Table_4_McMYB10 Modulates the Expression of a Ubiquitin Ligase, McCOP1 During Leaf Coloration in Crabapple.DOC

In higher plants, anthocyanins are protective secondary metabolites, which contribute to the color of leaves, stems, flowers, and fruits, and have been found to have an antioxidant role in human health. In this study, we determined the expression of McMYB10 and its specific E3 ubiquitin ligase, McCOP1, in crabapple leaves during the course of a day and in five leaf development stages. Interestingly, the results showed that the transcription level of McCOP1 genes was higher in daylight than at night, and the transcripts of McMYB10 presented a positive correlation with the transcription of McCOP1-1 and McCOP1-2 and anthocyanin accumulation in a crabapple cultivar with red-colored leaves. Several MYB transcription factor (TF) binding sites of the MYBCORE type were found in the McCOP1-1 and McCOP1-2 promoters, and we deduced that there may be a relationship between McMYB10 and McCOP1-1 and McCOP1-2 at the transcriptional level. Yeast one hybrid (Y1H) and electrophoretic mobility shift assays (EMSA) demonstrated that the McMYB10 TF binds specifically to the promoter of McCOP1-1 and McCOP1-2. Furthermore, increased levels of McMYB10 promoted anthocyanin biosynthesis and the expression level of McCOP1-1 and McCOP1-2 in crabapple leaves during continuous light treatments, and overexpression or silencing of McMYB10 in crabapple leaves and apple fruits also result in an increase or decrease, respectively, in the expression of McCOP1-1 and McCOP1-2 and in anthocyanin biosynthesis. Our results reveal a new self-regulation mechanism in where McMYB10 modulates its own expression by activating McCOP1-1 and McCOP1-2 expression to promote ubiquitination of the McMYB10 protein by McCOP1.

在高等植物中，花青素作为一种具有保护作用的次生代谢物，对叶片、茎、花朵及果实的颜色形成起着至关重要的作用，并被发现对人体健康具有抗氧化作用。在本研究中，我们测定了麦李（Malus crabapple）叶片在一天之中以及五个叶片发育阶段中McMYB10及其特异性的E3泛素连接酶McCOP1的表达情况。有趣的是，结果显示，McCOP1基因的转录水平在白天高于夜间，而McMYB10的转录本与McCOP1-1和McCOP1-2的转录以及一种红叶品种的麦李中的花青素积累呈正相关。在McCOP1-1和McCOP1-2启动子中发现了多个MYB转录因子（TF）结合位点，类型为MYBCORE，据此推断，McMYB10可能与McCOP1-1和McCOP1-2在转录水平上存在关联。酵母单杂交（Y1H）和电泳迁移率变动分析（EMSA）表明，McMYB10 TF特异性地结合于McCOP1-1和McCOP1-2的启动子。此外，在持续光照处理中，McMYB10水平增加促进了花青素的生物合成以及McCOP1-1和McCOP1-2在麦李叶片中的表达水平；而在麦李叶片和苹果果实中过表达或沉默McMYB10，分别导致McCOP1-1和McCOP1-2的表达以及花青素生物合成的增加或减少。我们的研究结果揭示了McMYB10通过激活McCOP1-1和McCOP1-2的表达来促进自身蛋白McMYB10的泛素化，从而实现自我调节的新机制。