Setaria viridis chlorotic and seedling-lethal mutants define critical functions for chloroplast gene expression

Deep insights into chloroplast biogenesis have been obtained by mutant analysis; however, in C₄ plants a relevant mutant collection has only been developed and exploited for maize. Here, we report the initial characterization of an ethyl methyl sulfonate-induced mutant population for the C₄ model Setaria viridis. Approximately 1000 M₂ families were screened for the segregation of pale-green seedlings in the M₃ generation, and a subset of these was identified to be deficient in post-transcriptional steps of chloroplast gene expression. Causative mutations were identified for three lines using deep sequencing-based bulked segregant analysis, and in one case confirmed by transgenic complementation. Using chloroplast RNA-sequencing and other molecular assays, we describe phenotypes of mutants deficient in PSRP7, a plastid-specific ribosomal protein, OTP86, an RNA editing factor, and cpPNP, the chloroplast isozyme of polynucleotide phosphorylase. The psrp mutant is globally defective in chloroplast translation, and has varying deficiencies in the accumulation of chloroplast-encoded proteins. The otp86 mutant, like its Arabidopsis counterpart, is specifically defective in editing of the rps14 mRNA; however, the conditional pale-green mutant phenotype contrasts with the normal growth of the Arabidopsis mutant. The pnp mutant exhibited multiple defects in 3' end maturation as well as other qualitative changes in the chloroplast RNA population. Overall, our collection opens the door to global analysis of photosynthesis and early seedling development in an emerging C₄ model.