Functional characterization of chloroplast J-like proteins OsV5a AND OsV5B in rice

Abstract

In plants, leaf chlorosis mutants are the most widely used materials for investigating biological functions of the genes involved in chlorophyll metabolism and chloroplast development. Many leaf chlorosis mutants have been characterized by morphological and physiological studies, however, molecular basis for some mutations was still unclear. Previously, a temperature conditional virescent mutant (osv5a) was identified by map-based cloning in our laboratory. The mutant displays leaf chlorosis phenotype at lower growth temperature (22℃) and green leaf at higher growth temperature (28℃). A close homolog of OsV5A, namely OsV5B, was also identified in rice and both OsV5A and OsV5B can regulate the level of protochlorophyllide oxidoreductase (POR) through their chaperone activity.

This study first demonstrated that OsV5A/OsV5B regulate abundances of OsPOR enzymes at early developmental stages of rice seedlings. Deficiencies of OsV5A and OsV5B at early developmental stages in the osv5a mutant seedling at 22℃ lead to reduced POR accumulation and chlorophyll contents. Furthermore, SWATH-MS quantitative proteomic analysis of pre-emergence 4th leaf (L4-1) in osv5a and wild type (WT) seedlings revealed that abundances of 481 proteins are significantly changed compared with WT. Among them, 288 proteins are up-regulated and 153 are down-regulated in the osv5a. A total of 141 proteins were predicted to be chloroplast-targeted, 72 were up-regulated and 59 were down-regulated. Several major metabolic pathways are highly affected in osv5a, such as protein metabolism, photosynthesis, chlorophyll biosynthesis, pyruvate and amino acid metabolism, glutathione metabolism and major heat shock-related proteins. Yeast two hybrid (Y2H) screening identified three novel OsV5A/OsV5B interacting proteins: Os06g04790, Os02g03330 and Os10g35140. Chloroplast localization experiments further demonstrated Os02g03330 is localized in thylakoid, Os06g04790 in stroma and envelope, and Os10g35140 in thylakoid and envelope.

To understand the biological roles of OsV5A/OsV5B-interacting proteins in plants, Os02g03330, which is the light harvesting complex-like protein3 (OsLIL3), was chosen for further investigations. Previously OsLIL3 was shown to interact with geranylgeranyl reductase (OsGGR), geranylgeranyl diphosphate synthase (OsGGPPS1) and geranylgeranyl diphosphate synthase recruiting protein (OsGRP), associating with these proteins to form a multiprotein complex in thylakoid membrane. Bimolecular fluorescent complementation (BiFC) and Y2H assay demonstrated OsV5A and OsV5B could also interact with OsGGR but not the other OsLIL3-interacting partners. In addition, deficiency of OsLIL3 results in reduced chlorophyll contents. At the same time, accumulation levels of OsV5A and OsV5B were lower in an oslil3 mutant when compared with that in WT. Mutation of OsLIL3 causes severe growth defects and low fertility rates. While in osv5a, accumulation levels of OsLIL3 and OsGGR were down-regulated as compared with WT at lower temperature (22℃) in L4-1. Meanwhile, blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis demonstrated that OsV5A and OsV5B are detected in a multiprotein complex consisting of OsPORB, OsLIL3 and OsGGR components in thylakoid membranes. Moreover, CPP1 (the chaperone-like protein of POR1) is an ortholog of OsV5A and OsV5B. A dexamethasone (DEX)-inducible CPP1 RNAi line has been acquired, and abundances of AtGGR and AtLIL3 were also reduced upon DEX induction. Overall, these findings suggest a new role of OsV5A and OsV5B in regulation of OsGGR/OsLIL3 through direct protein-protein interactions, and similar function is conserved in Arabidopsis. These chloroplast J-like proteins may play essential roles in chlorophyll and tocopherol biosynthesis