Transcriptome and Flavonoids Metabolomic Analysis Identifies Regulatory Networks and Hub Genes in Black and White Fruits of Lycium ruthenicum Murray.

Tingting Li,Yanlong Li,Guoxiu Li,Lin Tang,Fang Chen,Jingjin Wang,Huan Qin,Xiaoya Qin,Guoli Dai,Youlong Cao,Yunfang Fan,Yue Yin

doi:10.3389/fpls.2020.01256

Abstract

Lycium ruthenicum Murry. is a highly nutritional cash crop due to its fruit abundant anthocyanins. To understand the complex metabolic networks underlying the color formation in black and white fruits of L. ruthenicum, we conducted transcriptome and flavonoid metabolic profiling to identify the candidate genes possibly involved in flavonoid biosynthesis. As a result, 147 flavonoids were identified and there was almost no anthocyanin in white fruits, while luteolin, kaempferol, and quercetin derivatives showed markedly higher abundance. Furthermore, applying weighted gene co-expression network analyses, 3 MYB, 2 bHLH, 1WRKY and 1 NAC transcription factor, associated with anthocyanin biosynthesis were identified. A bHLH transcription factor, LrAN1b showed the greatest correlations with anthocyanin accumulation with no expression in white fruits. In addition, gene function analysis and qRT-PCR experiments identified a new activated anthocyanin MYB transcription factor designed as LrAN2-like. Yeast two-hybrid and transient tobacco overexpression experiments showed that LrAN1b could interact with LrAN2-like and LrAN11 to form MBW complex to activate the anthocyanin pathway. The yeast one-hybrid experiment indicated that LrAN2-like bonded anthocyanin structural gene LrDFR and LrANS promoters. Heterologous expression of LrAN1b in tobacco can significantly increase the anthocyanin content of tobacco florals and capsules, and activate anthocyanin synthesis related genes. Taken together, an anthocyanin regulatory network model in L. ruthenicum fruit was proposed firstly and we speculate that the white fruit phenotype was due to abnormal expression of LrAN1b. The findings provide new insight into the underlying mechanism of flavonoids, laying the foundation for future functional and molecular biological research in L. ruthenicum.

Highlights

Anthocyanins are an important subclass of water-soluble flavonoids pigments that can give plants a bright red to blue color
Almost no anthocyanins were detected in the white fruits, which is compatible with the phenotype (Figure 1B)
The results show that the black fruit of L. ruthenicum may be a good choice as an anthocyanin supplement for consumers, highlighting the prodigious differences in anthocyanin accumulation in black and white fruits of L. ruthenicum

Summary

Introduction

Anthocyanins are an important subclass of water-soluble flavonoids pigments that can give plants a bright red to blue color. Anthocyanin is a downstream product of the flavonoid pathway It takes 4-coumaroyl-CoA and malonyl-CoA as substrates; is synthesized by a series of enzymes encoded by the structural genes chalcone synthase (CHS), chalcone isomerase (CHI), flavonoid 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3’H), flavonoid 3′5′-hydroxylase (F3’5’H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS); and is transported to vacuoles after glycosylation, methylation, and acylation (Tanaka et al, 2010). In recent years, these structural genes and modification- and transport-related genes are mainly regulated by R2RMYB transcription factors, basic helix-loop-helix proteins (bHLH), and WD40 protein (MBW complexes) (Koes et al, 2005). For L. ruthenicum, an important economic crop as a source of anthocyanins, genetic control of the fruit color and exploration of germplasm resources are of particular concern

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Results

Conclusion