Gene cloning and functional characterization of a yellow-green leaf and reduced tillering mutant in rice

Leaf color mutants are valuable genetic resources for elucidating chloroplast development and photosynthetic regulation in rice. In this study, a stably inherited yellow-green leaf and reduced tillering mutant (ygrt) was isolated from tissue-cultured seedlings of the rice cultivar Zhonghua 11 (ZH11). ygrt plants exhibited a persistent yellow-green phenotype, reduced tiller number, and lower seed setting rate. I₂-KI staining revealed that ygrt pollen grains were more lightly stained than ZH11 and showed oval or irregular shapes. Semi-thin anther sections indicated delayed tapetum degradation and abnormal microspore development in ygrt. Moreover, chlorophyll contents and net photosynthetic rate were significantly decreased, accompanied by an increased intercellular CO₂ concentration in ygrt. Transmission electron microscopy showed defective chloroplast ultrastructure, including loosely distributed grana lamellae and numerous small vesicles. Through map-based cloning, the target gene was localized to the region between 42,443-42,635 kb region on chromosome 1, and LOC_Os01g73450 was identified as the candidate gene. CRISPR/Cas9 knockout of this gene reproduced the yellow-green leaf phenotype of the mutant, whereas reintroduction of the wild-type coding sequence restored normal leaf coloration. YGRT is predominantly expressed in leaves, with maximal expression observed at the booting stage. The ygrt plants displayed growth defects under both normal and low-light conditions, which were particularly pronounced under normal light, suggesting that YGRT is involved in the response to light intensity. Cloning and characterization of YGRT provide important genetic resources, insights into chloroplast development and photosynthetic regulation, and potential targets for rice yield improvement.