Flavonoids are an important class of secondary metabolites widely found in plants, contributing to plant growth and development and having prominent applications in food and medicine. The biosynthesis of flavonoids has long been the focus of intense research in plant biology. Flavonoids are derived from the phenylpropanoid metabolic pathway, and have a basic structure that comprises a C15 benzene ring structure of C6-C3-C6. Over recent decades, a considerable number of studies have been directed at elucidating the mechanisms involved in flavonoid biosynthesis in plants. In this review, we systematically summarize the flavonoid biosynthetic pathway. We further assemble an exhaustive map of flavonoid biosynthesis in plants comprising eight branches (stilbene, aurone, flavone, isoflavone, flavonol, phlobaphene, proanthocyanidin, and anthocyanin biosynthesis) and four important intermediate metabolites (chalcone, flavanone, dihydroflavonol, and leucoanthocyanidin). This review affords a comprehensive overview of the current knowledge regarding flavonoid biosynthesis, and provides the theoretical basis for further elucidating the pathways involved in the biosynthesis of flavonoids, which will aid in better understanding their functions and potential uses.

Double flower domestication is of great value in ornamental plants and presents an excellent system to study the mechanism of morphological alterations by human selection. The classic ABC model provides a genetic framework underlying the control of floral organ identity and organogenesis from which key regulators have been identified and evaluated in many plant species. Recent molecular studies have underscored the importance of C-class homeotic genes, whose functional attenuation contributed to the floral diversity in various species. Cultivated Camellia japonica L. possesses several types of double flowers, however the molecular mechanism underlying their floral morphological diversification remains unclear. In this study, we cloned the C-class orthologous gene CjAG in C. japonica. We analyzed the expression patterns of CjAG in wild C. japonica, and performed ectopic expression in Arabidopsis. These results revealed that CjAG shared conserved C-class function that controls stamen and carpel development. Further we analyzed the expression pattern of CjAG in two different C. japonica double-flower varieties, `Shibaxueshi’ and `Jinpanlizhi’, and showed that expression of CjAG was highly contracted in `Shibaxueshi’ but expanded in inner petals of `Jinpanlizhi’. Moreover, detailed expression analyses of B- and C-class genes have uncovered differential patterns of B-class genes in the inner organs of `Jinpanlizhi’. These results demonstrated that the contraction and expansion of CjAG expression were associated with the formation of different types of double flowers. Our studies have manifested two different trajectories of double flower domestication regarding the C-class gene expression in C. japonica.

Camellia nitidissima is a woody plant with high ornamental value, and its golden-yellow flowers are rich in a variety of bioactive substances, especially flavonoids, that are beneficial to human health. Chalcone isomerases (CHIs) are key enzymes in the flavonoid biosynthesis pathway; however, there is a scarcity of information regarding the CHI family genes of C. nitidissima. In this study, seven CHI genes of C. nitidissima were identified and divided into three subfamilies by phylogenetic analysis. The results of multiple sequence alignment revealed that, unlike CnCHI1/5/6/7, CnCHI2/3/4 are bona fide CHIs that contain all the active site and critical catalytic residues. Analysis of the expression patterns of CnCHIs and the total flavonoid content of the flowers at different developmental stages revealed that CnCHI4 might play an essential role in the flavonoid biosynthesis pathway of C. nitidissima. CnCHI4 overexpression significantly increased flavonoid production in Nicotiana tabacum and C. nitidissima. The results of the dual-luciferase reporter assay and yeast one-hybrid system revealed that CnMYB7 was the key transcription factor that governed the transcription of CnCHI4. The study provides a comprehensive understanding of the CHI family genes of C. nitidissima and performed a preliminary analysis of their functions and regulatory mechanisms.

