The function of tea plant CsMORF9.3 in chlorophyll biosynthesis and RNA editing

CsMORF9.3 regulates chlorophyll biosynthesis, affecting the RNA editing efficiency of matK-445, rpoA-200, ndhD-674 and ndhD-1310, and it interacts with CsMORFs, CsPPRs and CsCHLD proteins. Leaf color is an important factor affecting tea quality as well as the growth and development. It has been reported that several genes and transcription factors participated in chlorophyll metabolism in tea plants (Camellia sinensis). However, the role of chloroplast RNA editing factors in chlorophyll biosynthesis in C. sinensis remains poorly understood. In this study, multiple dysregulated RNA editing sites in the chloroplast genome were identified from etiolation and albino tea cultivars. Multiple organellar RNA editing factor 9.3 (CsMORF9.3), a core RNA editing factor localized in the chloroplasts, was identified as a candidate regulator of leaf coloration. Antisense oligonucleotide (AsODN) and virus-induced gene silencing (VIGS) confirmed that suppressing CsMORF9.3 expression reduces chlorophyll content, downregulates genes involved in chlorophyll biosynthesis and chloroplast development, as well as disrupts chloroplast RNA editing. Protein-protein interaction assays confirmed that CsMORF9.3 could form both homodimers and heterodimers with itself or other MORF proteins through yeast two-hybrid (Y2H), luciferase complementation imaging (LCI) assays, and bimolecular fluorescence complementation (BiFC) assays. Moreover, CsMORF9.3 was found to interact with multiple PLS-type pentatricopeptide repeat (PPR) proteins, including CsCRR21, CsCRR28, CsOTP84, and CsLPA66, as well as with CsCHLD, a key subunit of magnesium chelatase in chlorophyll biosynthesis. Our study provides the protein interaction of CsMORF9.3, demonstrating its potential role in regulating RNA editing and chlorophyll biosynthesis in C. sinensis. These results broaden the understanding of the regulatory mechanisms of chlorophyll biosynthesis and provide new insights into the breeding of etiolation and albino tea plant germplasm.