Dissecting The Molecular Mechanism Of Canola MPKa-WRKYa Cascade In Regulating ROS Accumulation And Cell Death

Canola(Brassica napus L.)is one of major oil crops with important economic value.However its yielding is often affected by biotic or abiotic stesses.Therefore,it is very important to explore new genes modulating stress tolerance and to dissect the underlying molecular mechanisms in canola.Mitogen-activated protein kinase(MAPK,MPK)signaling cascades have a pivotal role in plants’ against biotic,abotic stresses as well as in plant growth and development.Upon challenged by environment stresses,MAPK cascades in plants can transduce and amplify the signals producing from sensors/receptors to the downstream stress responsive genes through three titre phosphorylation,and finally lead to cellular response.WRKY transcription factors constitute one of the largest transcription factor families in plants.They usually fulfil their functions through binding to W-box elements present in the promoters of downstream target genes and modulating their transcription.Recent studies have demonstrated that WRKY activity can be regulated through phosphorylation by MPKs.Reactive oxygen species(ROS),an important type of signaling and toxic molecule of plants suffering environment stresses,can be regulated by MPKs and WRKYs.Both MPKs and WRKYs have a close relathionship with ROS accumulation.However,report about how an MPK regulates ROS through a WRKY is largely unknown.In our previous work,we found that transient overexpression of BnaMPKa and BnaWRKYa in tobacco(Nicotiana benthamiana)induced ROS accumulation and cell death.We also observed that BnaMPKa interacts with BnaWRKYa.Hence,we speculated that BnaMPKa may activate ROS accumulation through phosphrylating BnaWRKYa.Therefore,we furthermore investigated the function and underlying mechanism of BnaMPKa and BnaWRKYa.Our results showed that overexpression of BnaMPKa and BnaWRKYa in tobacco could cause accumulation of malondialdehyde(MDA),DNA fragmentation,and regulate the expression of a series of genes related to ROS homoestasis,cell death,leaf senescence and defense response.These results implied that BnaMPKa and BnaWRKYa may induce ROS accumulation and cell death by regulating ROS-related target genes.Furthermore,a dual luciferase assay showed that co-expression of BnaMPKa and BnaWRKYa,could increase the expression of NbGPX2 and NbGST than expressing BnaMPKa or BnaWRKYa alone.Using online bioinformatic tools,we found that classical W-box elements and phosphorylation sites exist in the promoters of NbGPX2,NbGST and protein sequence of BnaWRKYa,respectively.In vitro electrophoretic mobility shift assay(EMSA)confirmed that BnaWRKYa could bind to the W-box as well as promoters of NbGPX2 and NbGST.However,whether Bna MPKa could increase binding activity of BnaWRKYa is still under investigation.More interestingly,Arabidopsis leaves of BnaMPKa overexpression lines showed premature senescence phenotype than wildtype after treated with jasmonic acid(JA),whereas mutant mpka showed delayed senescence triggerred by JA.Our results indicate that BnaMPKa functions in JA-induced leaf senescence and further work will be needed to elucidate the mechanism.