Lignin production marked a milestone in vascular plant evolution, and the emergence of syringyl (S)-lignin is lineage-specific. S-lignin biosynthesis in angiosperms, mediated by ferulate 5-hydroxylase (F5H, CYP84A1), has been considered a recent evolutionary event. F5H uniquely requires the cytochrome b5 protein CB5D as an obligatory redox partner for catalysis. However, it remains unclear how CB5D functionality originated and whether it co-evolved with F5H. We reveal here the ancient evolution of CB5D-type function supporting F5H-catalyzed S-lignin biosynthesis. CB5D emerged in charophyte algae, the closest relatives of land plants, and is conserved and proliferated in embryophytes, especially in angiosperms, suggesting functional diversification of the CB5 family before terrestrialization. A sequence motif containing acidic amino residues in helix 5 of the CB5 heme-binding domain contributes to the retention of CB5D function in land plants but not in algae. Notably, CB5s in the S-lignin-producing lycophyte Selaginella lack these residues, resulting in no CB5D-type function. An independently evolved S-lignin biosynthetic F5H (CYP788A1) in Selaginella relies on NADPH-dependent cytochrome P450 reductase as sole redox partner, distinct from angiosperms. These results suggest that angiosperm F5Hs co-opted the ancient CB5D, forming a modern cytochrome P450 monooxygenase system for aromatic ring meta-hydroxylation, enabling the re-emergence of S-lignin biosynthesis in angiosperms.

中文翻译：

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绿色谱系中细胞色素b5的多样性先于紫丁香基木质素生物合成的进化

木质素的产生标志着维管植物进化的一个里程碑，紫丁香基（S）-木质素的出现具有谱系特异性。被子植物中由阿魏酸 5-羟化酶（F5H、CYP84A1）介导的 S-木质素生物合成被认为是最近的进化事件。 F5H 独特地需要细胞色素 b5 蛋白 CB5D 作为催化的强制性氧化还原伙伴。然而，目前尚不清楚 CB5D 功能是如何起源的以及它是否与 F5H 共同进化。我们在这里揭示了支持 F5H 催化的 S-木质素生物合成的 CB5D 型功能的古老进化。 CB5D出现在轮藻类（陆地植物的近亲）中，并在有胚植物中保守和增殖，特别是在被子植物中，这表明CB5家族在陆地化之前的功能多样化。 CB5 血红素结合结构域螺旋 5 中含有酸性氨基酸残基的序列基序有助于在陆地植物中保留 CB5D 功能，但在藻类中则不然。值得注意的是，产生 S-木质素的石松植物卷柏中的 CB5 缺乏这些残基，导致不具有 CB5D 型功能。卷柏中独立进化的 S-木质素生物合成 F5H (CYP788A1) 依赖于 NADPH 依赖性细胞色素 P450 还原酶作为唯一的氧化还原伙伴，与被子植物不同。这些结果表明，被子植物 F5Hs 吸收了古老的 CB5D，形成了用于芳环间羟基化的现代细胞色素 P450 单加氧酶系统，使得 S-木质素生物合成在被子植物中重新出现。