MiR156 regulates anthocyanin biosynthesis through SPL targets and other microRNAs in poplar

Yamei Wang,Zhubing Hu,Xinwei Wang,Wenwen Liu,Siyi Guo,Ruijuan Yang,Han Wang,Lei Wang,Jinxing Lin,Chunxiang Fu,Hailing Zhang,Zhenying Wu

doi:10.1038/s41438-020-00341-w

Abstract

Anthocyanins biosynthesized from the flavonoid pathway are types of pigments that are involved in the protection of poplar from biotic and abiotic stresses. Previous researchers studying anthocyanin-related transcription factors and structural genes in poplar have made significant discoveries. However, little is known about the regulatory role of microRNAs in anthocyanin biosynthesis in poplar. Here, we overexpressed miR156 in poplar to study the comprehensive effects of the miR156-SPL module on the biosynthesis of anthocyanins. Small RNA sequencing analysis revealed 228 microRNAs differentially expressed in transgenic poplar plants with dramatically increased miR156 levels. Furthermore, integrated microRNAomic and transcriptomic analysis suggested that two microRNAs, miR160h, and miR858, have the potential to affect anthocyanin accumulation in poplar by regulating auxin response factors and MYB transcription factors, respectively. Additionally, the accumulation of miR160h and miR858 displayed a positive correlation with miR156 levels, suggesting a possible interaction between the miR156-SPL module and these microRNAs in poplar. Last, metabolomics analysis revealed that the levels of anthocyanins, flavones, and flavonols were substantially elevated in transgenic poplar plants overexpressing miR156 compared with the wild type, whereas the total lignin content was reduced in the transgenic plants. Taken together, our results indicate that miR156 can fine tune the anthocyanin biosynthetic pathway via multiple factors, including microRNAs, transcription factors, and the levels of structural genes, in poplar. This provides additional clues for understanding the complex regulatory network of anthocyanin biosynthesis in woody plants.

Highlights

Anthocyanins, flavones, flavonols, and lignin are important metabolites derived from the essential phenylpropanoid pathway in higher plants[1]
Our work revealed the comprehensive influences of miR156 overexpression on other microRNAs and their targets related to anthocyanin biosynthesis based on the combined analysis of microRNAomic, transcriptomic and metabolomic data
Our results indicate that exogenous miR156b functioned efficiently in a dose-dependent manner during morphological changes of poplar

Summary

Introduction

Anthocyanins, flavones, flavonols, and lignin are important metabolites derived from the essential phenylpropanoid pathway in higher plants[1]. Structural genes encoding enzymes including phenylalanine ammonialyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol reductase (DFR), leucoanthocyanidin dioxygenase/anthocyanidin synthase (LDOX/ANS), glutathione S-transferase (GST), and UDP-glucose flavonoid 3-o-glycosyltransferase (UFGT) are responsible for the formation of anthocyanins[1,3]. These genes are primarily regulated by R2R3-type MYB, basic helix-loop-helix (bHLH), and WD40 repeat (WDR) transcription factors, which compose the MYB–bHLH–WDR (MBW) transcriptional complex[4]. MiR156-targeted SPL9, a plant-specific transcription factor, negatively regulates the expression of anthocyanin biosynthetic genes by impairing the stability of the MBW complex[8]

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Results

Conclusion