Key message

Isoflavones are not involved in rhizobial signaling in red clover, but likely play a role in defense in the rhizosphere.

Abstract

Red clover (Trifolium pratense) is a high-quality forage legume, well suited for grazing and hay production in the temperate regions of the world. Like many legumes, red clover produces a number of phenylpropanoid compounds including anthocyanidins, flavan-3-ols, flavanols, flavanones, flavones, and isoflavones. The study of isoflavone biosynthesis and accumulation in legumes has come into the forefront of biomedical and agricultural research due to potential for medicinal, antimicrobial, and environmental implications. CRISPR/Cas9 was used to knock out the function of a key enzyme in the biosynthesis of isoflavones, isoflavone synthase (IFS1). A hemizygous plant carrying a 9-bp deletion in the IFS1 gene was recovered and was intercrossed to obtain homozygous mutant plants. Levels of the isoflavones formononetin, biochanin A and genistein were significantly reduced in the mutant plants. Wild-type and mutant plants were inoculated with rhizobia to test the effect of the mutation on nodulation, but no significant differences were observed, suggesting that these isoflavones do not play important roles in nodulation. Gene expression profiling revealed an increase in expression of the upstream genes producing the precursors for IFS1, namely, phenylalanine ammonium lyase and chalcone synthase, but there were no significant differences in IFS1 gene expression or in the downstream genes in the production of specific isoflavones. Higher expression in genes involved in ethylene response was observed in the mutant plants. This response is normally associated with biotic stress, suggesting that the plants may have been responding to cues in the surrounding rhizosphere due to lower levels of isoflavones.

中文翻译：

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红三叶草异黄酮合酶基因CRISPR/Cas9缺失突变体的异黄酮水平、结瘤和基因表达谱

关键信息

异黄酮不参与红三叶草的根瘤菌信号传导，但可能在根际防御中发挥作用。

抽象的

红三叶草) 是一种优质豆类草料，非常适合世界温带地区的放牧和干草生产。像许多豆类一样，红三叶草产生许多苯丙烷类化合物，包括花青素、黄烷-3-醇、黄烷醇、黄烷酮、黄酮和异黄酮。由于具有药用、抗菌和环境影响的潜力，豆类中异黄酮生物合成和积累的研究已成为生物医学和农业研究的前沿。CRISPR/Cas9 用于敲除异黄酮生物合成中的关键酶异黄酮合酶 (IFS1) 的功能。回收在 IFS1 基因中带有 9-bp 缺失的半合子植物并进行杂交以获得纯合突变体植物。异黄酮的水平 biochanin A 和染料木黄酮在突变植物中显着减少。用根瘤菌接种野生型和突变型植物以测试突变对结瘤的影响，但没有观察到显着差异，表明这些异黄酮在结瘤中不起重要作用。基因表达谱显示产生 IFS1 前体的上游基因的表达增加，即苯丙氨酸铵裂解酶和查尔酮合酶，但没有显着差异IFS1基因表达或在下游基因中产生特异性异黄酮。在突变植物中观察到参与乙烯反应的基因的更高表达。这种反应通常与生物胁迫有关，这表明由于异黄酮水平较低，植物可能一直在对周围根际的线索做出反应。