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

Abstract

Deep insights into chloroplast biogenesis have been obtained by mutant analysis; however, in C 4 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 C 4 model Setaria viridis. Approximately 1000 M 2 families were screened for the segregation of pale-green seedlings in the M 3 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 chloroplastencoded 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 0 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 C 4 model.

CFM1, a member of the CRM‐domain protein family, functions in chloroplast group II intron splicing in Setaria viridis

“…Because the splicing of many chloroplast introns, and in particular those in subgroup IIA, is reduced when chloroplast translation is compromised (Jenkins et al ., 1997), we compared Sv ‐ cfm1 both with the WT and with several other Setaria mutants with global defects in chloroplast translation. These included Sv ‐ pnp1 and Sv ‐ psrp7 , which are defective in RNA maturation and translation initiation, respectively (Feiz et al ., 2020), as well as Sv ‐ 381 and Sv ‐ 800 , whose causative mutations have not been identified but which exhibit decreased abundance of 16S and 23S rRNAs (see ethidium bromide‐stained gel in Figure 6a), consistent with translation defects. Sv ‐ 381 is particularly useful because the magnitude of its rRNA deficiencies was similar to that of Sv ‐ cfm1 .…”

“…A mutation in the Setaria CFM1 gene ( Sv ‐ cfm1 ) arose in an ethyl methanesulfonate (EMS)‐induced mutant collection (Feiz et al ., 2020) and was first detected by its pale‐green/yellowish phenotype (Figure 1a). The chlorotic phenotype segregated as a single, recessive mutation, and mutant plants died 10–12 days after planting, presumably when their seed reserves were exhausted.…”

“…We sequenced DNA from bulked segregants to identify the causal mutation in Sv ‐ cfm1 . Our analytical pipeline (Feiz et al ., 2020) identifies homozygous EMS‐induced mutations that co‐segregate with the mutant phenotype; these are subsequently screened manually for those that are predicted to affect the expression or function of a protein potentially required for photosynthetic function. The candidate mutation best conforming to these criteria was a G to A transition at the splice acceptor site in the seventh intron of Si021265m, which encodes a previously uncharacterized CRM‐domain protein in the CRS1 subfamily (Figure 2).…”

“…Mutagenesis, transformation and growth of Setaria were performed as described (Feiz et al ., 2020). The maize mutant UFMu‐10982 was obtained from the UniformMu Transposon Resource (https://www.maizegdb.org/-uniformmu) and genotyped for the insertion MU1086839 in the last exon of the gene GRMZM5G813259 using primers Zm‐TIR 6 and GRMZM5G813259 F8, and for the WT copy using GRMZM5G813259 F8 and GRMZM5G813259 R7 (Table S1).…”