title: Proteomic evidence for genetic epistasis: ClpR4 mutations switch leaf variegation to virescence in Arabidopsis.
abstract: Chloroplast development in plants is regulated by a series of coordinated biological processes. In this work, a genetic suppressor screen for the leaf variegation phenotype of the thylakoid formation 1 (thf1) mutant combined with a proteomic assay was employed to elucidate this complicated network. We identified a mutation in ClpR4, named clpR4-3, which leads to leaf virescence and also rescues the var2 variegation. Proteomic analysis showed that the chloroplast proteome of clpR4-3 thf1 is dominantly controlled by clpR4-3, providing molecular mechanisms that cause genetic epistasis of clpR4-3 to thf1. Classification of the proteins significantly mis-regulated in the mutants revealed that those functioning in the expression of plastid genes are oppositely regulated while proteins functioning in antioxidative stress, protein folding, and starch metabolism are changed in the same direction between thf1 and clpR4-3. The levels of FtsHs including FtsH2/VAR2, FtsH8, and FtsH5/VAR1 are greatly reduced in thf1 compared with those in the wild type, but are higher in clpR4-3 thf1 than in thf1. Quantitative PCR analysis revealed that FtsH expression in clpR4-3 thf1 is regulated post-transcriptionally. In addition, a number of ribosomal proteins are less expressed in the clpR4-3 proteome, which is in line with the reduced levels of rRNAs in clpR4-3. Furthermore, knocking out PRPL11, one of the most downregulated proteins in the clpR4-3 thf1 proteome, rescues the leaf variegation phenotype of the thf1 and var2 mutants. These results provide insights into molecular mechanisms by which the virescent clpR4-3 mutation suppresses leaf variegation of thf1 and var2.
authors.affiliation: National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Wu
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhu
authors.forename: Zhaoxue
authors.initials: Z
authors.lastname: Ma
authors.forename: Yi
authors.initials: Y
authors.lastname: Sun
authors.forename: Quan
authors.initials: Q
authors.lastname: Quan
authors.forename: Peng
authors.initials: P
authors.lastname: Li
authors.forename: Pengzhan
authors.initials: P
authors.lastname: Hu
authors.forename: Tieliu
authors.initials: T
authors.lastname: Shi
authors.forename: Clive
authors.initials: C
authors.lastname: Lo
authors.forename: Ivan K
authors.initials: IK
authors.lastname: Chu
authors.forename: Jirong
authors.initials: J
authors.lastname: Huang
mesh_terms: D049069:ATP-Dependent Proteases / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics* / Q000254:growth & development / Q000502:physiology / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism*; D015536:Down-Regulation; D049071:Endopeptidase Clp / Q000235:genetics / Q000378:metabolism; D004843:Epistasis, Genetic; D018506:Gene Expression Regulation, Plant; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D018384:Oxidative Stress; D010641:Phenotype; D018515:Plant Leaves / Q000235:genetics / Q000254:growth & development / Q000502:physiology / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D017510:Protein Folding; D040901:Proteomics; D036226:Seedlings / Q000235:genetics / Q000254:growth & development / Q000502:physiology / Q000648:ultrastructure; D017422:Sequence Analysis, DNA; D013213:Starch / Q000378:metabolism; D020524:Thylakoids / Q000378:metabolism
chemical_list: D049069:ATP-Dependent Proteases; D029681:Arabidopsis Proteins; D049071:Endopeptidase Clp; D008565:Membrane Proteins; D008666:Metalloendopeptidases; D013213:Starch; C512069:THF1 protein, Arabidopsis; C402651:AT2G30950 protein, Arabidopsis; C000591696:ClpR4 protein, Arabidopsis; C580852:FtsH protein, Arabidopsis
keywords: Arabidopsis thaliana; ClpR4; THF1; chloroplast proteome; epistasis; leaf variegation; virescence