Phenotypic Characterization and Gene Mapping of the Lesion Mimic Mutant lmm28 in Rice

Rice lesion mimic mutants are important materials for studying the mechanisms of cell death. In-depth research on these mutants can provide insights into the molecular mechanisms underlying rice growth and development, offering a theoretical basis for crop improvement. In this study, rice variety Wuyunjing 21 (WYJ21) was mutagenized with ethyl methanesulfonate to obtain a lesion mimic mutant, lmm28. Unlike wild-type (WT) plants, the lmm28 mutant exhibits brown lesions on the leaves starting from the early tillering stage. The size of the lesions increases as the plant grows. Additionally, the lmm28 mutant shows significantly reduced plant height, tiller number, number of effective panicles, seed setting rate, and 1000-grain weight compared to the WT. Leaf staining of the mutant revealed an accumulation of reactive oxygen species and cell death in the lesion leaves. Transmission electron microscopy images showed that, in the lmm28 mutant, the nuclear boundaries in leaf cells became indistinct and damage to the chloroplast membrane structures was observed, with thylakoid disorganization occurring in some chloroplasts. Genetic analysis and map-based cloning localized the candidate gene of the mutant to a 167.79 kb region on chromosome 5. After analyzing the annotated genes within this region, the candidate gene was preliminarily identified as OsBON3. Sequencing analysis revealed that, in lmm28, a base change from GT to GC occurred at the 5′ splice junction of the 15th intron of OsBON3. Further analysis, using cDNA amplification of exons 14–16 followed by sequencing, showed that the mutation at the splice recognition site caused the incorrect splicing of OsBON3 pre-mRNA, leading to an increased number of transcripts in lmm28. The transcript containing an inserted intron is present at much higher levels than the normal transcript, which may lead to a reduction in the protein levels containing the functional vWA domain. Therefore, the vWA domain of OsBON3 is likely crucial for maintaining ROS homeostasis in rice and plays a key role in regulating its growth and development.