RESUMEN
Flavonols are widely synthesized throughout the plant kingdom, playing essential roles in plant physiology and providing unique health benefits for humans. Their glycosylation plays significant role in improving their stability and solubility, thus their accumulation and function. However, the genes encoding the enzymes catalyze this glycosylation remain largely unknown in apple. This study utilized a combination of methods to identify genes encoding such enzymes. Initially, candidate genes were selected based on their potential to encode UDP-dependent glycosyltransferases (UGTs) and their expression patterns in response to light induction. Subsequently, through testing the in vitro enzyme activity of the proteins produced in Escherichia coli cells, four candidates were confirmed to encode a flavonol 3-O-galactosyltransferase (UGT78T6), flavonol 3-O-glucosyltransferase (UGT78S1), flavonol 3-O-xylosyltransferase/arabinosyltransferase (UGT78T5), and flavonol 3-O-rhamnosyltransferase (UGT76AE22), respectively. Further validation of these genes' functions was conducted by modulating their expression levels in stably transformed apple plants. As anticipated, a positive correlation was observed between the expression levels of these genes and the content of specific flavonol glycosides corresponding to each gene. Moreover, overexpression of a flavonol synthase gene, MdFLS, resulted in increased flavonol glycoside content in apple roots and leaves. These findings provide valuable insights for breeding programs aimed at enriching apple flesh with flavonols and for identifying flavonol 3-O-glycosyltransferases of other plant species.
Asunto(s)
Flavonoles , Glicósidos , Glicosiltransferasas , Malus , Proteínas de Plantas , Malus/genética , Malus/metabolismo , Malus/enzimología , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Flavonoles/metabolismo , Flavonoles/biosíntesis , Glicósidos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , GlicosilaciónRESUMEN
MAIN CONCLUSION: CsNAC086 was found to promote the expression of CsFLS, thus promoting the accumulation of flavonols in Camellia sinensis. Flavonols, the main flavonoids in tea plants, play an important role in the taste and quality of tea. In this study, a NAC TF gene CsNAC086 was isolated from tea plants and confirmed its regulatory role in the expression of flavonol synthase which is a key gene involved in the biosynthesis of flavonols in tea plant. Yeast transcription-activity assays showed that CsNAC086 has self-activation activity. The transcriptional activator domain of CsNAC086 is located in the non-conserved C-terminal region (positions 171-550), while the conserved NAC domain (positions 1-170) does not have self-activation activity. Silencing the CsNAC086 gene using antisense oligonucleotides significantly decreased the expression of CsFLS. As a result, the concentration of flavonols decreased significantly. In overexpressing CsNAC086 tobacco leaves, the expression of NtFLS was significantly increased. Compared with wild-type tobacco, the flavonols concentration increased. Yeast one-hybrid assays showed CsNAC086 did not directly regulate the gene expression of CsFLS. These findings indicate that CsNAC086 plays a role in regulating flavonols biosynthesis in tea plants, which has important implications for selecting and breeding of high-flavonols-concentration containing tea-plant cultivars.
Asunto(s)
Camellia sinensis , Flavonoles , Regulación de la Expresión Génica de las Plantas , Nicotiana , Proteínas de Plantas , Camellia sinensis/genética , Camellia sinensis/metabolismo , Flavonoles/biosíntesis , Flavonoles/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Hojas de la Planta/metabolismo , Hojas de la Planta/genética , Plantas Modificadas GenéticamenteRESUMEN
Flavonol glycosides, contributing to the health benefits and distinctive flavors of tea (Camellia sinensis), accumulate predominantly as diglycosides and triglycosides in tea leaves. However, the UDP-glycosyltransferases (UGTs) mediating flavonol multiglycosylation remain largely uncharacterized. In this study, we employed an integrated proteomic and metabolomic strategy to identify and characterize key UGTs involved in flavonol triglycoside biosynthesis. The recombinant rCsUGT75AJ1 exhibited flavonoid 4'-O-glucosyltransferase activity, while rCsUGT75L72 preferentially catalyzed 3-OH glucosylation. Notably, rCsUGT73AC15 displayed substrate promiscuity and regioselectivity, enabling glucosylation of rutin at multiple sites and kaempferol 3-O-rutinoside (K3R) at the 7-OH position. Kinetic analysis revealed rCsUGT73AC15's high affinity for rutin (Km = 9.64 µM). Across cultivars, CsUGT73AC15 expression inversely correlated with rutin levels. Moreover, transient CsUGT73AC15 silencing increased rutin and K3R accumulation while decreasing their respective triglycosides in tea plants. This study offers new mechanistic insights into the key roles of UGTs in regulating flavonol triglycosylation in tea plants.