Molecular Mechanism By Which Arabidopsis BRCA1 Represses RRTF1-mediated ROS Production Under Dehydration Stress

Plants,which serve as producers in the ecosystem,play an irreplaceable role in the entire biosphere.Because of a sessile lifestyle,plants need to deal with complex and changeable environments,including stress environments that are harmful to plant growth and development.These adverse stress environments mainly include the following two categories:one is biological stress,including pathogenic microbial infection and herbivore feeding and etc.The other is abiotic stress,including drought stress,high temperature,cold damage,nutrient deficiency,salt stress,and soil pollution of toxic metals such as aluminum,arsenic and cadmium.Among them,drought stress is one of the common abiotic stress,which severely restricts the growth of plants.Plants change the rate of water transpiration via stomata to regulate drought stress tolerance.The opening and closing of stomata are regulated by different factors,including water potential,CO₂ concentration,light intensity,reactive oxygen species(ROS)and abscisic acid(ABA)content.As a central signaling molecule,ROS plays an important role in regulating the opening and closing of stomata.Breast cancer susceptibility gene 1(BRCA1)is widely studied in animals as a tumor suppressor.The research on its function in plants mainly focuses on the process of DNA damage repair.However,the function of BRCA1 gene in response to abiotic stress in plants is still unclear.In the previous research,it was found that the atbrca1 mutant showed a drought-resistant phenotype,and under normal conditions,the atbrca1 mutant accumulated more ROS in the plant compared with the wild type.In this research,we used a variety of analytical methods such as molecular biology,genetics,cell biology,plant physiology,and bioinformatics to explore the function and molecular mechanism of AtBRCA1 in response to drought stress.The main findings are as follows:(1)The atbrca1 mutants showed higher sensitivity to stomatal closure in the presence of H₂O₂ and ABA than Col-0 and the two complementation lines.(2)To measure ABA-induced ROS production,we treated with ABA to Col-0,atbrca1mutants and AtBRCA1 complementation lines.It was found that ABA-induced ROS production in atbrca1-1 and atbrca1-2 was higher than in Col-0 and complementation lines in guard cells.(3)By comparing the up-regulated ROS-related differentially expressed genes in atbrca1-1 and RRTF1OX(RRTF1 overexpression plants),it was found that 31%of them can overlap.This suggests that AtBRCA1 and RRTF1 together affect the expression of ROS-responsive genes.(4)Through short-term water loss and long-term drought experiments on Col-0,rrtf1mutants and RRTF1OX,it was found that compared with Col-0 seedlings,the rate of water loss was lower in RRTF1OX seedlings and higher in rrtf1 seedlings.Moreover,under long-term dehydration stress,RRTF1OX displayed higher survival rates and rrtf1 showed lower survival rates than Col-0.(5)To measure ABA-induced ROS production,we treated Col-0,rrtf1 mutants and RRTF1OX with ABA.The accumulation of ROS was enhanced in RRTF1OX seedlings but reduced in the rrtf1 mutant seedlings.(6)By comparing the survival rates under drought stress and ABA-induced ROS production in stomata of Col-0,atbrca1-1,rrtf1,and atbrca1-4 rrtf1 double mutants,the atbrca1-4 rrtf1 double mutant displayed similar survival rates and ROS accumulation to rrtf1.These results indicate that AtBRCA1 is epistasis to RRTF1.(7)Using co-immunoprecipitation(Co-IP),bimolecular fluorescence complementation experiment(Bi FC)and yeast two-hybrid experiment,it is found that AtBRCA1 and RRTF1can interact in vivo and in vitro,and AtBRCA1 can interact with RRTF1 through its PHD domain of C-terminal directly.In conclusion,the results of this study indicate that under drought stress conditions,AtBRCA1 and RRTF1 jointly regulate the expression of ROS response genes and affect intracellular ROS accumulation,thereby regulating stomatal closure and responding to drought stress.Overall,our study reveals a novel molecular function of AtBRCA1 in the transcriptional regulation of intracellular ROS homeostasis under dehydration stress.