OsWHY1/OsTRXz/OsMORFs complex is essential for RNA modification and early chloroplast development in rice

Abstract

AbstractWHIRLY (WHY) proteins are single-stranded DNA/RNA-binding proteins that play multifaceted roles in various plant species. The regulatory mechanisms of WHY proteins in rice remains blank. Here we demonstrate thatOsWHY1in rice is required for early chloroplast development. CRISPR/Cas9-generatedoswhy1knockout lines displayed albino seedling phenotypes, abnormal chloroplast structure and comprised redox balance in leaves. OsWHY1 interacts with multiple plastid proteins, including the thioredoxin OsTRXz and two multiple organellar RNA editing factors (OsMORF8 and OsMORF9) in chloroplasts. Accordingly, several plastid genes dependent on plastid-encoded RNA polymerase (PEP) in theoswhy1mutants were significantly depressed at both transcript and protein levels. The editing ofrps14transcripts and splicing ofrpl2,along with their protein expression, were defective in theoswhy1mutants. OsWHY1 exhibited RNA-binding activity, specifically binding torps14andrpl2precursor RNAs, which underscores its role as a post-transcriptional regulator essential for normal protein synthesis in chloroplasts. Loss-of- function mutants of eitherOsWHY1orOsMORF9andOsTRXzdisplayed albino phenotypes, disrupted H2O2homeostasis, and defective RNA processing inrps14andrpl2, suggesting the OsWHY1-OsTRXz-OsMORFs regulatory module is vital for maintaining chloroplast stability and integrity through its RNA-binding activity and its role in recruiting OsTRXz and OsMORFs to ensure proper RNA modification.One sentence summaryOsWHY1 is integral to chloroplast development in rice through its RNA-binding activity and its role in recruiting OsTRXz and OsMORFs to ensure proper RNA modification.