2001; Wood et al. 2000) and biogenesis and assembly of snoRNPs (small nucleolar ribonucleoproteins) for the generation of pre-rRNA (Zhao et al. 2008). It is a widely studied protein in other systems but is scarcely studied in plants with few reports on Arabidopsis (Holt et al. 2002;Schorova et al. 2019) and rice . In this study, we report the functional characterization of OsRuvBL1a, a rice homolog of RuvB1 which exhibits nucleic acid independent ATPase and nucleic acid unwinding activity (Sai et al. 2018).
Over-expression of OsRuvBL1a encoding gene in rice plant has been used for the functional validation of the gene. Several methods have been reported for Agrobacterium-mediated transformation of the Indica rice such as tissue culture ( (Dong et al. 2001). Callus regeneration method is the most widely used method for the rice transformation. However, the method is also beset with limitations such as, time consumption, somaclonal variations (Fukui 1983), genotype speci city and epigenetic changes (Smulders and de Klerk 2010) that reduce the rice transformation e ciency. This further reinforces the need for the development of a new transformation method (Hansen and Wright 1999).
In-planta seed transformation of rice is a new emerging and e cient method that overcomes the limitations of callus method (Bent 2000). In-planta rice transformation through seed was rst attempted by Supartana et al. (2005) by piercing the pre-soaked seed embryo using Agrobacterium tumefaciens dipped needle (Supartana et al. 2005). Although this method opened the avenues for new improved way for rice transformation, however, the method need to be improvised to make it more prompt and easy to carry out. We, in the present study, have therefore attempted to improvise the in-planta transformation protocol with improved e ciency. Moreover, the improvised method is faster and easy to perform than the previously reported in-planta transformation methods (Lin et al. 2009; Ratanasut et al. 2017; Supartana et al. 2005). Further, the study involved the detailed analysis of OsRuvBL1a overexpressing transgenic lines produced using this new improved method. The recombinant OsRuvBL1a overexpressing lines seemed to be more tolerant to salinity and drought stress as surmised by reactive oxygen species (ROS) level, membrane stability and chlorophyll retention. Further, Yeast two-hybrid analysis was also carried out to predict the interacting partners of the OsRuvBL1a that may play a part in imparting the stress tolerance to rice.
Experimental Procedure: OsRuvBL1a gene selection Preliminary studies involving stress responsive expression level analysis was performed for OsRuvBL1a gene for the selection of candidate RuvBL gene from RuvBL gene family (Sai et al. 2018).
Generation of OsRuvBL1a overexpressing rice transgenics with in-planta transformation method (i) Plant material and seed sterilization Healthy and mature seeds of IR64 variety of Oryza sativa were collected from IARI, New Delhi, India and dehusked seeds were sterilized as described by Sahoo and Tuteja (2012).
(ii) Preculture of seeds Surface-sterilized seeds were precultured in 100 mL of ½ MS liquid medium with 0.5 mg L -1 of GA 3 in a 250mL Erlenmeyer ask incubated in an orbital shaker (Kuhner, Switzerland) set at a constant temperature of 28 o C for different time periods i.e., 0, 3, 12, 18, 24, and 48 hrs. Optimized concentration as surmised from the optical density of Agrobacterium at 600 nm (OD = 0.4) and co-cultivation duration (up to 24 hrs) is given in the supplementary tables, S2 and S3.
(iii) Agrobacterium strains and binary vector Two strains of Agrobacterium tumifaciens (LBA4404 and EHA105) with gene cassette were used for rice seed transformation.
(iv) Preparation of Agrobacterium inoculum Agrobacterium cells showing absorbance of 0.6-0.8 at 600 nm were harvested at 3500 rpm for 20 min at 4 o C. The harvested bacterial cells were resuspended in co-cultivation medium (Table S7).
(vi) Effect of hygromycin concentration on germination percentage Germination percentage was calculated as the average number of seeds germinated over 5 to 10 days' period. The in uence of hygromycin was observed on seed germination ( Figure S1).

