Table 10_Integrated metabolomic and transcriptomic analysis of the mechanism underlying leaf variegation in Miscanthus sinensis ‘Zebrinus’.xls

IntroductionMiscanthus sinensis ‘Zebrinus’ is a landscape plant with high ornamental value, whose core ornamental feature is determined by the irregularly distributed yellow variegation on its leaves, supporting its extensive application in landscape design and configuration. M. sinensis ‘Zebrinus’, as a typical variegated-leaf gramineous plant, possesses a key phenotypic trait of leaf variegation that distinguishes it from ordinary Miscanthus species. However, up to the present moment, we know little about the molecular regulatory mechanism underlying this unique variegation, with relevant research carried out in the exploratory stage.
MethodsThis study was performed with the use of two leaf phenotypes [Yellow area of variegated leaves (YS) and Green area of variegated leaves (GS)] of M. sinensis ‘Zebrinus’. Differential metabolites between GS and YS leaf samples was conducted using the metabolomic analysis, with a focus on identifying key metabolites associated with leaf variegation. Furthermore, gene expression profiles of GS and YS leaves were acquired through transcriptome sequencing. With the screening of differentially expressed genes (DEGs), this study also carried out functional annotation and pathway enrichment analysis. Moreover, the expression levels of candidate genes in GS and YS leaves were measured via quantitative real-time polymerase chain reaction (qRT-PCR). In addition, a “gene-metabolite” regulatory network was constructed by integrating the metabolomic and transcriptomic data to screen out the key metabolites and core genes responsible for regulating leaf variegation in M. sinensis ‘Zebrinus’.
ResultsMetabolomic analysis identified 4,036 common metabolites in GS and YS samples, with major enrichment in the flavonoid biosynthesis pathway. Secondary classification of this pathway indicated that flavonoids had the highest content. Further comparison of the expression levels of key metabolites revealed that the accumulation patterns of neohesperidin, taxifolin, naringenin, and xanthohumol in YS were all higher than those in GS, with naringenin showing the most significant difference, suggesting that it might be the core metabolite regulating leaf spot formation. According to subsequent transcriptome sequencing, 5,252 DEGs were screened out from the YS and GS samples, which were mainly enriched in flavonoid biosynthesis phenylpropanoid biosynthesis and other pathways. qRT-PCR presented the highest expression level in chalcone synthase (CHS). Integration of metabolome and transcriptome demonstrated significant enrichment of differential metabolites and DEGs in the flavonoid biosynthesis pathway. Additionally, correlation network graph analysis suggested the highest correlation of naringenin with CHS.
DiscussionThis study identifies the core intrinsic regulatory mechanism underlying leaf variegation in M. sinensis ‘Zebrinus’ through integrated metabolomic and transcriptomic analysis. CHS has a strong correlation with naringenin, suggesting that the transcriptional regulation of the CHS gene may directly affect the biosynthesis of naringenin. The synergistic effect of the two may be one of the key molecular mechanisms underlying the formation of yellow leaf variegation.

引言
斑叶芒（Miscanthus sinensis ‘Zebrinus’）是一种极具观赏价值的园林植物，其核心观赏性状由叶片上不规则分布的黄色斑纹决定，这一特性使其在园林设计与配置中应用广泛。作为典型的斑叶禾本科植物，斑叶芒拥有区别于普通芒属植物的关键表型性状——叶片斑纹。然而截至目前，人们对这一独特斑纹形成的分子调控机制仍知之甚少，相关研究尚处于探索阶段。

材料与方法
本研究以斑叶芒的两种叶片表型为研究材料：斑叶黄色区域（YS）与斑叶绿色区域（GS）。通过代谢组学分析检测GS与YS叶片样本间的差异代谢物，旨在筛选与叶片斑纹形成相关的关键代谢物。此外，通过转录组测序获取GS与YS叶片的基因表达谱，经差异表达基因（DEGs）筛选后开展功能注释与通路富集分析。同时，采用实时荧光定量聚合酶链反应（qRT-PCR）检测候选基因在GS与YS叶片中的表达水平。此外，本研究整合代谢组与转录组数据构建“基因-代谢物”调控网络，以筛选调控斑叶芒叶片斑纹形成的关键代谢物与核心基因。

结果
代谢组学分析在GS与YS样本中共鉴定出4036种共有代谢物，主要富集于黄酮类生物合成通路。对该通路进行次级分类后发现，黄酮类物质含量最高。进一步对比关键代谢物的积累模式发现，新橙皮苷、花旗松素、柚皮素与黄腐醇在YS中的积累量均高于GS，其中柚皮素的差异最为显著，提示其可能是调控叶斑形成的核心代谢物。后续转录组测序共筛选得到5252个差异表达基因，主要富集于黄酮类生物合成、苯丙烷类生物合成等通路。qRT-PCR结果显示，查尔酮合酶（CHS）的表达水平最高。整合代谢组与转录组数据发现，差异代谢物与差异表达基因均显著富集于黄酮类生物合成通路。此外，相关网络图分析表明，柚皮素与CHS的相关性最高。

讨论
本研究通过整合代谢组与转录组分析，揭示了斑叶芒叶片斑纹形成的核心内在调控机制。CHS与柚皮素具有极强的相关性，提示CHS基因的转录调控可能直接影响柚皮素的生物合成，二者的协同效应可能是黄色叶斑形成的关键分子机制之一。