Transcriptome Analysis and HPLC Profiling of Flavonoid Biosynthesis in Citrus aurantium L. during Its Key Developmental Stages

SIMPLE SUMMARY: Flavonoid is an important secondary metabolite with rich biological activity and pharmacological activity, and Citrus aurantium L. is of value in this regard. Citrus aurantium L. fruits are rich in flavonoid, but research information on flavonoid biosynthesis of Citrus aurantium L. is rare. Therefore, analysis of the key developmental stage and genes for flavonoid biosynthesis is essential for breeding Citrus aurantium L. varieties with high flavonoid content. Here, we report the profile of flavonoid and key flavonoid biosynthesis genes in the growth period of Citrus aurantium L. based on transcriptome analysis. We found that total flavonoid content decreased gradually during the fruit development stage, and that neohesperidin was the main flavonoid in the early development stage but with the progression of the development stage, naringin content increased rapidly and became the main flavonoid component. In addition, the key genes related to flavonoid biosynthesis in Citrus aurantium L. were identified. These results will lay the foundation for the mechanism underlying flavonoid biosynthesis in Citrus aurantium L. fruits. ABSTRACT: Citrus aurantium L. (sour orange) is a significant Chinese medicinal and fruit crop rich in flavonoids. However, the pathways and genes involved in flavonoid biosynthesis at the key developmental stages of Citrus aurantium L. are not fully understood. This study found that the total flavonoid concentration gradually decreased as the fruit developed. Additionally, it showed that neohesperidin was the main flavonoid in the early stages of sour orange fruit development. However, as the development stage progressed, naringin content increased rapidly and emerged as the main flavonoid component. From 27 cDNA libraries, RNA sequencing yielded 16.64 billion clean bases, including 8989 differentially expressed genes. We identified 74 flavonoid related unigenes mapped to the phenylalanine, tyrosine, and phenylpropanoid biosynthesis pathways. A total of 152 UDP-glucuronosyltransferase genes (UGTs) were identified from C. aurantium L. transcriptome database, in which 22 key flavonoid-correlated UGTs were divided into five main AtGT groups: E, G, I, L, M. We observed that the ethylene responsive factors (ERF) and myeloblastosis (MYB) family mainly regulated the key genes involved in flavonoid biosynthesis. Overall, our study generated extensive and detailed transcriptome data on the development of C. aurantium L. and characterized the flavonoid biosynthesis pattern during its fruit developmental stages. These results will benefit genetic modification or selection to increase the flavonoid content in sour oranges.