Integration of Metabolome and Transcriptome Reveals the Relationship of Benzenoid-Phenylpropanoid Pigment and Aroma in Purple Tea Flowers.

Tea (Camellia sinensis) flowers are normally white, even though the leaves could be purple. We previously discovered a specific variety with purple leaves and flowers. In the face of such a phenomenon, researchers usually focus on the mechanism of color formation but ignore the change of aroma. The purple tea flowers contain more anthocyanins, which belong to flavonoids. Meanwhile, phenylalanine (Phe), derived from the shikimate pathway, is a precursor for both flavonoids and volatile benzenoid-phenylpropanoids (BPs). Thus, it is not clear whether the BP aroma was attenuated for the appearance of purple color. In this study, we integrated metabolome and transcriptome of petals of two tea varieties, namely, Zijuan (ZJ) with white flowers and Baitang (BT) with purple flowers, to reveal the relationship between color (anthocyanins) and aroma (volatile BPs). The results indicated that in purple petals, the upstream shikimate pathway promoted for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) was elevated. Among the increased anthocyanins, delphinidin-3-O-glucoside (DpG) was extremely higher; volatile BPs, including benzyl aldehyde, benzyl alcohol, acetophenone (AP), 1-phenylethanol, and 2-phenylethanol, were also enhanced, and AP was largely elevated. The structural genes related to the biosynthesis of volatile BPs were induced, while the whole flavonoid biosynthesis pathway was downregulated, except for the genes flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H), which were highly expressed to shift the carbon flux to delphinidin, which was then conjugated to glucoside by increased bronze-1 (BZ1) (UDP-glucose: flavonoid 3-O-glucosyltransferase) to form DpG. Transcription factors (TFs) highly related to AP and DpG were selected to investigate their correlation with the differentially expressed structural genes. TFs, such as MYB, AP2/ERF, bZIP, TCP, and GATA, were dramatically expressed and focused on the regulation of genes in the upstream synthesis of Phe (DAHPS; arogenate dehydratase/prephenate dehydratase) and the synthesis of AP (phenylacetaldehyde reductase; short-chain dehydrogenase/reductase), Dp (F3'H; F3'5'H), and DpG (BZ1), but inhibited the formation of flavones (flavonol synthase) and catechins (leucoanthocyanidin reductase). These results discovered an unexpected promotion of volatile BPs in purple tea flowers and extended our understanding of the relationship between the BP-type color and aroma in the tea plant.

A Comparative Metabolomic Analysis Reveals Difference Manufacture Suitability in "Yinghong 9" and "Huangyu" Teas (Camellia sinensis).

"Yinghong 9" is a widely cultivated large-leaf variety in South China, and the black tea made from it has a high aroma and strong sweet flavor. "Huangyu" is a light-sensitive tea variety with yellow leaves. It was cultivated from the bud-mutation of "Yinghong 9" and has a very low level of chlorophyll during young shoot development. Due to chlorophyll being involved in carbon fixation and assimilation, the changes in photosynthesis might potentially affect the accumulation of flavor metabolites, as well as the quality of "Huangyu" tea. Although "Huangyu" has a golden yellow color and high amino acid content, the mechanism underlying the formation of leaf color and drinking value remains unclear. The widely targeted metabolomics and GC-MS analysis were performed to reveal the differences of key metabolites in fresh and fermented leaves between "Yinghong 9" and "Huangyu." The results showed that tea polyphenols, total chlorophyll, and carotenoids were more abundant in "Yinghong 9." Targeted metabolomics analysis indicated that kaempferol-3-glycoside was more abundant in "Yinghong 9," while "Huangyu" had a higher ratio of kaempferol-3-glucoside to kaempferol-3-galactoside. Compared with "Yinghong 9" fresh leaves, the contents of zeaxanthin and zeaxanthin palmitate were significantly higher in "Huangyu." The contents of α-farnesene, ß-cyclocitral, nerolidol, and trans-geranylacetone, which were from carotenoid degradation and involved in flowery-fruity-like flavor in "Huangyu" fermented leaves, were higher than those of "Yinghong 9." Our results indicated that "Huangyu" was suitable for manufacturing non-fermented tea because of its yellow leaf and flowery-fruity-like compounds from carotenoid degradation.

Metabolome and Transcriptome Analysis Reveals Putative Genes Involved in Anthocyanin Accumulation and Coloration in White and Pink Tea (Camellia sinensis) Flower.

A variant of tea tree (Camellia sinensis (L.)) with purple buds and leaves and pink flowers can be used as a unique ornamental plant. However, the mechanism of flower coloration remains unclear. To elucidate the molecular mechanism of coloration, as well as anthocyanin accumulation in white and pink tea flowers, metabolite profiling and transcriptome sequencing was analyzed in various tea flower developmental stages. Results of metabolomics analysis revealed that three specific anthocyanin substances could be identified, i.e., cyanidin O-syringic acid, petunidin 3-O-glucoside, and pelargonidin 3-O-ß-d-glucoside, which only accumulated in pink tea flowers, and were not able to be detected in white flowers. RNA-seq and weighted gene co-expression network analysis revealed eight highly expressed structural genes involved in anthocyanin biosynthetic pathway, and particularly, different expression patterns of flavonol synthase and dihydroflavonol-4-reductase genes were observed. We deduced that the disequilibrium of expression levels in flavonol synthases and dihydroflavonol-4-reductases resulted in different levels of anthocyanin accumulation and coloration in white and pink tea flowers. Results of qRT-PCR performed for 9 key genes suggested that the expression profiles of differentially expressed genes were generally consistent with the results of high-throughput sequencing. These findings provide insight into anthocyanin accumulation and coloration mechanisms during tea flower development, which will contribute to the breeding of pink-flowered and anthocyanin-rich tea cultivars.

Comprehensive analysis of putative dihydroflavonol 4-reductase gene family in tea plant.

One identified dihydroflavonol 4-reductases (DFR) encoding gene (named as CsDFRa herein) and five putative DFRs (named as CsDFRb1, CsDFRb2, CsDFRb3, CsDFRc and CsDFRd) in tea (Camellia sinensis) have been widely discussed in recent papers concerning multi-omics data. However, except for CsDFRa, their function and biochemical characteristics are not clear. This study aims to compare all putative CsDFRs and preliminarily evaluate their function. We investigated the sequences of genes (coding and promoter regions) and predicted structures of proteins encoded, and determined the activities of heterologously expressed CsDFRs under various conditions. The results showed that the sequences of five putative CsDFRs were quite different from CsDFRa, and had lower expression levels as well. The five putative CsDFRs could not catalyze three dihydroflavonol substrates. The functional CsDFRa had the strongest affinity with dihydroquercetin, and performed best at pH around 7 and 35°C but was not stable at lower pHs or higher temperatures. Single amino acid mutation at position 141 modified the preference of CsDFRa for dihydroquercetin and dihydromyricetin, and also weakened its stability. These data suggest that only CsDFRa works in the pathway for generating anthocyanidins and catechins. This study provides new insights into the function of CsDFRs and may assist to develop new strategies to manipulate the composition of tea flavonoids in the future.