The AtRNC1-AtWTF1 protein complex is essential for chloroplast group Ⅱ intron splicing, ribosome biogenesis, and embryo development in Arabidopsis.

The splicing of group Ⅱ introns in chloroplasts is essential for photosynthesis and plant development and relies on a series of nucleus-encoded RNA-binding proteins. Here, we demonstrate that two Arabidopsis proteins, ribonuclease Ⅲ-domain protein AtRNC1 and the Plant organelle RNA recognition (PORR) protein AtWTF1, are essential for chloroplast function. Null mutants of AtRNC1 or AtWTF1 are embryo lethal. We rescued the null mutant by expressing wild-type AtRNC1 under the control of the seed-specific ABSCISIC ACID-INSENSITIVE3 (ABI3) promoter. We also created atrnc1 knockdown plants using artificial-microRNA (amiRNA). Both the rescued and knockdown atrnc1 plants exhibit chlorotic phenotype. Similarly, AtWTF1 hypomorphic mutants display variegated phenotypes. Both proteins are chloroplast-localized, with AtRNC1 residing in the stroma and AtWTF1 in both the stroma and thylakoids. Molecular phenotyping established that AtRNC1 and AtWTF1 are required for the efficient splicing of a specific, overlapping set of chloroplast group Ⅱ introns, including those in petB, petD, rpl2, rps12, and tRNAs. RNA immunoprecipitation confirmed the association of both proteins with their target introns in vivo, indicating direct roles in splicing. The knockdown of AtRNC1 or AtWTF1 in Arabidopsis impairs the chloroplast ribosome accumulation and accordingly reduced the efficiency of mRNA translation. Notably, protein interaction assays, including yeast two-hybrid, luciferase complementation image, and IP-MS indicate that AtRNC1 and AtWTF1 associate with each other, implying their cooperative function in a splicing complex. These findings establish AtRNC1 and AtWTF1 as key components of the chloroplast RNA splicing machinery, essential for the maturation of plastid transcripts and overall chloroplast homeostasis in Arabidopsis.