(vii) Seed germination, selection and acclimatization
Agro-infected seeds were inoculated on ½ MS Plates, and incubated in the dark at 27 o C. After 2 weeks, the germinated embryos with small shoots were transferred to jam bottles containing ½ MS media supplemented with hygromycin for selection and then transferred to vermiculite for hardening. Root morphology was closely observed in jam bottles. Putative positive plants which survived on hygromycin were transferred to vermiculite.
(viii) Screening of putative transformants (a) PCR analysis: The PCR reactions were carried out by using two primer combinations such as hptII speci c primers (F'-CAACCTTTTATGAAAAAGCCTGAACTCACCGC and R'-CCACTT AGTCTCGAGTCTATTTCTTTGCCCTCGGACGAG) and CaMV35S promoter forward and gene speci c reverse (ACGGATCCCTCGAGATGAGGATCGAGGAGGTGCAGTCGG).
(b) Histochemical analysis of Gus gene expression: Histochemical analysis of Gus gene expression was performed on leaf sheath from transgenic and WT leaf using the procedure described by Vitha et al. (1995).
(ix) Transformation e ciency Transformation e ciency was calculated based on the presence of the transgene/s in transgenic plants as described by Vitha et al. (1995).

(x) Optimization of co-cultivation media
To attain the higher transformation e ciency, we studied the effect of different Agrobacterium concentrations (corresponding to the Optical density 0.3, 0.4, 0.5 and 0.6), pH (5.0-6.0) of media, surfactant (Tween 20 and Triton X-100) and growth regulator (GA 3 ). The influence of Agrobacterium strains (LBA4404 and EHA105) on the in-planta transformation was also studied.

Copy number analysis
Copy number analysis was performed by using two different approaches of qPCR assay and then its con rmation with Southern hybridization analysis as reported by Passricha et al. (2016).

Analysis of transgenic plants (i) Expression analysis of OsRuvBL1a gene in transgenic plants
Transcript level of OsRuvBL1a gene in rice overexpressing transgenic lines was performed by qPCR method with WT plant as control and Actin as reference gene as described by Tuteja et al. (2013).
(ii) Transgene inheritance and homozygous line selection Transgene inheritance in OsRuvBL1a overexpressing transgenic lines was analyzed by PCR and Gus method. Positive plants were analyzed for homozygous lines by using qPCR approach as described by Passricha et al. (2016) in T 1 generation ). List of primers used in this experiment is given in Table S8.
Physiological analysis of OsRuvBL1a transgenic plants Different physiological analysis such as germination assay, root/shoot length, chlorophyll retention, dry weight and test grain weight were performed as described by (Garg et al. 2012) and Tuteja et al. (2013).

Biochemical analysis for salinity tolerance of transgenic plants
Biochemical analysis for transgenic lines and WT plants was performed as described by Garg et al. (2012) for salinity (200 mM NaCl) and drought (150 mM mannitol) stress.

In vivo stress tolerance
In vivo stress tolerance in OsRuvBL1a overexpression transgenic was analyzed as described by  for 200 mM NaCl.
Qualitative and quantitative measurement of ions Rice OsRuvBL1a overexpressing transgenic lines with WT control were grown for 15 days in normal tap water. The seedlings were then subjected to salinity stress by addition of 200 mM NaCl for 12 hrs duration. Crown roots of these plants (stressed and non-stressed) were used for the confocal imaging for Na + and Ca 2+ accumulation using the method described by Nath et al., (2016). The mean uorescence pixel intensity (MFPI) was calculated using ve independent measurements of the respective uorescent confocal images using ImageJ program (Collins 2007).
Cell Viability 15 days old seedlings of WT and OsRuvBL1a transgenics were subjected to salinity (200 mM NaCl) and drought (150 mM mannitol) stress for 12 hrs duration. Crown roots of these seedlings were analyzed for cell viability by staining of roots using propidium iodide (3 µg/mL; Hi Media) for 1 min as described by Nath et al. (2016).

