Cloning And Identification Of MAPKKK Genes From Rapeseed And Functional Characterization Of Bna MAPKKKx Modulating ROS Accumulation And Cell Death

Mitogen-activated protein kinase(MAPK/MPK)cascades are a highly conserved signal module in plants,which acts downstream of receptors/sensors and works as a hub to transduce and amplify the signals from extracellular into intracellular space and finally induces cellular responses to regulate plant growth and development as well as to combat with various stresses.MAPKKKs,being the uppermost components in the cascade,could phosphorylate downstream MKKs or other targets to fufil their functions.So far,only a few of MAPKKK members have been functionally studied even in the model plant Arabidopsis,due to possible functional redundancy of MAPKKK family.Rapeseed(Brassica napus L.)is one of the most important oil crops in China and worldwide.However,it is often threatened by environmental stresses and pathogens.Therefore,it is of great value to mine resistant genes so as to enhance rapeseed's resistance towards abiotic and biotic stresses.In the present work,we firstly cloned the cDNA sequences of a few members of MAPKKK family in rapeseed,and analyzed their sequences as well as assayed the interactions between BnaMAPKKs and downstream multiple BnaMKKs through yeast two-hybrid(Y2H)system.We identified quite a few different signaling modules compared to those of Arabidopsis.Further,we succeeded in identification of a novel member of BnaMAPKKKs family,namly,BnaMAPKKKx,expression of which elicited ROS(reactive oxygen species)accumulation and hypersensitive response(HR)-like cell death.This function was supported by DAB staining,quantification of malonaldehyde(MDA)and chlorophyll content,measurement of relative conductivity as well as evaluation of nuclear DNA fragmentation.In order to clarify the mechanism of BnaMAPKKKx,a Y2 H screening was performed using the full length and deletion form of BnaMAPKKKx as the baits and we found that a few proteins interacting with BnaMAPKKKx,including BnaMKK6/8,BnaMPK6 and Arabidopsis sucrose-nonfermenting related protein kinases 1.1/1.2(SnRK1.1/1.2)implicated in sugar signaling,AtAHG3/PP2 CA belonging to serine/threonine-protein phosphatase 2C negatively regulatig ABA signaling.Furthermore,the interactions between BnaMAPKKKx and BnaMKK6/8 were confirmed by biomolecular fluorescence complementation(BiFC).Furthermore,BnaMKK6 could be phosphorylated by BnaMAPKKKx in a protein phosphorylation assay.But it is not clear which stimuli could lead to the phosphoryation of BnaMAPKKKx.Afterwards,qRT-PCR was performed to investigate the change of transcript level of genes related to ROS homeostasis and defense response,and we observed that NbMEK2,MPK4,RbohB and WRKY33 were significantly up-regulated.Moreover,VIGS(Virus-Induced Gene Silencing)technology was employed to silence the possible downstream targets including NbMEK1,MEK2,SIPK,WIPK,NTF6,MPK4,RbohB and WRKY33 individually.The results showed that silencing of NbMEK2 could alleviate the ROS accumulation elicited by expression of BnaMAPKKKx,however,silencing of NbMPK4 accelerated ROS accumulation and cell death as a result of BnaMAPKKKx expression.We further studied function of BnaMAPKKKx in transgenic Arabidopsis through manipulating the expression level of BnaMAPKKKx using constitutive and inducible expression system.After the constitutive expression and inducible overexpression lines have been generated,we subjected them to phenotypic assay.We found that growth of most constitutive expression seedlings of T1 generation was arrestted,which could not bolt or to be harvested.Similarly,expression of BnaMAPKKKx in inducible overexpression lines seriously influenced the growth and development after sprayed with ?-estradiol and especially the rosetta leaves showed ROS accumulation and cell death.On the contrary,the growth of Arabidopsis T-DNA insertion mutant-mapkkkx is better than wild type,especially with bigger cotyledons.Therefore,our reasearch will not only help us to know the characteristic of BnaMAPKKK family comprehensively,but also lay a solid foundation for more detailed characterization of MAPKKK genes in rapeseed.Moreover,our study about the regulation of ROS accumulation and cell death by BnaMAPKKKx will have important theoretical and practical guiding value for improving yielding and quality as well as resistance towards pathogens in rapeseed.