Abstract

MicroRNA172 (miR172) plays a role in regulating a diverse range of plant developmental processes, including flowering, fruit development and nodulation. However, its role in regulating flavonoid biosynthesis is unclear. In this study, we show that transgenic apple plants over-expressing miR172 show a reduction in red coloration and anthocyanin accumulation in various tissue types. This reduction was consistent with decreased expression of APETALA2 homolog MdAP2_1a (a miR172 target gene), MdMYB10, and targets of MdMYB10, as demonstrated by both RNA-seq and qRT-PCR analyses. The positive role of MdAP2_1a in regulating anthocyanin biosynthesis was supported by the enhanced petal anthocyanin accumulation in transgenic tobacco plants overexpressing MdAP2_1a, and by the reduction in anthocyanin accumulation in apple and cherry fruits transfected with an MdAP2_1a virus-induced-gene-silencing construct. We demonstrated that MdAP2_1a could bind directly to the promoter and protein sequences of MdMYB10 in yeast and tobacco, and enhance MdMYB10 promotor activity. In Arabidopsis, over-expression of miR172 reduced flavonoid (including anthocyanins and flavonols) concentration and RNA transcript abundance of flavonoid genes in plantlets cultured on medium containing 7% sucrose. The anthocyanin content and RNA abundance of anthocyanin genes could be partially restored by using a synonymous mutant of MdAP2_1a, which had lost the miR172 target sequences at mRNA level, but not restored by using a WT MdAP2_1a. These results indicate that miR172 inhibits flavonoid biosynthesis through suppressing the expression of an AP2 transcription factor that positively regulates MdMYB10.

中文翻译：

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microRNA172 靶向 APETALA2 以调节苹果 (Malus domestica) 中的类黄酮生物合成。

摘要

MicroRNA172 ( miR172 ) 在调节多种植物发育过程中发挥作用，包括开花、果实发育和结瘤。然而，其在调节类黄酮生物合成中的作用尚不清楚。在这项研究中，我们表明过表达miR172的转基因苹果植物在各种组织类型中显示出红色和花青素积累的减少。这种降低与APETALA2同源物MdAP2_1a（一种miR172靶基因）、MdMYB10和 MdMYB10 靶点的表达降低一致，如 RNA-seq 和 qRT-PCR 分析所示。MdAP2_1a的积极作用过表达MdAP2_1a的转基因烟草植物中花瓣花青素积累增强，以及用MdAP2_1a病毒诱导的基因沉默构建体转染的苹果和樱桃果实中花青素积累减少，支持了调节花青素生物合成。我们证明 MdAP2_1a 可以直接与酵母和烟草中MdMYB10的启动子和蛋白质序列结合，并增强MdMYB10启动子的活性。在拟南芥中， miR172的过表达在含有 7% 蔗糖的培养基上培养的小植株中，类黄酮（包括花青素和黄酮醇）浓度和 RNA 转录本丰度降低。花青素基因的花青素含量和 RNA 丰度可以通过使用MdAP2_1a的同义突变体部分恢复，该突变体在 mRNA 水平上丢失了miR172靶序列，但使用 WT MdAP2_1a不能恢复。这些结果表明miR172通过抑制正调节 MdMYB10 的 AP2 转录因子的表达来抑制类黄酮的生物合成。