Characterization of a novel allele encoding pheophorbide a oxygenase in rice

We identified a rapid cell death 2 (rcd2) mutant from an indica cultivar Zhongjian100 mutant bank. The red-brown lesions appeared firstly on young seedling leaves, then gradually merged and the leaves completely withered at the late tillering stage. rcd2 displayed apparent cell death at/around the lesions, accumulation of superoxide anion (O₂^-) and disturbed ROS scavenging system, impaired photosynthetic capacity with significantly reduced chlorophyll content. The lesion formation was controlled by a single recessive nuclear gene and induced by natural light as well as mechanical wounding. A single base mutation (A1726T) at the 6th exon of OsMH_03G0040800 resulted in I576F substitution in the encoding protein, pheophorbide a oxygenase (PAO). Functional complementation could rescue the mutant phenotype and PAO-knockout lines exhibited the similar phenotype to rcd2. The activity of PAO decreased significantly while the content of PAO substrate, pheophorbide a, increased apparently in rcd2. The expression of chlorophyll synthesis/degradation-related genes and the contents of metabolic intermediates were largely changed. Furthermore, the level of chlorophyllide a, the product of chlorophyllase, increased significantly, indicating chlorophyllase might play a role in chlorophyll degradation in rice. Our results suggested that the I576F substitution disrupted PAO function, leading to O₂^- accumulation and chlorophyll degradation breakdown in rice.