Fan Yuxin, Peng Jiayu, Wu Jiacheng, Zhou Ping, He Ruijie, Allan Andrew C, Zeng Lihui
Institute of Genetics and Breeding in Horticultural Plants, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
BMC Plant Biol. 2021 Jun 16;21(1):275. doi: 10.1186/s12870-021-03050-1.
Flavonoid biosynthesis in plants is primarily regulated at the transcriptional level by transcription factors modulating the expression of genes encoding enzymes in the flavonoid pathway. One of the most studied transcription factor complexes involved in this regulation consists of a MYB, bHLH and WD40. However, in Chinese Narcissus (Narcissus tazetta L. var. chinensis), a popular monocot bulb flower, the regulatory mechanism of flavonoid biosynthesis remains unclear.
In this work, genes related to the regulatory complex, NtbHLH1 and a R2R3-MYB NtMYB6, were cloned from Chinese Narcissus. Phylogenetic analysis indicated that NtbHLH1 belongs to the JAF13 clade of bHLH IIIf subgroup, while NtMYB6 was highly homologous to positive regulators of proanthocyanidin biosynthesis. Both NtbHLH1 and NtMYB6 have highest expression levels in basal plates of Narcissus, where there is an accumulation of proanthocyanidin. Ectopic over expression of NtbHLH1 in tobacco resulted in an increase in anthocyanin accumulation in flowers, and an up-regulation of expression of the endogenous tobacco bHLH AN1 and flavonoid biosynthesis genes. In contrast, the expression level of LAR gene was significantly increased in NtMYB6-transgenic tobacco. Dual luciferase assays showed that co-infiltration of NtbHLH1 and NtMYB6 significantly activated the promoter of Chinese Narcissus DFR gene. Furthermore, a yeast two-hybrid assay confirmed that NtbHLH1 interacts with NtMYB6.
Our results suggest that NtbHLH1 may function as a regulatory partner by interacting directly with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus.
植物中的类黄酮生物合成主要在转录水平上受到转录因子的调控，这些转录因子调节类黄酮途径中编码酶的基因的表达。参与这种调控的研究最多的转录因子复合物之一由一个MYB、bHLH和WD40组成。然而，在一种流行的单子叶球根花卉中国水仙（Narcissus tazetta L. var. chinensis）中，类黄酮生物合成的调控机制仍不清楚。
在这项研究中，从中国水仙中克隆了与调控复合物相关的基因NtbHLH1和一个R2R3-MYB基因NtMYB6。系统发育分析表明，NtbHLH1属于bHLH IIIf亚组的JAF13分支，而NtMYB6与原花青素生物合成的正调控因子高度同源。NtbHLH1和NtMYB6在中国水仙的基部鳞片中表达水平最高，那里有原花青素的积累。在烟草中异位过量表达NtbHLH1导致花朵中花青素积累增加，以及内源性烟草bHLH AN1和类黄酮生物合成基因的表达上调。相反，在NtMYB6转基因烟草中LAR基因的表达水平显著增加。双荧光素酶测定表明，NtbHLH1和NtMYB6的共浸润显著激活了中国水仙DFR基因的启动子。此外，酵母双杂交试验证实NtbHLH1与NtMYB6相互作用。
我们的结果表明，NtbHLH1可能通过与NtMYB6直接相互作用作为调控伙伴，以增强中国水仙中原花青素的积累。
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