Regulation Of Arabidopsis Matrix Metalloproteinases By MAP Kinases And Their Function In Leaf Senescence

Leaf senescence is a programmed cell death process influenced by a variety of endogenous signals and environmental factors.MAPK cascades are major pathways by which extracellular stimuli are transduced into cellular response in eukaryotic cells.In Arabidopsis,MKK4/MKK5-MPK3/MPK6 module has been shown to involve in plant growth,developmental,and immunity.The expression profiling data in DD transgenic plants after DEX treatment reveals matrix metalloproteinases?MMPs?are one of the highest induced genes.MMP is a family of zinc-and calcium-dependent endopeptidase had been reported in plant.In this study,we investigate the functions of MPK3/MPK6 signaling cascade and At-MMPs gene during leaf senescence,the results are shown as below:1.Induction of constitutive active MAPKKs leads to MPK3-and MPK6-dependent leaf senescenceWe previously generated conditional gain-of-function system by expressing the constitutively active MAPKK(GVG:AtMKK4^DD,GVG:AtMKK5^DD,GVG:NtMEK2^DD)variants under the control of a steroid-inducible promoter.Full induction of transgene expression after higher concentrations DEX treatment induces high-level activation of endogenous MPK3/MPK6,which triggers hypersensitive response?HR?-like cell death.However,when these plants are treated with lower concentrations of DEX,the plants show leaf-yellowing phenotype similar to senescence.The measurement of typical senescence-associated physiological markers and the transcript level of SAG12suggest DD-induced leaf yellowing is a senescence process,which is dependent on functional downstream MPK3 and MPK6.Furthermore,MPK3 plays more important role than MPK6 in this process.2.MKK4/MKK5 functions upstream of MPK3/MPK6 during leaf senescenceLeaf senescence is significantly delayed in mkk4 mkk5 double mutant compared to Col-0 under continuous light.The phosphorylation activation of MPK3 and MPK6during different leaf senescence stage suggests that MKK4 and MKK5 are involved in plant leaf senescence and play redundant function upstream of MPK3/MPK6 during leaf senescence.3.Expression of MMP genes are highly induced after gain-of-function activation of MPK3/MPK6 and during senescenceIn Arabidopsis,the MMP consists of five members named At1-MMP to At5-MMP.RT-qPCR assay reveals At2-MMP and At3-MMP expression are highly induced in DD plants after DEX treatment.However,in mpk3 and mpk6 single mutant backgrounds,At2-MMP and At3-MMP induction are compromised,correlating with the delayed leaf senescence.Also,RT-qPCR analysis reveals that the levels of all MMPs except At5-MMP transcript increased gradually during the progression of leaf development and senescence.Based on these results,we speculate that Arabidopsis MMPs are senescence-associated genes?SAGs?and may be involved in regulating leaf senescence downstream of MPK3/MPK6 cascade.4.Both constitutive and inducible overexpression of At-3MMP accelerate leaf senescenceConstitutive of At-3MMP exhibits an early-senescence phenotype compared to Col-0 in age-dependent manner.Precocious leaf yellowing and leaf death also are observed in the GVG:At-3MMP-dHA after treated with DEX.Furthermore,there are a good correlation between the expression levels of At-3MMP protein and the severity of leaf senescence.5.High-order mmp mutant fails to show delayed leaf senescenceNo obvious growth and developmental or senescence phenotype are observed in at1-mmp to at5-mmp single mutant,at2;3-mmp double mutants,at2;3;5-mmp triple mutants and at1;2;3;4;5-mmp pentuple mutants,suggesting although Arabidopsis MMPs are involved in regulating leaf senescence,there are other redundant pathway?s?that are sufficient to signal leaf senescence in the absence of At-MMPs6.WRKY33 is involved in MPK3/MPK6-induced At3-MMP genes activation and leaf senescenceWRKY33 is a substrate of MPK3/MPK6 in the activation of plant defense genes and ethylene production.RT-qPCR assay demonstrates the induction of At2-MMP and At3-MMP genes expression by the gain-of-function DD transgenic plants are compromised in wrky33 mutant background.Associated with this,the leaf senescence induced by DD is also inhibited.Furthermore,WRKY33 directly binds to the W-boxes in the promoter of At3-MMP gene based on chromatin-immunoprecipitation?ChIP?-qPCR assay.Based on these findings,we conclude that MPK3/MPK6 cascade,its downstream WRKY33 transcription factor,and At-3MMP target gene further downstream are involved in regulating leaf senescence in Arabidopsis.