Data Sheet 5_Integrative transcriptomic and metabolomic analysis reveals the molecular basis of leaf variegation in Cymbidium ensifolium.pdf

IntroductionLeaf variegation is a key ornamental trait of Cymbidium ensifolium (Jianlan); however, the molecular mechanisms underlying leaf color mutations in variegated sectors remain poorly understood. Elucidating the regulatory networks associated with pigment variation is essential for both basic research and ornamental improvement.
MethodsWild-type plants and two leaf color mutants—spot variegation type (BanYi) and line variegation type (XianYi)—were analyzed. Samples were divided into five groups based on leaf origin and color: CK (wild type), B (yellow sectors of BanYi), BL (green sectors of BanYi), X (yellow sectors of XianYi), and XL (green sectors of XianYi). Integrated transcriptomic and metabolomic analyses were performed. Differential expression analysis was conducted across four comparison groups (B vs BL, B vs CK, X vs CK, and X vs XL). Co-expression analysis, metabolite profiling, correlation analysis, and weighted gene co-expression network analysis (WGCNA) were used to identify key regulatory genes and modules associated with pigment accumulation.
ResultsA total of 6,716 differentially expressed genes (DEGs) were identified, with 141 shared among all comparison groups. These DEGs were significantly enriched in phenylpropanoid biosynthesis, zeatin biosynthesis, and flavonoid biosynthesis pathways. Twenty-four DEGs involved in flavonoid biosynthesis, including structural genes such as CCOAMT, TT4, and HCT, showed elevated expression in variegated leaf sectors. In addition, 80 transcription factors from the MYB, bHLH, and WRKY families were co-expressed with 50 pigment-related DEGs, suggesting coordinated transcriptional regulation. Metabolomic analysis identified 3,024 differentially accumulated metabolites (DAMs), of which 56 were shared across all groups. Correlation analysis revealed strong associations between co-DEGs and co-DAMs. WGCNA further identified key modules, including the “MEtan” module, which contained 83 genes significantly correlated with pigment-related metabolites such as 1′-Hydroxy-γ-carotene and violaxanthin.
DiscussionThese results demonstrate that leaf variegation in C. ensifolium is regulated by coordinated transcriptional and metabolic networks, particularly involving flavonoid and carotenoid-related pathways. The identified structural genes, transcription factors, and co-expression modules provide novel insights into the genetic basis of leaf color variation and offer valuable candidate targets for the ornamental improvement of Jianlan.

引言：叶斑彩变是建兰（Cymbidium ensifolium）的关键观赏性状，但目前对其彩变斑块中叶片颜色突变的分子机制仍知之甚少。阐明与色素变异相关的调控网络，对基础研究与观赏性状改良均具有重要意义。
方法：本研究分析了野生型植株及两种叶色突变体——斑点彩变型（斑艺，BanYi）与线条彩变型（线艺，XianYi）。根据叶片来源与颜色将样本分为5组：CK（野生型）、B（斑艺黄色斑块）、BL（斑艺绿色斑块）、X（线艺黄色斑块）及XL（线艺绿色斑块）。采用整合转录组与代谢组分析策略，对4组比较组合（B vs BL、B vs CK、X vs CK及X vs XL）开展差异表达分析。通过共表达分析、代谢物谱分析、相关性分析及加权基因共表达网络分析（Weighted Gene Co-expression Network Analysis, WGCNA），筛选与色素积累相关的关键调控基因及模块。
结果：本研究共鉴定得到6716个差异表达基因（Differentially Expressed Genes, DEGs），其中141个在所有比较组合中共享。这些DEGs显著富集于苯丙烷生物合成、玉米素生物合成及黄酮类生物合成通路。24个参与黄酮类生物合成的DEGs（包括CCOAMT、TT4、HCT等结构基因）在彩变叶片斑块中表达上调。此外，MYB、bHLH及WRKY家族的80个转录因子与50个色素相关DEGs共表达，提示存在协同转录调控。代谢组分析鉴定得到3024个差异积累代谢物（Differentially Accumulated Metabolites, DAMs），其中56个在所有组别中共享。相关性分析显示共表达DEGs与共积累DAMs之间存在显著关联。WGCNA进一步筛选得到关键模块，其中“MEtan”模块包含83个基因，与1'-羟基-γ-胡萝卜素、紫黄质等色素相关代谢物显著相关。
讨论：上述结果表明，建兰的叶斑彩变受协同转录与代谢网络调控，尤其涉及黄酮类与类胡萝卜素相关通路。本研究鉴定得到的结构基因、转录因子及共表达模块，为叶片颜色变异的遗传基础提供了新见解，也为建兰的观赏性状改良提供了极具价值的候选靶点。