Table_1_Single Nucleotide Mutagenesis of the TaCHLI Gene Suppressed Chlorophyll and Fatty Acid Biosynthesis in Common Wheat Seedlings.xls

Wheat (Triticum aestivum L.) is one of the most important crops in the world. Chlorophyll plays a vital role in plant development and crop improvement and further determines the crop productivity to a certain extent. The biosynthesis of chlorophyll remains a complex metabolic process, and fundamental biochemical discoveries have resulted from studies of plant mutants with altered leaf color. In this study, we identified a chlorophyll-deficiency mutant, referred to as chli, from the wheat cultivar Shaannong33 that exhibited an obvious pale-green leaf phenotype at the seedling stage, with significantly decreased accumulation of chlorophyll and its precursors, protoporphyrin IX and Mg-protoporphyrin IX. Interestingly, a higher protoporphyrin IX to Mg-protoporphyrin IX ratio was observed in chli. Lipid biosynthesis in chli leaves and seeds was also affected, with the mutant displaying significantly reduced total lipid content relative to Shaanong33. Genetic analysis indicated that the pale-green leaf phenotype was controlled by a single pair of recessive nuclear genes. Furthermore, sequence alignment revealed a single-nucleotide mutation (G664A) in the gene TraesCS7A01G480700.1, which encodes subunit I of the Mg-chelatase in plants. This single-nucleotide mutation resulted in an amino acid substitution (D221N) in the highly conserved domain of subunit I. As a result, mutant protein Tachli-7A lost the ability to interact with the normal protein TaCHLI-7A, as assessed by yeast two-hybrid assay. Meanwhile, Tachli-7A could not recover the chlorophyll deficiency phenotype of the Arabidopsis thaliana SALK_050029 mutant. Furthermore, we found that in Shaannong33, the protoporphyrin IX to Mg-protoporphyrin IX ratio was growth state-dependent and insensitive to environmental change. Overall, the mutation in Tachli-7A impaired the function of Mg-chelatase and blocked the conversion of protoporphyrin IX to Mg- protoporphyrin IX. Based on our results, the chli mutant represents a potentially useful resource for better understanding chlorophyll and lipid biosynthetic pathways in common wheat.

普通小麦（Triticum aestivum L.）是全球最重要的粮食作物之一。叶绿素（chlorophyll）在植物生长发育与作物遗传改良中发挥关键作用，且在一定程度上决定作物生产力。叶绿素的生物合成是一个复杂的代谢过程，对叶色变异植物突变体的研究曾推动诸多基础生物化学发现。本研究从小麦品种陕农33中筛选得到一个叶绿素缺失突变体，将其命名为chli。该突变体在苗期表现出显著的淡绿叶表型，其叶绿素及其生物合成前体——原卟啉IX（protoporphyrin IX）和镁原卟啉IX（Mg-protoporphyrin IX）的积累量均显著降低。值得注意的是，chli突变体中原卟啉IX与镁原卟啉IX的比值显著升高。此外，chli突变体的叶片与种子的脂质生物合成也受到影响，其总脂质含量相较于陕农33显著降低。遗传分析表明，该淡绿叶表型受一对隐性核基因控制。进一步序列比对发现，基因TraesCS7A01G480700.1存在单核苷酸突变（single-nucleotide mutation，G664A），该基因编码植物镁螯合酶（Mg-chelatase）的I亚基。该单核苷酸突变导致I亚基高度保守结构域内发生氨基酸替换（D221N）。经酵母双杂交实验（yeast two-hybrid assay）验证，突变体蛋白Tachli-7A丧失了与正常蛋白TaCHLI-7A的互作能力。同时，Tachli-7A无法恢复拟南芥（Arabidopsis thaliana）SALK_050029突变体的叶绿素缺失表型。此外，本研究发现，在陕农33中，原卟啉IX与镁原卟啉IX的比值依赖于植株生长状态，且不受环境变化影响。综上，Tachli-7A上的突变损害了镁螯合酶的功能，阻断了原卟啉IX向镁原卟啉IX的转化。基于本研究结果，chli突变体可作为一种潜在的有用资源，用于深入解析普通小麦的叶绿素与脂质生物合成通路。