State Key Laboratory of Crop Genetics and Germplasm Enhancement and Utilization, Ministry of Agriculture and Rural Affairs Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, China.
Key Laboratory of Horticultural Crop Biology and Germplasm Innovation, Ministry of Agriculture and Rural Affairs, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
Plant J. 2023 Aug;115(4):986-1003. doi: 10.1111/tpj.16275. Epub 2023 May 19.
The accumulation of carotenoids, such as xanthophylls, lycopene, and carotenes, is responsible for the color of carrot (Daucus carota subsp. sativus) fleshy roots. The potential role of DcLCYE, encoding a lycopene ε-cyclase associated with carrot root color, was investigated using cultivars with orange and red roots. The expression of DcLCYE in red carrot varieties was significantly lower than that in orange carrots at the mature stage. Furthermore, red carrots accumulated larger amounts of lycopene and lower levels of α-carotene. Sequence comparison and prokaryotic expression analysis revealed that amino acid differences in red carrots did not affect the cyclization function of DcLCYE. Analysis of the catalytic activity of DcLCYE revealed that it mainly formed ε-carotene, while a side activity on α-carotene and γ-carotene was also observed. Comparative analysis of the promoter region sequences indicated that differences in the promoter region may affect the transcription of DcLCYE. DcLCYE was overexpressed in the red carrot 'Benhongjinshi' under the control of the CaMV35S promoter. Lycopene in transgenic carrot roots was cyclized, resulting in the accumulation of higher levels of α-carotene and xanthophylls, while the β-carotene content was significantly decreased. The expression levels of other genes in the carotenoid pathway were simultaneously upregulated. Knockout of DcLCYE in the orange carrot 'Kurodagosun' by CRISPR/Cas9 technology resulted in a decrease in the α-carotene and xanthophyll contents. The relative expression levels of DcPSY1, DcPSY2, and DcCHXE were sharply increased in DcLCYE knockout mutants. The results of this study provide insights into the function of DcLCYE in carrots, which could serve as a basis for creating colorful carrot germplasms.
类胡萝卜素（如叶黄素、番茄红素和胡萝卜素）的积累是造成胡萝卜（Daucus carota subsp. sativus）肉质根颜色的原因。本研究使用橙色和红色肉质根的品种，调查了编码与胡萝卜根颜色相关的番茄红素 ε-环化酶的 DcLCYE 的潜在作用。在成熟阶段，红色胡萝卜品种中 DcLCYE 的表达明显低于橙色胡萝卜。此外，红色胡萝卜积累了更多的番茄红素和更少的 α-胡萝卜素。序列比较和原核表达分析表明，红色胡萝卜中的氨基酸差异不影响 DcLCYE 的环化功能。DcLCYE 催化活性分析表明，它主要形成 ε-胡萝卜素，同时也观察到对 α-胡萝卜素和 γ-胡萝卜素的侧活性。启动子区序列的比较分析表明，启动子区的差异可能影响 DcLCYE 的转录。在 CaMV35S 启动子的控制下，红色胡萝卜‘Benhongjinshi’中过表达 DcLCYE。转基因胡萝卜根中的番茄红素发生环化，导致 α-胡萝卜素和叶黄素积累增加，而 β-胡萝卜素含量显著降低。类胡萝卜素途径中其他基因的表达水平也同时上调。CRISPR/Cas9 技术敲除橙色胡萝卜‘Kurodagosun’中的 DcLCYE 导致 α-胡萝卜素和叶黄素含量降低。DcLCYE 敲除突变体中 DcPSY1、DcPSY2 和 DcCHXE 的相对表达水平急剧增加。本研究结果为 DcLCYE 在胡萝卜中的功能提供了深入了解，为创造彩色胡萝卜种质资源提供了依据。
