YDA-MKK4/MKK5-MPK3/MPK6 Cascade Regulates Cuticle Formation In Arabidopsis

The mitogen-activated protein kinase（MAPK）cascades are conserved signal transduction pathways in eukaryotes.The MAPK signal pathways are involved in many physiological processes of plant growth,development and disease resistance.After plants are stimulated by internal and external signals,a series of signal transduction pathways in the cells are activated,which in turn initiates the expression of downstream genes in response to the stimuli.The cuticle is a layer of hydrophobic waxy substance on the top surface of the plant,which has the functions of retaining moisture and resisting pathogenic bacteria.Many genes related to cuticle formation and immunity have been reported.Loss-of-function mutants of these genes will show changes in resistance to pathogens,which indicates that the normal development of the cuticle is very important for plants to resist pathogens.There are many studies on the MAPK cascades regulation of plant growth,development and resistance.We must first understand the relationship between MAPKs and cuticle formation.This project is dedicated to discovering the mechanism of MAPK cascade regulating cuticle morphogenesis and disease resistance.We found that a conservative MAPK cascade can positively regulate the formation of Arabidopsis cuticle.Loss function of any member of this pathway will lead to defects in the cuticle of Arabidopsis leaves.In this paper,we mainly discuss that the YDA-MKK4/5-MPK3/6 cascade regulates Arabidopsis cuticle formation.The main results are as follows:1.The YDA-MKK4/MKK5-MPK3/MPK6 cascade participates in the regulation of the formation of the cuticle in Arabidopsis cotyledon.Cotyledons are the first pair of leaves formed during embryonic development of plants.Cotyledon defects are generally caused by problems in embryonic development.Through toluidine blue（TB）staining experiments,it was found that the loss of function of MPK3/MPK6,MKK4/MKK5 and YDA all caused the defects of the cuticle of Arabidopsis cotyledons.Among them,the phenotypes of mpk3 and mpk6 single mutants are not obvious,while the defective phenotypes of MPK3SR and MPK6SR treated with NA-PP1 and mkk4mkk5 double mutants and yda mutants are very obvious.After the cotyledons are stained with TB,a high proportion of the leaves are distributed in blue-black spots,and are damaged to varying degrees,while the wild-type cotyledons can hardly be stained by toluidine blue.Therefore,we found that the YDA-MKK4/MKK5-MPK3/MPK6 cascade plays an important role in the development of the Arabidopsis cotyledon cuticle.2.The YDA-MKK4/MKK5-MPK3/MPK6 cascade also regulates the development of the cuticle in Arabidopsis true leaf.Most of the existing studies on the development of the cuticle are aimed at the embryonic development stage of plants,and there are few studies on the development of the cuticle of the true leaf.Through toluidine blue staining experiments,it was found that the loss of function of YDA,MKK4/MKK5 and MPK3/MPK6 would all lead to defects in the cuticle of Arabidopsis true leaves.After the true leaves are stained by TB,the blue-black spots are distributed in dense dots.Therefore,the YDA-MKK4/5-MPK3/6 cascade will also regulate the formation of the Arabidopsis true leaf cuticle.Moreover,the mechanism of cuticle defects in true leaves may be different from that of cotyledons.In the course of the experiment,we also found that MAPKs regulates the formation of the cuticle with spatiotemporal characteristics.The cuticle defects of MAPKs mutant leaves of different growth days and different parts of the same leaf are different.3.YDA-MKK4/MKK5-MPK3/MPK6 cascade partly regulates the formation of the cuticle of Arabidopsis cotyledon downstream of GSO1/GSO2.GSO1 and GSO2 are a pair of LRR-RLKs,which together regulate the formation of the cuticle.The gso1 gso2 double mutant exhibits a very serious phenotype of cuticle defect.Many RLKs function upstream of MAPKs,so we guess that the YDA-MKK4/5-MPK3/6cascade regulates the formation of the cuticle downstream of GSO1/GSO2.In order to confirm the relationship between GSO1/GSO2 and MAPKs,we used GSO1 promoter and GSO2 promoter to drive the expression of MKK4^DD and MKK5^DD respectively to restore the severely cuticle defective phenotype of the gso1 gso2 double mutant.it is found that GSO1and GSO2 promoters driven MKK4^DD and MKK5^DD can partially restore the cuticle defect phenotype of gso1 gso2 double mutants.And treatment of Arabidopsis with the ligand CIF2of GSO1 and GSO2 can activate the phosphorylation of MPK3 and MPK6 in vivo.Therefore,we preliminarily concluded that the Arabidopsis YDA-MKK4/5-MPK3/6 cascade regulates the formation of the cuticle in cotyledon in the downstream part of GSO1/GSO2.In addition,it has been reported that GSO1/GSO2 participates in the formation of cuticle only in the cotyledon.We found that both GSO1 and GSO2 can be highly expressed when the first pair of true leaves are just formed.As the true leaves mature,their expression rapidly decreases.The true leaves of gso1 gso2 double mutants can also be stained by TB when the first pair of true leaves are just born.Therefore,we speculate that GSO1 and GSO2 also have an effect on the formation of Arabidopsis true leaf cuticle.