花青素生物合成的代谢组和转录组分析揭示了红茎紫花苜蓿中的关键代谢物和候选基因 （<i>紫苜蓿</i>)

背景：紫花苜蓿 （<i>Medicago sativa </i>L.） 是一种重要的优质饲料资源，尤其是在缺乏富含蛋白质的饲料的热带和亚热带地区。太空突变紫花苜蓿茎的红色色素沉着主要是由花青素积累引起的。然而，对控制紫花苜蓿茎中花青素生物合成机制的研究一直很少。结果： 在本研究中，我们对两种类型的苜蓿茎进行了转录组和代谢组的联合分析： 空间突变红茎紫花苜蓿和非空间突变绿茎紫花苜蓿 （对照）。花青素代谢组分析揭示了 45 种与花青素生物合成相关的代谢物，其中矢车菊素-3-O-葡萄糖苷、天竺葵素-3-O-阿拉伯糖苷、飞燕草素-3-O-（6-O-乙酰）-葡萄糖苷和山奈酚-3-O-芸香糖苷被鉴定为红茎苜蓿的主要花青素。转录组分析显示 72 个与花青素生物合成途径相关的差异表达基因，其中 54 个基因在红色茎中高表达，包括 12 个 PAL （苯丙氨酸解氨酶）、22 个 4CLs （4-香豆酰：CoA 连接酶）、8 个 CHS （查尔酮合酶）、3 个 F3Hs （黄烷酮 3-羟化酶）、2 个 ANRs （花青素还原酶）、3 个 DFRs （二氢黄酮醇-4-还原酶）、3 个 ANS （花青素合酶） 和 1 个 FLS （黄酮醇合酶） 基因。这些基因可能是红茎花青素生物合成的关键。差异表达基因和差异表达花青素相对含量的共表达分析表明，每个花青素与多个基因密切相关，花青素积累过程受多个基因调控。这些基因的表达与矢车菊素-3-O-葡萄糖苷、天竺葵素-3-O-阿拉伯糖苷和山奈酚-3-O-芦丁的相对含量呈显著正相关。结论： 总体而言，<i>PAL</i>、 <i>4CL</i>、 <i>CHS</i>、 <i>F3H</i>、 <i>ANR</i>、 <i>DFR</i>、 <i>ANS</i>和 <i>FLS</i>与花青素的组成和含量密切相关。不同的花青素积累模式可能导致紫花苜蓿的茎颜色不同。这些发现为红茎紫花苜蓿中花青素生物合成的分子机制提供了全面的见解。<br>Background: Alfalfa (Medicago sativa L.) is an important high-quality forage resource, especially in tropical and subtropical regions where protein-rich forages are scarce. The red pigmentation of stems in space-mutated alfalfa is mainly caused by anthocyanin accumulation. However, studies on the mechanisms governing anthocyanin biosynthesis in alfalfa stems have been relatively scarce. Results: In this study, we performed combined transcriptomic and metabolomic analyses of two alfalfa stem types: space-mutated red-stem alfalfa and non-space-mutated green-stem alfalfa (control). Anthocyanin metabolomic analysis revealed 45 metabolites related to anthocyanin biosynthesis, among which cyanidin-3-O-glucoside, pelargonidin-3-O-arabinoside, delphinidin-3-O-(6-O-acetyl)-glucoside, and kaempferol-3-O-rutinoside were identified as the major anthocyanins in red-stem alfalfa. Transcriptomic analysis identified 72 differentially expressed genes (DEGs) related to the anthocyanin biosynthesis pathway, of which 54 genes were highly expressed in red stems, including 12 <i>PAL</i> (phenylalanine ammonia-lyase) genes, 22 <i>4CL</i> (4-coumarate:CoA ligase) genes, 8 <i>CHS</i> (chalcone synthase) genes, 3 <i>F3H</i> (flavanone 3-hydroxylase) genes, 2 <i>ANR</i> (anthocyanidin reductase) genes, 3 <i>DFR</i> (dihydroflavonol 4-reductase) genes, 3 <i>ANS</i> (anthocyanidin synthase) genes, and 1 <i>FLS</i> (flavonol synthase) gene. These genes may be key regulators of anthocyanin biosynthesis in red-stem alfalfa. Co-expression analysis of DEGs and the relative contents of differentially expressed anthocyanins showed that each anthocyanin was closely associated with multiple genes, and the anthocyanin accumulation process was regulated by multiple genes. The expression of these genes was significantly positively correlated with the relative contents of cyanidin-3-O-glucoside, pelargonidin-3-O-arabinoside, and kaempferol-3-O-rutinoside. Conclusion: Overall, <i>PAL</i>, <i>4CL</i>, <i>CHS</i>, <i>F3H</i>, <i>ANR</i>, <i>DFR</i>, <i>ANS</i>, and <i>FLS</i> are closely associated with the composition and content of anthocyanins. Different anthocyanin accumulation patterns may lead to variations in stem color of alfalfa. These findings provide comprehensive insights into the molecular mechanisms underlying anthocyanin biosynthesis in red-stem alfalfa.