Abstract

Isoflavonoids are commonly found in leguminous plants. Glycitein is one of the isoflavones produced by soybean. The genes encoding the enzymes in the isoflavone biosynthetic pathway have mostly been identified and characterized. However, the gene(s) for isoflavone O-methyltransferase (IOMT), which catalyzes the last step of glycitein biosynthesis, has not yet been identified. In this study, we conducted multi-omics analyses of fungal-inoculated soybean and indicated that glycitein biosynthesis was induced in response to biotic stress. Moreover, we identified a unique type of IOMT, which participates in glycitein biosynthesis. Soybean seedlings were inoculated with Aspergillus oryzae or Rhizopus oligosporus and sampled daily for 8 d. Multi-omics analyses were conducted using liquid chromatography–tandem mass spectrometry and RNA sequencing. Metabolome analysis revealed that glycitein derivatives increased following fungal inoculation. Transcriptome co-expression analysis identified two candidate IOMTs that were co-expressed with the gene encoding flavonoid 6-hydroxylase (F6H), the key enzyme in glycitein biosynthesis. The enzymatic assay of the two IOMTs using respective recombinant proteins showed that one IOMT, named as GmIOMT1, produced glycitein. Unlike other IOMTs, GmIOMT1 belongs to the cation-dependent OMT family and exhibited the highest activity with Zn²⁺ among cations tested. Moreover, we demonstrated that GmIOMT1 overexpression increased the levels of glycitein derivatives in soybean hairy roots when F6H was co-expressed. These results strongly suggest that GmIOMT1 participates in inducing glycitein biosynthesis in response to biotic stress.

中文翻译：

---

基于生物胁迫下大豆的多组学分析，鉴定了一种独特类型的异黄酮 O-甲基转移酶 GmIOMT1。

 抽象的

异黄酮类化合物常见于豆科植物中。黄豆黄素是大豆产生的异黄酮之一。编码异黄酮生物合成途径中的酶的基因大部分已被鉴定和表征。然而，催化黄豆黄素生物合成最后一步的异黄酮O-甲基转移酶 (IOMT) 基因尚未确定。在这项研究中，我们对接种真菌的大豆进行了多组学分析，结果表明黄豆黄素生物合成是针对生物胁迫而诱导的。此外，我们还发现了一种独特类型的 IOMT，它参与黄豆黄素生物合成。将大豆幼苗接种米曲霉或少孢根霉，并每天取样，持续 8 天。使用液相色谱-串联质谱和 RNA 测序进行多组学分析。代谢组分析显示，接种真菌后黄豆黄素衍生物增加。转录组共表达分析确定了两个候选 IOMT，它们与编码类黄酮 6-羟化酶 (F6H)（黄豆黄素生物合成中的关键酶）的基因共表达。使用各自的重组蛋白对两种 IOMT 进行酶促分析表明，一种名为 GmIOMT1 的 IOMT 产生黄豆黄素。与其他 IOMT 不同，GmIOMT1 属于阳离子依赖性 OMT 家族，在测试的阳离子中表现出最高的 Zn ²⁺活性。此外，我们证明，当 F6H 共表达时，GmIOMT1 过表达会增加大豆毛状根中黄豆黄素衍生物的水平。这些结果强烈表明，GmIOMT1 参与诱导黄豆黄素生物合成以应对生物胁迫。