Cloning and Characterization of a Flavonoid 3′-Hydroxylase Gene from Tea Plant (Camellia sinensis)

Abstract

Tea leaves contain abundant flavan-3-ols, which include dihydroxylated and trihydroxylated catechins. Flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21) is one of the enzymes in the establishment of the hydroxylation pattern. A gene encoding F3′H, designated as CsF3′H, was isolated from Camellia sinensis with a homology-based cloning technique and deposited in the GenBank (GenBank ID: KT180309). Bioinformatic analysis revealed that CsF3′H was highly homologous with the characterized F3′Hs from other plant species. Four conserved cytochrome P450-featured motifs and three F3′H-specific conserved motifs were discovered in the protein sequence of CsF3′H. Enzymatic analysis of the heterologously expressed CsF3′H in yeast demonstrated that tea F3′H catalyzed the 3′-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent Km values for these substrates were 17.08, 143.64 and 68.06 μM, and their apparent Vmax values were 0.98, 0.19 and 0.44 pM·min−1, respectively. Transcription level of CsF3′H in the new shoots, during tea seed germination was measured, along with that of other key genes for flavonoid biosynthesis using real-time PCR technique. The changes in 3′,4′-flavan-3-ols, 3′,4′,5′-flavan-3-ols and flavan-3-ols, were consistent with the expression level of CsF3′H and other related genes in the leaves. In the study of nitrogen supply for the tea plant growth, our results showed the expression level of CsF3′H and all other tested genes increased in response to nitrogen depletion after 12 days of treatment, in agreement with a corresponding increase in 3′,4′-catechins, 3′,4′,5′-catechins and flavan 3-ols content in the leaves. All these results suggest the importance of CsF3′H in the biosynthesis of 3′,4′-catechins, 3′,4′,5′-catechins and flavan 3-ols in tea leaves.

Transcriptome and phytochemical analyses provide new insights into long non-coding RNAs and characteristic secondary metabolites of oolong tea (Camellia sinensis) in solar-withering

“…Other 2 genes in the flavonoid metabolism were F3H and F3'H. Previous studies have shown that the catechins content in tea plant were positively correlated with the transcript levels of F3H and F3'H [88,89]. In this research, there was a negative correlation between the expression levels lncRNA and these two genes.…”

Transcriptome and Phytochemical Analyses Provide New Insights Into Long Non-Coding RNAs Modulating Characteristic Secondary Metabolites of Oolong Tea (Camellia sinensis) in Solar-Withering

“…The F3H and F3'H genes also influence flavonoid metabolism. Previous studies revealed that the catechin content of tea plants are positively correlated with the F3H and F3'H transcript levels (Zhou et al, 2016;Guo et al, 2019b). In the current study, we observed a negative correlation between the expression levels of lncRNAs and these two genes.…”

Transcriptomic Analysis Reveals the Molecular Adaptation of Three Major Secondary Metabolic Pathways to Multiple Macronutrient Starvation in Tea (Camellia sinensis)

“…Tea is rich in many secondary metabolites associated with tea quality, such as catechin derivatives (catechins), caffeine, and L-theanine [25][26][27]. Catechins that are known as flavan-3-ols are derived from phenylpropanoid and then flux into the flavonoid pathway via the catalyzation of chalcone synthase (CHS) to chalcone [28,29].…”