Molecular and biochemical rhythms in dihydroflavonol 4-reductase-mediated regulation of leucoanthocyanidin biosynthesis in Carthamus tinctorius L
Molecular and biochemical rhythms in dihydroflavonol 4-reductase-mediated regulation of leucoanthocyanidin biosynthesis in Carthamus tinctorius L
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DOI：
10.1016/j.indcrop.2020.112838
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发表时间：
2020-11-15
影响因子：
5.9
通讯作者：
Li Haiyan
中科院分区：
文献类型：
--
作者：
Ahmad, Naveed;Li Tian;Li Haiyan
黄酮类化合物是一类独特的酚类化合物，已知参与植物生长、发育和花色素沉着。二氢黄酮醇-4-还原酶（dihydroflavonol-4-reductase）是天然苯丙氨酸途径中催化二氢黄酮醇立体专一性还原为白花色素的第一个关键酶，但对红花二氢黄酮醇-4-还原酶的进化和功能的研究较少。本研究首次在全基因组范围内对红花二氢黄酮醇4-还原酶进行了鉴定，并对其功能进行了分析。在红花基因组中共鉴定出20个CtCYP 45082 C酶编码基因。系统发育分析表明，CtCYP 45082 C序列聚类成5个主要的分支，说明整个植物界的进化分歧的显着影响。进一步的计算机模拟分析表明，所有编码CtCYP 45082 Cs的酶都含有基本的顺式调节单元和蛋白质结构域/基序，这些结构域/基序在整个真核细胞CYP 450中特异性保守。此外，与绿色荧光蛋白融合的CtCYP 45082 C1在洋葱表皮细胞和烟草叶片中的瞬时表达证实了明显不同的质膜亚细胞定位。CtCYP 45082 C1的异源蛋白表达分析表明，CtCYP 45082 C1能有效地催化cis-3，4-leucopelargonidin和dihydromyricycline在leucoanthocyanidin生物合成中的还原。此外，还采用实时荧光定量PCR技术对20个红花CYP 45082 C基因的转录表达进行了分析。总体而言，在所有研究的组织中均检测到每个mRNA转录本，但除了CtCYP 45082 C17 -20之外，具有不同的模式，表明CtCYP 45082 C基因家族在红花次生代谢物生物合成中的潜在作用。此外，CtCYP 45082 C1和两个下游类黄酮途径调控基因在茉莉酸甲酯、冷、H（2）0（2）和热辐射诱导下的表达表明，CtCYP 45082 C1、CtCHI和CtFLS的mRNA表达对各种环境变化敏感。该研究为进一步研究CtCYP 45082 C编码酶的功能特性提供了有意义的见解，这些酶也可能参与黄酮类化合物的生物合成。
Flavonoids are a distinctive class of phenolic compounds known to be involved in plant growth, development and floral pigmentation. During the natural phenylalanine pathway, dihydroflavonol-4-reductase is the first committed enzyme that catalyzes the stereo-specific reduction of dihydroflavonols into leucoanthocyanidins.Howsoever, less attention has been given to studies explaining the evolution and function of dihydroflavonol-4-reductases in Carthamus tinctorius L. This study explains the first comprehensive genome-wide identification and functional characterization of putative dihydroflavonol 4-reductase in Carthamus tinctorius L. Altogether, 20 CtCYP45082C enzyme encoding genes have been identified in the Carthamus tinctorius genome. Phylogeney analysis revealed the clustering of CtCYP45082C sequences into five major clades illustrating the significant effects of evolutionary divergence across the plant kingdom. Further in silico analyses indicated that all enzyme-encoding CtCYP45082Cs contain fundamental cis-regulatory units and protein domains/motifs that are specifically conserved throughout eukaryotic CYP450 s. In addition, the transient expression of CtCYP45082C1 fused with green fluorescent protein in onion epidermal cells and tobacco leaves confirmed a clearly distinct subcellular localization to plasma membrane. Biochemical characterization of CtCYP45082C1 using the heterologous protein expression assay indicated that CtCYP45082C1 effectively catalyzes the reduction of cis-3,4-leucopelargonidin and dihydromyricetin in leucoanthocyanidin biosynthesis. Moreover, the transcript expression of 20 Carthamus tinctorius derived CYP45082C genes has also been analyzed by real-time quantitative PCR. Each mRNA transcript was detected, in general, in all the investigated tissues, but with different patterns except for CtCYP45082C17-20, indicating the potential role of CtCYP45082C gene family in the secondary metabolite biosynthesis of Carthamus tinctorius. Additionally, the expression of CtCYP45082C1 and two downstream flavonoid pathway regulatory genes following induction of methyl jasmonate, cold, H(2)0(2) and heat irradiation suggested that mRNA expressions of CtCYP45082C1, CtCHI and CtFLS are susceptible to various environmental changes. This study provides meaningful insights for further functional characterization studies of the CtCYP45082C encoding enzymes which may also be involved in biosynthesis of flavonoids.