Transcriptome and Flavonoids Metabolomic Analysis Identifies Regulatory Networks and Hub Genes in Black and White Fruits ofLycium ruthenicumMurray

Lycium ruthenicumMurry. 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 ofL. 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,LrAN1bshowed 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 asLrAN2-like. Yeast two-hybrid and transient tobacco overexpression experiments showed thatLrAN1bcould interact withLrAN2-likeandLrAN11to form MBW complex to activate the anthocyanin pathway. The yeast one-hybrid experiment indicated thatLrAN2-likebonded anthocyanin structural geneLrDFRandLrANSpromoters. Heterologous expression ofLrAN1bin 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 inL. ruthenicumfruit was proposed firstly and we speculate that the white fruit phenotype was due to abnormal expression ofLrAN1b. The findings provide new insight into the underlying mechanism of flavonoids, laying the foundation for future functional and molecular biological research inL. ruthenicum.