The accumulation of various pigments leads to the formation of different flower colors in plants. However, the regulation mechanism of yellow flower formation and flower color differences between Camellia nitidssima C.W.Chi (CN) and its hybrids C. ‘Zhenghuangqi’ (ZHQ), C. ‘Huangxuanlv’ (HXL), and C. ‘Xinshiji’ (XSJ), remains largely unknown. Here, we showed that the content of two flavonols, quercetin-7-O-glucoside (Qu7G) and quercetin-3-O-glucoside (Qu3G), was positively correlated with the yellow degree of petals in CN and its three hybrids. Additionally, we performed a comparative transcriptomic analysis of petals of the four yellow camellia plants, which revealed 322 common upregulated and 866 common downregulated DEGs (differentially expressed genes) in the CN vs. ZHQ, CN vs. HXL, and CN vs. XSJ comparison groups. Their regulatory pathway analysis showed that flavonol biosynthesis genes (FLSs and GTs) and transcriptional regulatory genes MYBs were all expressed higher in CN than its three hybrids, which corresponded to differences in the flavonol content among the four yellow camellias. Further, two ethylene synthesis genes (ACSs, ACO) and three ethylene signaling genes (EIN2s, EIN3, ERFs) were all upregulated in the yellow petals of CN. In conclusion, the expression of flavonol-related genes and flavonols (Qu7G and Qu3G) accumulation could play a key role in the formation of yellow flowers in camellia, and the ethylene pathway might be involved in the regulation of yellow flower formation of camellias. This work describes the possible regulatory pathway of yellow camellia, thereby laying a foundation for future verification of genes linked to flower coloring and the breeding of yellow camellia.

Plant secondary metabolism is complex in its diverse chemical composition and dynamic regulation of biosynthesis. How the functional diversification of enzymes contributes to the diversity is largely unknown. In the flavonoids pathway, dihydroflavonol 4-reductase (DFR) is a key enzyme mediating dihydroflavanol into anthocyanins biosynthesis. Here, the DFR homolog was identified from Camellia nitidissima Chi. (CnDFR) which is a unique species of the genus Camellia with golden yellow petals. Sequence analysis showed that CnDFR possessed not only conserved catalytic domains, but also some amino acids peculiar to Camellia species. Gene expression analysis revealed that CnDFR was expressed in all tissues and the expression of CnDFR was positively correlated with polyphenols but negatively with yellow coloration. The subcellular localization of CnDFR by the tobacco infiltration assay showed a likely dual localization in the nucleus and cell membrane. Furthermore, overexpression transgenic lines were generated in tobacco to understand the molecular function of CnDFR. The analyses of metabolites suggested that ectopic expression of CnDFR enhanced the biosynthesis of polyphenols, while no accumulation of anthocyanins was detected. These results indicate a functional diversification of the reductase activities in Camellia plants and provide molecular insights into the regulation of floral color.

Camellia nitidissima is famous for its golden flowers. Its flowers are rich in secondary metabolites, and they have ornamental, medicinal, and edible value. Pigment composition and regulation has been studied in the golden petals, but there has been little research on pigment composition or the molecular mechanism underlying yellow stamens in C. nitidissima. To explore the molecular mechanism of yellow stamen formation, three developmental stages (S0, S1, and S2) were used for transcriptome and pigment analyses. Pigment analysis showed that the flavonoid content increased sharply from the S0 to S1 stage and decreased from the S1 to S2 stage, and the carotenoid content increased sharply during yellow stamen formation (from the S1 to S2 stage). RNA-seq analysis showed that a total of 20,483 differentially expressed genes (DEGs) were identified. KEGG and heatmap analyses showed that flavonoid and carotenoid biosynthesis pathways were enriched, and we identified 14 structural genes involved in flavonoid biosynthesis and 13 genes involved in carotenoid biosynthesis and degradation. In addition, the expression of carotenoid- and flavonoid-related genes was consistent with carotenoid and flavonoid content. In addition, correlation network analysis indicated that the WARYK, MYB, bHLH, and AP2/ERF transcription factor families were screened for involvement in the biosynthesis of flavonoids and carotenoids. In this study, we describe the pathway associated with color formation in the stamens of C. nitidissima.