Grains of tetraploid wheat (Triticum turgidum L.) mainly accumulate the non-provitamin A carotenoid lutein—with low natural variation in provitamin A β-carotene in wheat accessions necessitating alternative strategies for provitamin A biofortification. Lycopene ɛ-cyclase (LCYe) and β-carotene hydroxylase (HYD) function in diverting carbons from β-carotene to lutein biosynthesis and catalyzing the turnover of β-carotene to xanthophylls, respectively. However, the contribution of LCYe and HYD gene homoeologs to carotenoid metabolism and how they can be manipulated to increase β-carotene in tetraploid wheat endosperm (flour) is currently unclear. We isolated loss-of-function Targeting Induced Local Lesions in Genomes (TILLING) mutants of LCYe and HYD2 homoeologs and generated higher order mutant combinations of lcye-A, lcye-B, hyd-A2, and hyd-B2. Hyd-A2 hyd-B2, lcye-A hyd-A2 hyd-B2, lcye-B hyd-A2 hyd-B2, and lcye-A lcye-B hyd-A2 hyd-B2 achieved significantly increased β-carotene in endosperm, with lcye-A hyd-A2 hyd-B2 exhibiting comparable photosynthetic performance and light response to control plants. Comparative analysis of carotenoid profiles suggests that eliminating HYD2 homoeologs is sufficient to prevent β-carotene conversion to xanthophylls in the endosperm without compromising xanthophyll production in leaves, and that β-carotene and its derived xanthophylls are likely subject to differential catalysis mechanisms in vegetative tissues and grains. Carotenoid and gene expression analyses also suggest that the very low LCYe-B expression in endosperm is adequate for lutein production in the absence of LCYe-A. These results demonstrate the success of provitamin A biofortification using TILLING mutants while also providing a roadmap for guiding a gene editing-based approach in hexaploid wheat.

中文翻译：

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番茄红素ε-环化酶和β-胡萝卜素羟化酶2同源物的突变组合增加了四倍体小麦（Triticum turgidum L.）籽粒胚乳中β-胡萝卜素的积累

四倍体小麦 ( Triticum turgidum L.) 的谷物主要积累非维生素 A 原类胡萝卜素叶黄素——小麦种质中维生素 A 原β-胡萝卜素的自然变异较低，因此需要替代维生素 A 原生物强化策略。番茄红素 ɛ-环化酶 (LCYe) 和 β-胡萝卜素羟化酶 (HYD) 分别起到将碳从 β-胡萝卜素转移到叶黄素生物合成和催化 β-胡萝卜素转化为叶黄素的作用。然而，LCYe和HYD基因同源物对类胡萝卜素代谢的贡献以及如何操纵它们以增加四倍体小麦胚乳（面粉）中的β-胡萝卜素目前尚不清楚。我们分离了LCYe的靶向基因组局部损伤 (TILLING) 突变体的功能丧失和HYD2同源物并产生lcye-A、lcye-B、hyd-A2和hyd-B2的更高阶突变组合。Hyd-A2 hyd-B2、lcye-A hyd-A2 hyd-B2、lcye-B hyd-A2 hyd-B2和lcye-A lcye-B hyd-A2 hyd-B2显着增加了胚乳中的β-胡萝卜素，其中lcye-A hyd-A2 hyd-B2表现出与对照植物相当的光合性能和光响应。类胡萝卜素谱的比较分析表明，消除HYD2同源物足以防止 β-胡萝卜素在胚乳中转化为叶黄素，而不会影响叶片中叶黄素的产生，并且 β-胡萝卜素及其衍生的叶黄素可能在营养组织和谷物中受到不同的催化机制。类胡萝卜素和基因表达分析还表明，在没有LCYe-A的情况下，胚乳中极低的LCYe-B表达足以产生叶黄素。这些结果证明了使用 TILLING 突变体进行维生素 A 生物强化的成功，同时也为指导六倍体小麦中基于基因编辑的方法提供了路线图。