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

Abstract

<i>Lycium ruthenicum</i> 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 <i>L. ruthenicum</i>, 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, <i>LrAN1b</i> 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 <i>LrAN2-like</i>. Yeast two-hybrid and transient tobacco overexpression experiments showed that <i>LrAN1b</i> could interact with <i>LrAN2-like</i> and <i>LrAN11</i> to form MBW complex to activate the anthocyanin pathway. The yeast one-hybrid experiment indicated that <i>LrAN2-like</i> bonded anthocyanin structural gene <i>LrDFR</i> and <i>LrANS</i> promoters. Heterologous expression of <i>LrAN1b</i> 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 <i>L. ruthenicum</i> fruit was proposed firstly and we speculate that the white fruit phenotype was due to abnormal expression of <i>LrAN1b</i>. The findings provide new insight into the underlying mechanism of flavonoids, laying the foundation for future functional and molecular biological research in <i>L. ruthenicum</i>.