背景1：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. 

背景2：Lycopene ε-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. 

提出问题：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. 

研究方法：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.

结果1-高阶突变体：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. 

结果2-HYD2：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. 

结果3-LCYe-B和LCYe-A：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. 

结论：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.

 摘 要 

四倍体小麦的籽粒主要积累的是非维生素原A的类胡萝卜素叶黄素，而维生素原A（即β-胡萝卜素）含量的自然变异较低，因此需要选择维生素原A生物强化的育种策略。番茄红素ε-环化酶（LCYe）主要作用于将番茄红素催化形成α-胡萝卜素，然后进入叶黄素（lutein）的生物合成途径；另一条支路是由番茄红素β-环化酶催化，将番茄红素转变为β-胡萝卜素，然后经过β-胡萝卜素羟化酶（HYD）催化形成玉米黄素（一种xanthophylls，即叶黄素类物质）。但是，LCYe和HYD基因的部分同源基因（homoeologs，同一物种中、不同亚基因组间的直系同源基因，如LCYe-A和LCYe-B）对四倍体小麦中类胡萝卜素代谢的贡献，及其如何能够提高四倍体小麦胚乳中β-胡萝卜素的含量仍不清楚。本文中，作者利用定向诱导基因组局部突变技术（TILLING），获得了LCYe和HYD部分同源基因的功能缺失突变体，并获得了lcye-A、lcye-B、hyd-A2和hyd-B2之间组合的高阶突变体。Hyd-A2 hyd-B2双突、lcye-A hyd-A2 hyd-B2三突、lcye-B hyd-A2 hyd-B2三突以及lcye-A lcye-B hyd-A2 hyd-B2四突小麦植株的胚乳中的β-胡萝卜素含量显著增加，而lcye-A hyd-A2 hyd-B2三突小麦植株与对照相比，具有相似的光合性能和光响应。类胡萝卜素图谱的比较分析发现，HYD2部分同源基因足以防止β-胡萝卜素向叶黄素类物质的转变，但是叶片中的叶黄素类含量并未发生太大变化，说明小麦营养器官和籽粒中β-胡萝卜素及其衍生的叶黄素类物质的催化机制是不同的。类胡萝卜素和基因表达分析显示，在缺少LCYe-A基因的情况下，尽管LCYe-B基因的表达量很低，担其仍然能够形成足够多的叶黄素（lutein）。本文的研究结果揭示了利用TILLING技术进行四倍体小麦籽粒维生素原A生物强化的可行性，同时也为未来六倍体小麦基于基因编辑的育种改良提供了一种潜在策略。

p.s. 类胡萝卜素（Carotenoids）主要包括两大类：胡萝卜素（Carotenes）和叶黄素类（Xanthophylls），其中叶黄素类主要包括叶黄素（Lutein）、玉米黄素（Zeaxanthin）、新黄质（neoxanthin）和紫黄质（Violaxanthin）等物质。