Table 3_Stage-specific metabolic divergence in flavonoid biosynthesis correlates with embryogenic capacity in rubber tree (Hevea brasiliensis).xlsx

IntroductionSomatic embryogenesis (SE) is an essential propagation technology for Hevea brasiliensis, yet its application remains limited by the strong genotype dependence of embryogenic capacity.
MethodsTo elucidate the metabolic basis of this variation, we conducted integrated metabolomic and transcriptomic analyses across four SE developmental stages in a high-embryogenic (HE) and a low-embryogenic (LE) genotype, including explants, induced callus, non-embryogenic / embryogenic callus, and cotyledonary embryos (HE-specific).
ResultsA total of 1,383 metabolites belonging to 11 major classes were identified, with flavonoids, phenolic acids, and amino acids being the predominant groups. PCA and hierarchical clustering revealed that metabolic variation was driven primarily by developmental stage rather than genotype. Differential metabolite profiling revealed strong stage specificity, with the callus-to-differentiation transition (LE-C vs. HE-EC) exhibiting the greatest metabolic divergence between genotypes. KEGG enrichment consistently highlighted flavonoid biosynthesis as a key differentiating pathway. Comparative analyses revealed a conserved-to-divergent pattern of metabolic regulation. During the explant-to-callus transition, both genotypes exhibited highly conserved flavonoid biosynthesis responses, with 67.5% of genes and 85.7% of metabolites showing concordant regulation (either both up-regulated or both down-regulated). In contrast, during the callus-to-differentiation transition, pronounced metabolic divergence emerged, with only 37.5% of genes and 6.7% of metabolites showing concordant regulation, and 11 flavonoid-related genes displaying opposite regulatory directions between genotypes. Notably, the HE genotype exhibited coordinated repression of CHS, CHI, F3H, UFGT, and anthocyanin biosynthesis, accompanied by decreased accumulation of naringenin and glycosylated flavonoids, along with an overall attenuation of dihydroflavonol accumulation. Conversely, the LE genotype maintained relatively active flavonoid biosynthesis and glycosylation, along with increased amino sugar and nucleotide sugar metabolism.
DiscussionOur results provide comprehensive metabolomic evidence for stage-dependent metabolic reprogramming during SE in H. brasiliensis. The contrasting patterns of flavonoid metabolism between genotypes at the callus-to-differentiation transition—systematic downregulation in the HE genotype versus sustained activation in the LE genotype—are consistent with the hypothesis that a timely reallocation of metabolic flux from secondary to primary metabolism may favor somatic embryo development. This study identifies the callus-to-differentiation transition as a critical metabolic checkpoint and suggests flavonoid biosynthesis genes, particularly CHS and glycosyltransferases, as potential targets for improving SE efficiency in recalcitrant genotypes.

引言：体细胞胚胎发生（Somatic embryogenesis, SE）是巴西橡胶树（Hevea brasiliensis）的重要繁殖技术，但其应用仍受限于胚胎发生能力的强基因型依赖性。
方法：为阐明该变异的代谢基础，本研究针对高胚胎发生型（high-embryogenic, HE）和低胚胎发生型（low-embryogenic, LE）两个橡胶树基因型，在体细胞胚胎发生的4个关键发育阶段开展整合代谢组与转录组分析，所涵盖的样本类型包括外植体、诱导愈伤组织、非胚性/胚性愈伤组织以及子叶胚（仅高胚胎发生型特有）。
结果：本研究共鉴定出1383种代谢物，隶属于11个主要类别，其中黄酮类、酚酸类及氨基酸类为优势代谢物类别。主成分分析（PCA）与层次聚类结果显示，代谢变异主要受发育阶段调控，而非基因型差异。差异代谢物分析揭示了显著的阶段特异性，其中愈伤组织向分化阶段的转变（低胚胎发生型愈伤组织 vs 高胚胎发生型胚性愈伤组织，即LE-C vs HE-EC）呈现出基因型间最大的代谢差异。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析一致表明，黄酮类生物合成是关键的分化通路。比较分析揭示了代谢调控从保守到分化的转变模式：在外植体向愈伤组织转变阶段，两个基因型的黄酮类生物合成响应高度保守，67.5%的基因与85.7%的代谢物呈现协同调控（均上调或均下调）；而在愈伤组织向分化阶段，代谢差异显著凸显，仅37.5%的基因与6.7%的代谢物呈现协同调控，且有11个黄酮类相关基因在两个基因型间呈现相反的调控方向。值得注意的是，高胚胎发生型基因型表现出查尔酮合酶（Chalcone Synthase, CHS）、查尔酮异构酶（Chalcone Isomerase, CHI）、黄烷酮3-羟化酶（Flavanone 3-Hydroxylase, F3H）、类黄酮糖基转移酶（UDP-Flavonoid Glucosyltransferase, UFGT）及花青素生物合成的协同抑制，伴随柚皮素与糖基化黄酮的积累减少，同时整体二氢黄酮醇的积累水平降低。相反，低胚胎发生型基因型则维持了相对活跃的黄酮类生物合成与糖基化过程，同时氨基糖与核苷酸糖代谢水平升高。
讨论：本研究结果为巴西橡胶树体细胞胚胎发生过程中依赖于发育阶段的代谢重编程提供了全面的代谢组学证据。在愈伤组织向分化阶段，两个基因型的黄酮类代谢模式呈现显著差异：高胚胎发生型基因型呈现系统性下调，而低胚胎发生型基因型则持续激活，这与"代谢流及时从次级代谢向初级代谢重新分配可能有利于体细胞胚胎发育"的假说一致。本研究明确了愈伤组织向分化阶段为关键的代谢检查点，并提出黄酮类生物合成基因（尤其是CHS与糖基转移酶）可作为提升顽拗型基因型体细胞胚胎发生效率的潜在靶点。