Study On The Function Of Arabidopsis AtSIS Gene Under Abiotic Stress

Various abiotic stresses seriously affect the growth and development of plants.At present,a variety of molecular mechanisms of plants against stress are revealed,and inherently disordered proteins play an important role in this process.AtSIS encodes an inherently disordered protein that participates in plant response to stress,but its specific function remains unknown.In this paper,the target protein was identified by prokaryotic expression and mass spectrometry,and the spatial structure of AtSIS protein was predicted by using bioinformatics software;pCsGFPBT-AtSIS and AtSIS-pCCFP plant expression vectors were constructed to analyze the subcellular localization of AtSIS protein under abiotic stress conditions.At the germination,seedling and bolting stages of Arabidopsis,salt,drought,heat,cold,exogenous ABA and oxidative stress treatment were performed on the T-DNA insertion mutants,overexpressors and wild type of AtSIS,respectively.Physiological characteristics and the transcription level of AtSIS and related genes were analyzed to reveal the function of AtSIS under different abiotic stresses.The main findings are as follows:A prokaryotic expression vector AtSIS-pET22b was constructed,and two bands of similar size to the target protein were obtained by induced expression,separation and purification.Mass spectrometry identified both bands as AtSIS protein.SOPMA and SSpro software were used to predict the structure of AtSIS:the secondary structure was random coil mainly,which cannot form a stable tertiary structure,confirming that AtSIS is an inherently disordered protein.Laser confocal microscopy found that under normal growth conditions,AtSIS protein is localized in the nucleus and guard cells,however,AtSIS protein is mainly localized in guard cells under NaCl,drought,heat,cold,exogenous ABA and H₂O₂ stress treatment.AtSIS overexpression affects transpiration rate,stomatal density and stomatal opening of Arabidopsis thaliana leaves.AtSIS is involved in the regulation of stomata opening and closing under stress in an ABA-dependent manner.AtSIS promoted stomatal closure under salt,drought and cold stress;AtSIS inhibited stomatal closure under heat,exogenous ABA and oxidative stress.Analysis of AtSIS T-DNA insertion mutants,overexpressors,and wild-type phenotypes revealed that AtSIS negatively regulated Arabidopsis tolerance to salt,drought,and cold,while positively regulated Arabidopsis tolerance to heat and oxidative stress during germination.AtSIS positively regulated Arabidopsis tolerance to salt,drought and cold,and negatively regulated Arabidopsis resistance to high temperature and oxidative stress at Arabidopsis seedling stage by a comprehensive analysis of phenotype,stomatal opening,SOD,POD activity,MDA content.Analysis of phenotype,SOD,POD activity,and MDA content showed that AtSIS negatively regulated Arabidopsis tolerance to salt,drought and heat,and positively regulated Arabidopsis tolerance to cold and oxidation stress during Arabidopsis bolting period.Analysis of the data from exogenous ABA treatment showed that AtSIS was sensitive to ABA at the germination and bolting stages of Arabidopsis,and AtSIS might play a negative regulatory role in the ABA signaling pathway at the seedling stage.The gene expression results showed that the alternative splices of AtSIS.1 and AtSIS.2 were involved in abiotic stress response.AtSIS gene expression was induced under NaCl,drought,heat,cold,exogenous ABA and H₂O₂ stress treatment.AtSIS upregulated AtNAP expression under salt and heat stress,while AtSIS downregulated AtNAP expression under cold stress,indicating that AtSIS may be an upstream regulator of AtNAP in ABA-dependent signaling pathway.Expression analysis of stress-responsive genes?RD29A,COR15B,HSP101?and ABA synthesis and ABA signaling pathway-related genes?NCED3,SnRK2.3,SnRK2.6?showed that AtSIS upregulated SnRK2.3 and SnRK2.6 expression under NaCl treatment;AtSIS downregulated the expression of NCED3 under drought stress;AtSIS upregulated the expression of NCED3 and SnRK2.6 under heat stress;AtSIS upregulated the expression of RD29A,COR15b,NCED3,and SnRK2.6 under cold stress;AtSIS upregulated the expression of NCED3 and Sn RK2.3 under oxidative stress;AtSIS downregulated the expression of NCED3 and upregulated the expression of SnRK2.3under exogenous ABA treatment.The results indicated that AtSIS participated in ABA synthesis and ABA-dependent pathways in response to abiotic stress.In summary,AtSIS protein was mainly localized in guard cells under stress.AtSIS participated in the regulation of stomata opening and closing in ABA-dependent under stress.AtSIS affected ABA synthesis by regulating the expression of NCED3,and AtSIS responded to abiotic stress in the ABA-dependent pathway via regulating the expression of SnRK2.3 and SnRK2.6.AtSIS affected plant stress response by promoting the expression of downstream transcription factors RD29A and COR15b under cold stress.