Identi cation of interacting partners
Interacting partners of OsRuvBL1a were identi ed by yeast two-hybrid (Y2H) assay. The ORF of OsRuvBL1a and another member of this family, OsRuvBL2a were cloned in frame in both the pGBKT7 and pGADT7 vectors at Nde I and EcoR I restriction sites. Rice cDNA library was cloned in pGADT7 vector. The yeast two-hybrid assay was performed as described by Fields and Song (1989. Few interacting partners from Y2H results were validated with Bimolecular uorescence complementation (BiFC) assay for one-to-one interaction as well as for authenticity of Y2H results. Selected interacting partners were cloned in pSPYNE vector and OsRuvBL1a was cloned in pSPYCE vector for BiFC. Abaxial surface of Nicotiana benthamiana leaves were co-transformed with Agrobacterium cells containing pSPYCE and pSPYNE constructs having OsRuvBL1a and selected interacting partners along with p19 RNAi suppressor gene construct as described by Passricha et al. 2019. Transformed plants incubated at 22°C for 2 days and then screened with uorescence microscopy for interactions. pSPYCE and pSPYNE empty vectors were used as negative control.
Agrobacterium cells containing each of the OsRuvBL1a-pSPYCE construct & LecRLK-SPYNE construct were inoculated in 5mL LB containing rifampicin, Kanamycin and Chloramphenicol. Agrobacterium containing p19 RNAi suppressor gene construct were similarly inoculated in 5mL LB media containing rifampicin, gentamycin, and kanamycin using a 2mL syringe, the Agrobacterium cocktail was injected into the abaxial surface of a leaf of Nicotiana benthamiana. Plants were then kept at 22°C and screened after 2 days using a uorescence microscope (ZEISS AXIO Imager.Z2 microsystems from Germany).

Statistical analysis
Average 50 seeds, in three replications were used for each combination and data represented in Table S2 and S4 is the mean±SE calculated by using MSTAT computer program. Least significant difference (LSD) among the means at P>0.05 level of probability was considered as significant. For confocal study, data are presented as relative units of pixel intensities and the average uorescence intensity was calculated via three independent measurements of confocal images using ImageJ software (NIH) in arbitrary units. In addition, respective back-ground pixel intensity (unstained area) of the confocal image was also considered to calculate the nal mean uorescence pixel intensity using ImageJ (NIH). For all graphs, data were analyzed by ANOVA (Analysis of Variance). Dunnett's multiple comparison test was used to test the signi cance between WT and transgenic plants by using GraphPad Prism software version 6.0. The statistical signi cance was represented as "*" and "**" for p<0.05 and p<0.01 respectively.

Gene Selection and characterization
OsRuvBL1a gene has been selected based on our preliminary studies in which it was found to be an abiotic stress-responsive gene (Sai et al. 2018) and thus, make it a potential candidate gene mediating abiotic stress tolerance in rice. OsRuvBL1a encodes for a 55 kDa protein ( Fig. 1 a & b). Recently, it has been reported that puri ed recombinant protein showed nucleic acid independent ATPase ( Development of in-planta transformation method for rice with mature seeds OsRuvBL1a overexpressing rice transgenics were produced by using improved in-planta transformation method, which is less labour-intensive, easy and fast, using the mature rice seeds as explant. This report brie y describes the OsRuvBL1a overexpression transgenic of rice by using this improved in-planta method and its optimization for various factors. (i) Effect of in-planta transformation and hygromycin concentration on germination percentage The hygromycin concentration of 20 mg/L was observed as lethal (Minimum Inhibitory Concentration, MIC) as more than 50% of seeds failed to germinate (Table S1). Later same concentration (20 mg/L) was used for selecting the putative transformed seedlings (Fig. 2a). Wild type control and non-transgenic seedlings died on hygromycin selection ( Fig. 2ai and 2aiii) whereas putative transgenic plants survived and were selected ( Fig. 2aii and 2aiv).  (Table S2). Similarly, for Agrobacterium strain EHA105 mediated transformation, the same concentration of acetosyringone (200 mg/L) resulted in maximum transformation of 36.0±2.0% when incubated for 24 hrs (Table S3).
(iii) In uence of pre-culture duration on in-planta transformation e ciency The transformation e ciency was observed for different durations of pre-culture (0, 3, 12, 18, 24 and 48 hrs). A pre-culture period of 24 hrs was found to be the most suitable for T-DNA delivery, resulting in a signi cant increase in the transformation frequency from 32.7±0.9% to 41.33±3.5% for LBA4404 strain (Table S4).
(iv) Optimization of co-cultivation media for in-planta transformation method This in-planta method is strain-independent (Table S5) because both the strains of Agrobacterium (LBA4404 and EHA105) showed maximum transformation e ciency by 24 hrs co-cultivation. We have optimized that rice seeds pre-cultured for 24 hrs in MS medium with pH-5.8 and co-cultivated with Agrobacterium cells (at O.D. = 0.4) for 24 hrs in the presence of 200 mg/L acetosyringone showed maximum transformation e ciency. The transformed seeds were selected on 20 mg/L hygromycin supplemented MS medium to narrow down the selection process.

