Identification Of Functional Guard Cell SnRK2 Genes And Redox-regulated Sites Of BnSnRK2.6-2C In Brassica Napus

In plants,stomata consisting of guard cells in the leaf epidermal layer forms pores for controlling gas exchange,CO2 uptakes,water loss and microorganism invasion defense by modulating the aperture.Thus,stomatal movement plays a critical role in response to drought,pathogen invasion and phytohormones like abscisic acid(ABA)and methyl jasmonate(MeJA).Plants have adapted to adverse environmental conditions,such as drought,salinity,toxicity,and high/low temperatures,by developing adaptive features at morphological,physiological,and molecular levels.Among these,signaling pathways are essential for plant growth and development since they sense and relieve environmental stresses.In particular,protein phosphorylation and dephosphorylation by protein kinases and phosphatases,respectively,play a crucial role in stress-induced signal transduction.Sucrose non-fermenting-1(SNF1)-related protein kinases(SnRKs)are classified as SnRK1,SnRK2,and SnRK3 in plants.Among three SnRKs,SnRK2 is the most well studied group in plants because of their major roles in abscisic acid signaling and plant responses to osmotic stress,and members of SnRK2 play key roles in ABA-induced stomatal closure pathways.In the pathways which SnRK2s participated in guard cells,redox reagents and environmental condition changes regulate their activity,and then interfere stomatal movements.Brassica napus is an important oil crop,its tetraploid genome and close relationship with model plant Arabidopsis thatliana provide advantages of studying polypoid plants adapting to environments.Furthermore,the technique of extracting guard cells protoplasts of B.napus is also well studied,providing convenience for researching mechanisms of plants adaption to environment through stomatal movements.In this study,mechanism of redox regulation to funcational SnRK2s in B,napus guard cells was analyzed,results of this study are as following:First,thirty SnRK2 genes were predicted in Brassica napus genome using bioinformatics methods by comparing sequence information from B.rapa,B.oleracea and Arabidopsis thaliana.Among them,coding sequences of nine SnRK2s were cloned from guard cell protoplasts(GCPs)and identified by sequencing.Second,qPCR was used for analyzing expression patterns of 14 BnSnRK2s including the 9 genes identified from GCPs.Twelve of the 14 genes were detected in tissues and organs of different growth periods,meanwhile drought stress in leaf tissue and ABA treatment in GCPs can induce up-regulation of these genes.Among them,BnSnRK2.6-2C performed best specificity in GCPs and robust response to ABA treatment.Third,BnSnRK2.6-2C expressed and purified from prokaryotic system showed autophosphrylation activity in vitro.Kinase activity of BnSnRK2.6-2C was revisibly inhibited by GSNO and GSSG in a dose-dependent manner.Oxidation mofication level of BnSnRK2.6-2C can be also detected increased in a revesible way by GSNO treatment.Fourth,in the mass spectrometry identification of redox-regulated cysteine residues by mBBr reverse labeling strategy,reversible oxidation modifications were identified formed by GSNO and GSSG.Cysteine residue C159 perfomed the best redox sensitivity among the 6 cysteine residues in BnSnRK2.6-2C.Furthermore,the irreversible oxidation sulfonic acid was identified,glutathionylated cysteine residues were identified cannot be totally reduced in GSNO,GSSG or reduced gluthione treatments,indicating the kinase activity inhibition of BnSnRK2.6-2C caused by GSNO and GSSG was mainly from redox environmental chages but not directly cysteine modifications.Finally,tyrosine residues with mBBr labeling were identified in the mass spectrometry identification analysis,this discovery connected knowledge gap through researches between decades.Results in this study complemented information of SnRK2s in B.napus and their responses in guard cells under stress treatments,provided experimental bases for further researches of polypoid crops adaption to environments trough stomatal movements,and supports to studies about redox-regulated stomatal movements through SnRK2s in the future.