Molecular Mechanism Of Arabidopsis MKKK19 In Regulating Leaf Senescence

Senescence is an important stage of plant development,which is of great significance for plants to successfully complete their life cycle.However,premature leaf senescence often affects the yield of crops.In addition,as sessile organisms,plants live in constantly changing environments that are often unfavorable for growth and development,including biotic stress and abiotic stress.These stress conditions often induce premature leaf senescence,leading to the loss of the crop yield.Therefore,studying the molecular mechanism of senescence in plants is important for both basic study and agricultural application in improving crop performance.The mitogen-activated protein kinase?MAPK?signal transduction pathways are evolutionarily conserved in all eukaryotic organisms.They transduce internal and external signals and regulate the celluar response to the developmental and environmental cues.Here,a mitogen-activated protein kinase kinase kinase 19 is identified in Arabidopsis thaliana,to be involved in the regulation of both normal aging/senescence and stress-induced premature leaf aging/senescence under adverse growth conditions.Compared to wild type?WT?,MKKK19 T-DNA insertion mutants,mkkk19 shows delaying aging,but MKKK19 overexpression plants,MKKK19OEs display premature leaf senescence.Expressing the c DNA of MKKK19 under the control of the native promoter of MKKK19 in mkkk19-1?Complimentation lines,COMs?rescues the phenotype of mkkk19-1,indicating that loss function of MKKK19 indeed causes delaying senescence.Real time quantitative PCR?q RT-PCR?analysis reveals that the expression levels of SAGs in mkkk19 are decreased significantly compared with WT,while the expression levels of SAGs in MKKK19OE plants are increased significantly compared to both mkkk19 and WT.However,the expression levels of SAGs are comparable in WT and COMs.These data suggest that MKKK19 is involved in the regulation of plant aging process,and the expression levels of SAGs are related to the expression level of MKKK19.C-terminal GFP-fused MKKK19 protein is localized to the plasma membrane,cytoplasm,and the nucleus under confocal laser scanning microscopy.And tissue-specific expression analysis reveals that MKKK19 is expressed in the leaf,root,and flower,with the highest expression level in the mature rosette leaves.The tissue specific expression pattern of MKKK19 is similar to that of SAG12,both of them are expressed in mature and aging leaves.The expression level of MKKK19is increased with the leaf age,but barely detected in young leaves,suggesting that MKKK19participate in age-dependent leaf senescence.Moreover,some stress-related factors,which induce aging process,such as ethylene,jasmonic acid,abscisic acid,salicylic acid,hydrogen peroxide and darkness,enhance the transcriptional expression of MKKK19 gene.Thus,here it is speculated that MKKK19 may also participate in the stress-induced premature leaf senescence.The kinase activity of MKKK19 was examined using the MKKK19 purifiedfrom prokaryotic expression system and myelin-basic protein?MBP?as a substrate.Results show that MKKK19kinase activity is low in terms of autophosphorylation and MBP phosphorylation.However,in vivo phosphorylation assay using 35S:MKKK19-FLAG transgenic Arabidopsis plants indicates that the MKKK19 kinase activity is associated with leaf age.MKKK19 kinase activity is low in young leaf,and increased in mature leaves,and reaches highest activity in the leaves starting senescence,indicating that the kinase activity of MKKK19 is associated with the degree of leaf aging.Furthermore,a conservative ATP-binding site of MKKK19 mutated form MKKK19^K37Mshows no kinase activity in vitro.And the MKKK19^K37M can not complement mkkk19-1 mutant,showing the delayed aging phenotype that is similar to mkkk19-1.Furthermore,MKKK19^K37Moverexpressing plants show similar phenotype to WT,but different from MKKK19OE-1.These results indicate that the kinase activity of MKKK19 is required for its function in control of the aging process.Identification of MKKs downstream of MKKK19 was performed based on the expression of10 MKKs in aging leaves.Among 10 MKKs,the expression level of MKK1,MKK3,MKK5 and MKK9 are induced by senescence.Moreover,among the 10 MKKs overexpression plants,compared with WT,only MKK1,MKK3,MKK5,and MKK9 overexpression plants show premature leaf senescence phenotype.Furthermore,MKK3,MKK4,MKK5,MKK6,MKK9 and MKK10 interact with MKKK19 in yeast two hybrid?Y2H?assay and bimolecular fluorescence complementary?Bi FC?assay.The interaction of MKKK19 with MKK3,MKK5 and MKK9 were further verified in vivo with the Co-IP experiments.These data suggest that MKK3,MKK5 and MKK9 may play a role downstream of MKKK19 and participate in the regulation of plant aging process.In addition,in vitro phosphorylation experiments show that MKKK19 phosphorylates MKK5.Phenotypic analysis exhibits that mkk3-1,mkk5-1 and mkk9-1 mutants display similar phenotypes to mkkk19-1,showing delayed leaf senescence.Whereas,MKK3,MKK5 and MKK9overexpression plants show premature leaf senescence.Further phenotypic analysis shows that overexpressing MKKK19 in mkk3-1,mkk5-1 and mkk9-1 mutant background respectively,have no premature leaf senescence phenotype,indicating that mkk3-1,mkk5-1 and mkk9-1 mutations can partially rescue the phenotype of MKKK19OE.These results suggest that MKK3,MKK5 and MKK9 are downstream of MKKK19,which are involved in the leaf development and senescence process,and that MKK3,MKK5 and MKK9 may have functional redundancy,because mutation of MKK3,MKK5 and MKK9 can only partially complement the phenotype of the MKKK19OE.MPK6 is a common substrate for MKK3,MKK5 and MKK9,participating in the regulation of different biological processes.Aging process induces the expression level of MPK6.Phenotypic analysis shows that mpk6-1 mutant exhibits delayed-senescence phenotype,which is similar to the mkkk19-1,mkk3-1,mkk5-1 and mkk9-1 mutant phenotypes.Moreover,MPK6 mutation can only partially complement the phenotype in the MKKK19OE,MKK3OE,MKK5OE and MKK9OE transgenic plants.These results suggest that MPK6 is involved in the regulation of senescence process,and it acts as downstream component of MKKK19-MKK3/5/9.In summary,MAPK kinase cascade pathway,MKKK19-MKK3/5/9-MPK6,plays a role in both normal leaf aging process and stresses induced leaf premature senescence in Arabidopsis thaliana plants.