Copy number
Results of both the qPCR and Southern blot hybridization methods for copy number analysis of putative transgenic lines (L-1 to L-5) showed that L-1, L-3, L-4 and L-5 have single copy insertion whereas L-2 showed two copies of target gene in rice genome ( Fig. 2d and 2e). Based on these results single copy

Transgene inheritance
The segregation pattern for T-DNA was tested as a single dominant Mendelian gene segregation method. About 45 seeds were taken from each line (L-1, L-4 and L-5) and the results were validated using the classical chi-square ( 2) test taking 1 as degree of freedom. According to χ 2 test value T 1 population did not follow the Mendelian segregation pattern because average chi square value was much higher than table value (20.90) (Table S6). Gus histochemical assay was also performed in T 1 seeds and seedlings for the positive transgenics and it showed the formation of blue coloration in transgenic seeds and seedlings (Fig. 2g).

Physiological analysis of OsRuvBL1a transgenic plants
To examine the role of overexpression of OsRuvBL1a gene in conferring the abiotic stress tolerance in rice, comparative analysis of several physiological tests was performed in WT type and OsRuvBL1a overexpressing transgenic lines (L1, L4 and L5) under salinity and drought stress conditions. Germination assay revealed that under stress-free control conditions there was no signi cant difference in germination pattern in WT as compared to OsRuvBL1a transgenic lines ( Fig. 3a-d), however, when grown under salinity (200 mM NaCl) and drought (150 mM mannitol) stress the transgenic lines were germinated earlier than the corresponding WT. The seeds exposed to salinity stress had the germination percentage of ~60%, 75% and 55% (Fig. 3d); whereas, under drought stress the germination rate was 68%, 80% and 62% for lines L-1, L-4 and L-5, respectively (Fig. 3d). The WT seeds exhibited only ~15% germination under both the stress conditions (Fig. 3d). High root/shoot ratio and strong root architecture helps plant to overcome the salinity (Fig. 3e) and drought stress conditions (Fig. 3f). Transgenic lines (L-1, L-4 and L-5) showed root/shoot ratio of 1.2, 1.3 and 1.5, respectively under salinity stress (Fig. 3g) and 0.7, 0.8 and 0.8, respectively under drought stress (Fig. 3g). Whereas WT plants showed root/shoot ratio of 0.2 and 0.6 under salinity and drought stress (Fig. 3g). Higher dry weight of plant indicates lower water retention capacity. Lines L-1, L-4 and L-5 showed 1.7, 1.8 and 1.5-fold lesser dry weight, respectively than WT plants under the salinity stress conditions (Fig 3h). Under drought stress, all the three transgenic lines showed 1.8-fold lesser dry weight as compared to WT plants (Fig. 3h), whereas under the normal control conditions, transgenic lines and WT plants did not have signi cant difference in their respective dry weight (Fig. 3h). Stress conditions affected the photosynthetic machinery of plants and caused chlorophyll degradation (Fig. 3i-l). Total chlorophyll content measured in WT and OsRuvBL1a transgenic lines showed about 5-fold high chlorophyll content in all the three transgenic lines (L-1, L-4 and L-5) as compared to WT plants during salinity stress (Fig. 3j). Under drought stress, lines L-1and L-5 showed about 2.5-fold higher retention of chlorophyll content as compared to WT plants, whereas L-4 line showed 1.5-fold higher chlorophyll retention (Fig. 3l). showing maximum accumulation of proline under both salinity (Fig. 4c) and drought stresses (Fig. 4i). All transgenic lines showed higher electrolyte retention as compared to the WT plants with line L-4 showing maximum electrolyte retention under salinity stress (Fig. 4d) and line L-1 showed maximum electrolyte retention under drought stress (Fig. 4j). Overall these observations suggest the reduced effect of stress and damage to cell