SiTCD1 encodes a P-type PPR protein that affects early chloroplast development at low temperatures in foxtail millet

Chloroplast gene expression relies on nucleus-encoded factors for RNA metabolic processing, but the mechanisms under cold stress remain poorly understood. In this study, we isolated and characterized a foxtail millet (Setaria italica) mutant, temperature-sensitive chlorophyll-deficient (sitcd1), which exhibited reduced chlorophyll content and abnormal chloroplasts, resulting in an albino phenotype during early leaf development at low temperatures (20℃ during the day and 18℃ at night, L20/D18). Map-based cloning revealed that SiTCD1 encodes a P-type PPR protein localized in chloroplasts. In sitcd1 background, transgenic lines overexpressing SiTCD1 had nearly normal green leaves under L20/D18 conditions. SiTCD1 was especially expressed in the earlier development of leaves at low temperatures. In addition, SiTCD1 bound directly to the plastid gene atpF in vitro, so it might participate in the splicing of plastid gene atpF at low temperatures. RNA-seq results indicated that the expression of genes related to metabolism (such as porphyrin, chlorophyll and glutathione metabolism), which require ATP for energy, was down-regulated in sitcd1, resulting in reductions in chlorophyll content, reduced glutathione (GSH), and its redox couple (GSH/oxidized glutathione (GSSG)) at low temperatures. As sitcd1 exhibited greater sensitivity at the bud bursting stage than germination or the seedling stage under cold stress, we identified two haplotypes of SiTCD1 (SiTCD1^Hap1 and SiTCD1^Hap2) in 195 accessions, and found that accessions carrying the SiTCD1^Hap2 allele were more tolerant to cold stress than those with the SiTCD1^Hap1 allele at the bud bursting stage. In summary, our results suggest that SiTCD1 is essential for early chloroplast development at low temperatures in foxtail millet.