Plant Physiol |浙大滕元文教授研究团队揭示光诱导PpbHLH64促进梨花青素的生物合成和COP1介导的降解

光对红梨(Pyrus Pyrifolia)花青素的积累是必不可少的。花青素的生物合成是由结构基因编码的一系列酶催化的，这些酶受MYB-Basic/Helix/loop/Helix-WD Repeat(MYB-bHLH-WDR，MBW)复合物的调控。(SG)IIIF亚群的bHLH蛋白被认为参与了花色苷积累的调节。

在本研究中，我们揭示了属于SGIIIb的梨PpbHLH64对花青素的生物合成有正向调节作用，并且在转录和翻译后水平上都受到光的调节。具体地说，光照诱导了PpbHLH64在梨果实和愈伤组织中的表达和花青素的积累。在光照条件下，过量表达PpbHLH64的梨愈伤组织呈红色，而PpbHLH64-RNA干扰愈伤组织的花青素积累减少。另外，PpbHLH64在梨果皮中的瞬时过表达增加了花青素的积累，而病毒诱导的PpbHLH64的基因沉默则起到了相反的作用。

进一步分析表明，PpbHLH64是一种转录激活剂，直接与UDP葡萄糖-黄酮-3-O-GLYCOSYLTRANFERase启动子结合，上调表达。

此外，PpbHLH64与PpMYB10相互作用，但不与PpMYB114相互作用，形成显著诱导花色苷积累的MBW复合体。此外，PpbHLH64在黑暗中被组成型光形态发生蛋白1（COP1）靶向，随后被26S蛋白酶体降解。

遗传分析表明，PpbHLH64在光信号转导途径B-box 18下游发挥作用。B-box 18是光信号转导途径的一个组成部分。然而，我们无法检测到PpbHLH64和PpBBX18之间的直接相互作用。

本研究中PpbHLH64的特性突出了SGIIIb bHLH蛋白对光诱导的花青素积累的重要性。

Light is indispensable for the anthocyanin accumulation of red pear (Pyrus pyrifolia). Anthocyanin biosynthesis is catalyzed by a series of enzymes encoded by structural genes, which are regulated by MYB-basic/helix/loop/helix-WD repeat (MYB-bHLH-WDR, MBW) complex. The bHLH proteins of subgroup (SG) IIIf are believed to be involved in the regulation of anthocyanin accumulation. In this study, we revealed that pear PpbHLH64, which belongs to SGIIIb, positively regulates anthocyanin biosynthesis and is regulated by light at the transcriptional and post-translational levels. Specifically, an exposure to light induced PpbHLH64 expression and anthocyanin accumulation in pear fruit and calli. Under light conditions, pear calli overexpressing PpbHLH64 exhibited enhanced red coloration, whereas the anthocyanin accumulation decreased in the PpbHLH64-RNA-interference calli. Additionally, the transient overexpression of PpbHLH64 in pear fruit peel increased anthocyanin accumulation, whereas the virus-induced gene silencing of PpbHLH64 had the opposite effect. Further analyses indicated that PpbHLH64 is a transcriptional activator that directly binds to the promoter of UDP GLUCOSE-FLAVONOID-3-O-GLYCOSYLTRANFERASE, to up-regulate expression. Moreover, PpbHLH64 interacted with PpMYB10, but not with PpMYB114, to form an MBW complex that significantly induces the accumulation of anthocyanins. Furthermore, PpbHLH64 was targeted by CONSTITUTIVE PHOTOMORPHOGENIC 1 in darkness for subsequent degradation by the 26S proteasome. A genetic analysis indicated that PpbHLH64 functions downstream of B-BOX 18, a component of the light signal transduction pathway. However, we were unable to detect the direct interaction between PpbHLH64 and PpBBX18. The characterization of PpbHLH64 in this study highlights the importance of SGIIIb bHLH proteins for light-induced anthocyanin accumulation.

Ruiyan Tao, Wenjie Yu, Yuhao Gao, Junbei Ni, Lei Yin, Xiao Zhang, Hongxu Li, Dongsheng Wang, Songling Bai, Yuanwen Teng

Plant Physiology Oct 2020, pp.01188.2020; DOI: 10.1104/pp.20.01188

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