membrane in transgenic lines and con rm the role of OsRuvBL1a in maintenance of the cell membrane stability under the abiotic stresses. The activity of ROS scavenging enzymesascorbate peroxidase (APX) and catalase were also estimated. Both the enzymes APX and catalase use H 2 O 2 as substrate to reduce it into water. Under salinity stress condition transgenic line L-4 showed higher APX and catalase activities as compared to WT plants (Fig. 4 e & f). Under drought stress the transgenic line L-4 showed maximum activity of APX and catalase (Fig. 4 k & l). These biochemical analyses showed improved ROS scavenging machinery and maintenance of cell integrity in transgenic lines as compared to WT plant under stress conditions.
Determination of ion content and cell death in response to abiotic stresses The quanti cation of ions accumulation and cell death in plant tissue is another method to observe the effect of stress conditions on plants. In this study, we measured the effect of salinity stress and drought stress on rice seedlings. Sodium ion (Na + ) accumulation in root tissue during salinity stress was studied as a method for quanti cation of Na + ion imbalance in plants during stress conditions. CoroNa green dye was used for the non-destructive monitoring of relative accumulation of Na + ion in roots of WT and OsRuvBL1a overexpressing transgenic lines by using the confocal microscopy. The transgenic line L-5 showed the least uorescence and lines L-1 and L-4 also showed lesser uorescence as compared to WT roots under salinity stress (Fig. 5 a & b). These results suggest that more Na + ion accumulation occurred inside WT roots during salinity stress as compared to the transgenic lines. For the study of calcium ion (Ca 2+ ) accumulation an esteri ed form of Fluo-4, Fluo4-AM was used. Fluo4-AM is a Ca 2+ sensitive uorescent probe indicator which shows increase in uorescence upon binding with cytosolic Ca 2+ .
Confocal microscopy of WT and transgenic roots under salinity stress was used to observe the cytosolic Ca 2+ accumulation. Higher uorescence in WT roots as compared to transgenic line L-1 suggested an accumulation of more cytosolic Ca 2+ in WT roots under stress conditions (Fig. 5 c & d). Stress conditions also cause ionic imbalance and oxidative stress that make the cells inviable. Cell viability can be studied in plant root in non-destructive manner by using propidium iodide (PI) dye. PI is a membrane impermeant dye and hence viable cells show less or no uorescence whereas it penetrates in dead cells and intercalates in double stranded DNA and provides orescence on excitation. The cell viability study of rice root tissue under salinity and drought conditions showed higher uorescence in roots of WT plants as compared to transgenic lines under salinity stress (Fig. 5 e & f) and drought stress (Fig. 5 g & h). These results showed that stress conditions caused higher cell death in WT plants as compared to overexpressing transgenic lines. These studies suggest the role of OsRuvBL1a in providing stress tolerance under stress conditions at ionic and cell viability level.

Salinity and drought stress tolerance under in vivo conditions
WT and the transgenic plants (L-1, L-4 and L-5) overexpressing OsRuvBL1a gene showed signi cantly different behaviors under salinity and drought stress (Fig. 6). At day-1, all the plants of same age were exposed to salinity stress (200 mM NaCl) as well as drought stress (non-availability of water) as shown in Fig. 6 a & c. On day 20 salinity stress, the WT plant could not survive salinity stress till 20 days, on the other hand, the OsRuvBL1a transgenic plants withstood the stressed condition (Fig. 6b). Among the transgenics, the L-4 line was found to be the most tolerant line. For drought stress, WT plant died after 15 days while OsRuvBL1a-overexpressing transgenic plants were thriving (Fig. 6d).

