Roles Of BCAT4 And RGA1 In Rice Response To Drought Stress

Drought is an important factor affecting the growth and yield of rice,which reduces the water potential in soil environment and causes water loss and oxidative damage of plant cells.It’s of importance to search for drought-resistant genes and study their functions,and cultivate water-saving and drought-resistant rice varieties to ensure stable and high yield of rice.Previous studies have shown that rice Ga protein(RGA1)is involved in the response to drought stress,but its response mechanism is still unclear.In our previous study,we identified BCAT4,a RGA1 interacting protein,from a rice cDNA library.In this study,the interaction between RGA1 and BCAT4 was verified by bimolecular fluorescence complementation technique.In this study,the interaction between RGAl and BCAT4 was confirmed using bimolecular fluorescence complementation technology.The materials used in the experiment included wild type NIP,BCAT4 mutant B1,and RGA1 mutant R5 that were retained in the laboratory.A Double mutant(BR)was obtained by crossing with B1 and R5,and an overexpressing strain of RGA1(ROE)and BCAT4(BOE)were obtained by transgenic technology,was used PEG to simulate drought stress at seedling stage and natural drought stress was used at tillering stage to observe phenotypes of different rice genotypes(wild type NIP,single mutant B1 and R5,double mutant BR,overexpressed strains BOE and ROE),and physiological and molecular indexes related to drought resistance and yield were determined to study the mechanism of BCAT4 and RGA1 in response to drought stress.The results were as follows:(1)Biogenic analysis showed that there were multiple ABRE dehydration response elements in the promoter sequence of BCAT4 gene,which might be induced by ABA.BCAT4 was expressed in the whole growth stage of rice,and the expression level was higher in the middle vegetative growth stage and early reproductive growth stage.The interaction network of BCAT4 and RGA1 analyzed by STRING included Gα,Gβ and five enzymes involved in BCAAs metabolism.BiFC technology was used to verify the interaction between BCAT4 and RGA1 proteins on the cell membrane in tobacco leaf cells.(2)BCAT4 overexpressing strain BOE and RGA1 overexpressing strain ROE were obtained by transgenic technology,and qPCR results showed that the relative expression of BCAT4 in BOE was increased by 7.89 times,and the relative expression of RGA1 in ROE was increased by 3.65 times.BCAT4 and RGA1 double mutant BR was obtained by crossing with B1 and R5 as parents.Sequencing showed that both BCAT4 and RGA1 genes of BR were mutated.(3)Under controlled conditions,RGA1 expression significantly decreased in BOE and remained unchanged in B1,while BCAT4 expression significantly increased in R5 and did not change in ROE compared to NIP.Following drought stress treatment,both RGA1 and BCAT4 gene expressions were found to increase significantly.Compared with NIP,the expression of RGA1 increased in B1 but decreased in BOE;whereas the expression of BCAT4 increased significantly in R5,but did not change significantly in ROE..(4)The results of PEG simulated drought test in rice seedlings showed that the loss of BCAT4 significantly decreased the BCAT enzyme activity,the content of BCAAs,the content of endogenous ABA,the expression of stress-related genes and physiological indexes of rice under drought stress,and the drought survival rate of rice seedlings decreased by 38.52%.Overexpression of BCAT4 improved drought resistance of rice seedlings.Loss of RGA1 decreased seedling height,increased root-shoot ratio,and changed plant configuration to enhance drought tolerance,while overexpression of RGA1 had no significant effect on most of the above indexes.The results of principal component analysis on morphological and physiological indices related to stress resistance and the comprehensive factor scores for drought tolerance in different rice genotypes showed that compared to NIP,B1’s factor score decreased by 5.78%,BOE’s increased by 5.21%,R5’s increased by 10.2%,BR’s increased by 11.45%and ROE’s remained similar to NIP’s.The impact of RGA1 on rice drought tolerance surpasses that of BCAT4.(5)Compared to NIP,B1’s growth was significantly hindered and its yield decreased by 12.56%after alternate dry and wet irrigation at tillering stage.BOE’s aboveground and root dry weight as well as total leaf area index increased by 5.71%,14.58%,and 7.33%,but its yield did not show significant changes.The growth and yield of single mutant R5 and double mutant BR were both significantly inhibited,while ROE’s growth and yield remained relatively stable.In conclusion,RGA1 negatively regulates rice response to drought by affecting seedling height and root-shoot ratio,while BCAT4 positively regulates rice response to drought stress.The principal component analysis revealed that RGA1 had a stronger negative impact on rice drought resistance than the positive effect of BCAT4 and the two proteins resist the response of rice to drought stress.BCAT4 and RGA1 may interact on cell membranes,and be related through the metabolic process of BCAAs.These findings provide a theoretical and practical basis for further research on the role of BCAT4 and RGA1 proteins in rice breeding for improved drought resistance.