Lesion mimic mutants (LMMs) are advantageous materials for studying programmed cell death (PCD). Although some rice LMM genes have been cloned, the diversity of functions of these genes indicates that the mechanism of cell death regulation in LMMs needs further study. In this study, we identified a rice light-dependent leaf lesion mimic mutant 4 (llm4) that showed abnormal chloroplast structure, photoinhibition, reduced photosynthetic protein levels, massive accumulation of reactive oxygen species (ROS), and PCD. Map-based cloning and complementation testing revealed that LLM4 encodes zeaxanthin epoxidase (ZEP), an enzyme involved in the xanthophyll cycle, which functions in plant photoprotection, ROS scavenging, and carotenoid and abscisic acid (ABA) biosynthesis. The ABA content was decreased, and the contents of 24 carotenoids differed between the llm4 mutant and the wild type (WT). The llm4 mutant showed reduced dormancy and greater sensitive to ABA than the WT. We concluded that the mutation of LLM4 resulted in the failure of xanthophyll cycle, in turn causing ROS accumulation. The excessive ROS accumulation damaged chloroplast structure and induced PCD, leading eventually to the formation of lesion mimics.

The overuse of nitrogen (N) fertilizer in fields has increased production costs and raised environmental concerns. Increasing the N use efficiency (NUE) of rice varieties is crucial for sustainable agriculture. Here we report the cloning and characterization of OsNPF3.1, a gene that controls rice NUE. An amino acid mutation in the OsNPF3.1 coding region caused different NUEs in wild and cultivated rice. OsNPF3.1, which is expressed mainly in the aerial parts of rice, also affects rice plant height, heading date, and thousand-grain weight. The OsNPF3.1 protein is located in the plasma membrane. When OsNPF3.1 was subjected to artificial selection, two naturally varying loci were associated with NUE, of which OsNPF3.1^Chr6_8741040 differed between indica and japonica rice. OsNPF3.1 can be used as a new target gene for breeding rice varieties with high NUE.