Isolation and identi cation of interacting partners of OsRuvBL1a through yeast two-hybrid method
Physiological and biochemical analysis of overexpressing transgenic lines showed better performance as compared to WT under salinity and drought stress. To understand the working mechanism of OsRuvBL1a, its interacting partners were identi ed by using yeast two-hybrid method. Sequential selection of transformed yeast on two drop outs (2-DO) (-Leu, -Trp), 3-DO (-His, -Leu, -Trp) and 4-DO (-Ade, -His, -Leu, -Trp) media plates followed by lter lift assay showed positive interacting clones. The colonies found positive on X-gal assay were sequenced and analyzed using rice genome to nd the interacting partners of OsRuvBL1a enlisted as Table 1. Y2H (one-to-one interaction) as well as BiFC studies showed that OsRuvBL1a does not self-interact whereas, it exhibits strong interaction with another member of its family, OsRuvBL2a. The OsRuvBL1a-OsRuvBL2a interaction led to the formation of a hetro-oligomeric structure by these proteins. BiFC based one-to-one interaction study of OsRuvBL1a with few selected partners showed positive interactions and validate the Y2H results ( Figure S2). These validated interacting partners may have some direct or indirect role in conferring the stress tolerance to the plants.  The transformation e ciency is a factor of media composition, incubation period, acetosyringone concentration and seed treatments (Mayavan et al. 2013). Our method is based on the principle that transformation of meristematic cells at an early stage makes the inheritance stable from the parent generation to the next generation (Sajib et al. 2008). In this method, rice seeds were allowed to germinate in MS liquid medium with Agrobacterium to facilitate the integration of transgene during germination, as meristematic cells are rapidly dividing and highly receptive at this stage (  (Sangwan et al. 1991). In addition, pre-culture also prevents the negative effect of Agrobacteria and selection agent in the seed transformation. Similarly, previous reports also claimed an improvement of transformation e ciency due to pre-culturing (Mariashibu et al. 2013). After optimization of several factors, we conclude that this newly developed in-planta transformation method is better than previous methods because it is less labor-intensive, less time-consuming, strain-independent and cost-effective as well.
Salinity and drought inhibit seed germination and cause retarded root/shoot growth due to the osmotic imbalance in plant cells ( ions in WT roots than the transgenic lines grown in identical conditions. These results can be correlated with higher rate of cell deaths in WT plants grown under stress conditions. These results further reinforces that OsRuvBL1a is involved in regulation of many cellular activities since the Ca 2+ ion ux has been reported to initiate onset of various cellular pathways, which initially protect cells from stress but eventually lead to cell death under continuous stress conditions (Lin et al. 2008).
Hybridization studies revealed that OsRuvBL1a interacts with OsRuvBL2a, which is another member of the RuvB family, however no evidence of inter-molecular interaction is found between two OsRuvBL1a molecules. RuvB1 and RuvB2 interactions are also been reported earlier that results in the formation of The interacting partners of OsRuvBL1a that might play some direct or indirect role in the stress tolerance have been listed in Table 1 However, no such study has been carried out in the plant system, and our study may provide a lead to further explore the role of OsRuvBL1a as a part of some chromatin remodelling complex and therby effectuating the stress tolerance in rice. This study focused on the detailed characterization of rice homolog of RuvBL1 (OsRuvBL1a).
OsRuvBL1a exhibits ATPase and helicase activities that could be essential to perform cellular functions. Transgenic Rice plants over-expressing OsRuvBL1a encoding gene were developed for its functional validation through an e cient Agrobacterium-mediated in-planta transformation method. This study expounds a highly e cient method for rice transformation. OsRuvBL1a interacts with different proteins which directly or indirectly are involved in providing stress tolerance to the plant as shown in working mechanism (Fig. 7). Multiple physiological and biochemical analysis showed the better performance of OsRuvBL1a overexpressing transgenic lines under salinity and drought stress as compared to WT plants.
This is the rst report that details the role of RuvBL1 in plants and the information of its characteristics, highlighted through the present study, lays the foundation for the potential use of this enzyme in crop improvement. Furthermore, its role in mitigating the negative effects of abiotic stress in plants indicate its potential use in engineering plants for overcoming abiotic stress. Declarations:

Con ict of interest
We do not have any con ict of interest to declare.

Author Contribution Statement
SKS and NP conducted the experiment, NT and MN designed the study and SSG help in writing the manuscript. RT and NT thoroughly reviewed the manuscript.     were determined by two way analysis of variance (ANOVA) using Graphpad PRISM. The statistical signi cance was represented as "*" and "**" for p<0.05 and p<0.01 respectively.