The RAF22-MKK7/MKK9-MPK3/MPK6 Cascade Regulates Plant Growth Via Sucrose Metabolism In Arabidopsis

Mitogen-activated protein kinases(MAPKs)cascades are ubiquitous signaling modules in eukaryotes,which play important roles in plant growth,development and stress responses.Each MAPK cascade,which is minimally composed of MAPKKK-MAPKK-MAPK module,transfers signals from receptors/sensor by sequentially phosphorylation thereby activation.In Arabidopsis,there are approximately 80 putative MAPKKKs,however,the research of their function is quite limited in spite of their larger amount than MAPKK and MAPK.Especially for the RAF-like MAPKKK,which consists of 48 members,there is no clear conclusion for their function and position in MAPK cascade for now.Here,the function of the MAPK cascade modulated by RAF22 in the regulation of plant growth and development is analyzed in Arabidopsis,which provides important evidence to reveal the function of RAF-like MAPKKK gene family in plants.The results of GUS histochemical staining showed that RAF22 was expressed in almost all tissues and organs.Kinase assay revealed that RAF22 had both autophosphorylation and transphosphorylation activity.And the phenotype of raf22 mutant implied the function of RAF22 in the regulation of root growth.Meanwhile,the Arabidopsis RAF22WT transgenic plants showed severe growth inhibition both in shoot and root,while as a negative control,RAF22KR(an inactive allele of RAF22)transgenic seedlings exhibited normal growth.These results revealed that the expression of RAF22WT induced the growth inhibition,which was dependent on its kinase activity.Among all ten MKKs,MKK7 and MKK9 were demonstrated to interact with RAF22 by yeast two-hybrid,and the interaction was confirmed by Pull-down and Co-IP assays.Furthermore,the result of in vitro kinase assay revealed that MKK7 and MKK9 could be phosphorylated by RAF22.In order to prove RAF22 is the upstream of MKK7 and MKK9 exactly,the cross material RAF22WT mkk7,RAF22WT mkk9 and RAF22WT mkk7 mkk9 were generated,and the phenotype observation revealed that the growth inhibition of RAF22WT could be rescued obviously by the knock-out of MKK7 and MKK9.MPK3 and MPK6 are the known downstream of MKK7 and MKK9.In order to prove MPK3 and MPK6 are also the substrates of RAF22-MKK7/MKK9,in vitro kinase assay was carried out,and the results indicated that MPK3 and MPK6 could be phosphorylated by MKK7 and MKK9 activated by RAF22.Meanwhile,RAF22WT mpk3 and RAF22WT mpk6 rescued the growth defect in RAF22WT,which suggested that MPK3 and MPK6 were required in the growth inhibition regulated by RAF22.The above evidence demonstrated that RAF22-MKK7/MKK9-MPK3/MPK6 regulated Arabidopsis growth negatively.We also explored the mechanism in the process of plant growth regulated by RAF22-MKK7/MKK9-MPK3/MPK6.By exogenous applying different kinds of sugar,we found that the retarded phenotype in RAF22WT could be rescued in a large degree by sucrose and its hydrolysis products.And RAF22 transgenic seedlings were hypersensitive to sucrose while raf22 mutant showed less sensitive to sucrose.The results of transcript analysis of the sucrose metabolic enzymes coded genes showed that RAF22-MKK7/MKK9-MPK3/MPK6 cascade probably participated in the sucrose hydrolysis.Furthermore,the metabolomics data indicated that RAF22 probably regulated the metabolic balance of multiple substances;detection of embryo pattern suggested that RAF22 and RAF28 were involved in the the process of Arabidopsis embryonic developmentIn conclusion,RAF22-MKK7/MKK9-MPK3/MPK6 consist a full MAPK cascade,which regulates plant growth probably by involving in the sucrose metabolism in Arabidopsis.