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.

中文翻译：

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水稻中编码脱镁叶酸加氧酶的新型等位基因的表征

我们从籼稻品种Zhongjian100 突变体库中鉴定出快速细胞死亡2 (rcd2) 突变体。红褐色病斑首先出现在幼苗的叶片上，然后逐渐合并，在分蘖后期叶片完全枯萎。rcd2 在病变处/周围显示出明显的细胞死亡、超氧阴离子 (O2 -) 的积累和 ROS 清除系统受到干扰，光合能力受损，叶绿素含量显着降低。病变形成由单个隐性核基因控制，并由自然光和机械损伤诱导。OsMH_03G0040800 第 6 个外显子的单碱基突变 (A1726T) 导致编码蛋白脱镁叶酸加氧酶 (PAO) 中的 I576F 取代。功能互补可以挽救突变表型，并且 PAO 敲除系表现出与 rcd2 相似的表型。rcd2中PAO的活性显着降低，而PAO底物脱镁叶绿素a的含量明显增加。叶绿素合成/降解相关基因的表达和代谢中间体的含量发生了很大变化。此外，叶绿素酶产物叶绿素a的水平显着增加，表明叶绿素酶可能在水稻叶绿素降解中发挥作用。我们的结果表明，I576F 取代破坏了 PAO 功能，导致水稻中 O2 - 积累和叶绿素降解分解。叶绿素合成/降解相关基因的表达和代谢中间体的含量发生了很大变化。此外，叶绿素酶产物叶绿素a的水平显着增加，表明叶绿素酶可能在水稻叶绿素降解中发挥作用。我们的结果表明，I576F 取代破坏了 PAO 功能，导致水稻中 O2 - 积累和叶绿素降解分解。叶绿素合成/降解相关基因的表达和代谢中间体的含量发生了很大变化。此外，叶绿素酶产物叶绿素a的水平显着增加，表明叶绿素酶可能在水稻叶绿素降解中起作用。我们的结果表明，I576F 取代破坏了 PAO 功能，导致水稻中 O2 - 积累和叶绿素降解分解。