Functional characterization of chloroplast-targeted RbgA GTPase in higher plants.

Abstract

Plant RbgA GTPase is targeted to chloroplasts and co-fractionated with chloroplast ribosomes, and plays a role in chloroplast rRNA processing and/or ribosome biogenesis. Ribosome Biogenesis GTPase A (RbgA) homologs are evolutionarily conserved GTPases that are widely distributed in both prokaryotes and eukaryotes. In this study, we investigated functions of chloroplast-targeted RbgA. Nicotiana benthamiana RbgA (NbRbgA) and Arabidopsis thaliana RbgA (AtRbgA) contained a conserved GTP-binding domain and a plant-specific C-terminal domain. NbRbgA and AtRbgA were mainly localized in chloroplasts, and possessed GTPase activity. Since Arabidopsis rbgA null mutants exhibited an embryonic lethal phenotype, virus-induced gene silencing (VIGS) of NbRbgA was performed in N. benthamiana. NbRbgA VIGS resulted in a leaf-yellowing phenotype caused by disrupted chloroplast development. NbRbgA was mainly co-fractionated with 50S/70S ribosomes and interacted with the chloroplast ribosomal proteins cpRPL6 and cpRPL35. NbRbgA deficiency lowered the levels of mature 23S and 16S rRNAs in chloroplasts and caused processing defects. Sucrose density gradient sedimentation revealed that NbRbgA-deficient chloroplasts contained reduced levels of mature 23S and 16S rRNAs and diverse plastid-encoded mRNAs in the polysomal fractions, suggesting decreased protein translation activity in the chloroplasts. Interestingly, NbRbgA protein was highly unstable under high light stress, suggesting its possible involvement in the control of chloroplast ribosome biogenesis under environmental stresses. Collectively, these results suggest a role for RbgA GTPase in chloroplast rRNA processing/ribosome biogenesis, affecting chloroplast protein translation in higher plants.