Table_3_McMYB10 Modulates the Expression of a Ubiquitin Ligase, McCOP1 During Leaf Coloration in Crabapple.DOCX

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产物，对叶片、茎、花朵及果实的色泽形成起着至关重要的作用，并且研究发现其在人类健康中具有抗氧化功能。在本研究中，我们测定了麦红叶海棠（Malus domestica Borkh. 'McCrab') 叶片中 McMYB10 及其特异性的 E3 泛素连接酶 McCOP1 在一天之内以及五个叶片发育阶段中的表达情况。有趣的是，研究结果显示，McCOP1 基因的转录水平在白天高于夜晚，而 McMYB10 的转录物与 McCOP1-1 和 McCOP1-2 的转录以及具有红色叶片的海棠品种中的花青素积累呈现出正相关。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，MYBCORE 类型，我们据此推断 McMYB10 可能与 McCOP1-1 和 McCOP1-2 在转录水平上存在某种关联。酵母单杂交（Y1H）和电泳迁移率变动分析（EMSA）证实了 McMYB10 TF 能够特异性地结合至 McCOP1-1 和 McCOP1-2 的启动子。此外，在持续光照处理过程中，McMYB10 水平的提高促进了海棠叶片中花青素的生物合成以及 McCOP1-1 和 McCOP1-2 的表达水平。在海棠叶片和苹果果实中过表达或沉默 McMYB10，分别导致 McCOP1-1 和 McCOP1-2 以及花青素生物合成的表达水平上升或下降。我们的研究揭示了 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。