The Regulatory Mechanism Of PAD4 In MPK4-regulated Plant Growth And Development In Arabidopsis

MAPK cascades are composed of three protein kinases,a MAPK kinase kinase(MAPKKK),a MAPK kinase(MAPKK)and a MAP kinase(MAPK).By sequential phosphorylation,the downstream substrates of MAPK cascades are activated.MAPK cascades are highly conserved signal modules and are involved in the regulation of plant growth,development and responses to environmental stimuli in eukaryotes.There are 80 MAPKKKs,10 MAPKKs and 20 MAPKs in Arabidopsis.However,only a few of them are researched.Analysis of knock-out mutants revealed that some MAPK cascades members defective mutants exhibit the dwarf morphology,including MPK4.MPK4 whose defective mutant is the first reported as it possesses dwarf morphology,which plays a key role in plant immunity.Comprehensive analysis of previous studies shows that there is no causal relationship between MPK4-regulated immune response and plant development.Therefore,it has important scientific significance to screen mpk4 partially or completely rescued mutant and explore the function mechanism for understanding the function of MPK4 and MAPK cascade in the regulation of plant development.The mpk4 mutant is dwarf and sterile in the Col-0 background.In this study,we crossed some mutants with mpk4 heterozygote mutant to generate a segregating F2 population.For example,mpk4 NahG and mpk4 sid2-2 which associated with salicylic acid(SA)route;mpk4 pad3 and mpk4 wrky33 which associated with camalexin synthetic and regulation pathway;mpk4 pad4-1 and mpk4 NahG pad3 which associated with both SA synthetic and camalexin pathway.Plants homozygous were identified by PCR genotyping and used for phenotype analysis including the above-ground and under-ground parts of the plant,SA production and camalexin production analysis.Under the same condition,the mpk4 dwarf phenotype was partially rescued in the mpk4 NahG,mpk4 wrky33,mpk4 pad4-1 and mpk4 NahG pad3 mutants,but the production change of SA and camalexin was not the cause of plant developmental defect.To determine the way of MPK4 regulating the plant growth and development,the single or double mutants were further analyzed.By comparing the RNA-Seq-based transcriptome of Col-0,mpk4 and MPK4com,the action of MPK4 was further characterized.MPK4 deficiency led to ninety-six percent of lipid localization-related genes,of which seventy-four percent were lipid transport proteins,differential expression and significant enrichment.Next,certain carbon metabolism-and photosynthesis-related genes were differentially expressed and significantly enriched.By applying 2-D electrophoresis and the matrix-assisted laser desorption ionization time-of-flight mass spectrometry method,proteomic analysis was performed in the mpk4,mpk4 NahG,mpk4 pad4-1,mpk4 pad3 and mpk4 wrky33.The expression level change of photosynthesis-,oxidative stress-and disease-resistant-related proteins had common characters in all mpk4 partially rescued mutants.The results indicated that the phenotype of mpk4 was related to unusual photosynthesis and oxidation-reduction reaction.Analysis of under-ground parts of mpk4 NahG?mpk4 wrky33 and mpk4 pad4-1 mutants morphology revealed that mpk4 pad4-1 not only partially rescued the above-ground parts of mpk4 phenotype,but also partially rescued the root hair development of mpk4.PAD4 encodes a lipase-like protein which is triglyceride lipase,while,lipase could act directly on the substrate.Using the mpk4 pad4-1 mutant could help us to understand the regulation of lipid-regulated plant growth and development,so we also compared the plant fertility of mpk4 and mpk4 pad4-1.The results showed that pollen germination rate was increased in the mpk4 pad4-1 mutant compared with the mpk4 mutant.PAD4 interacts with MPK4 in yeast,and may act as the substrate of MPK4 involving in regulating pollen germination.In conclusion,the mpk4 dwarfism morphology is independent on SA and camalexin accumulation.PAD4 plays a key role in participating in MPK4-regulated that may be concerned with its enzymatic activity.This study provided a new theoretical basis for revealing the mechanism of MPK4-regulated plant growth and development in Arabidopsis.