CsUGT89A2 enhances tea plant resistance to Toxoptera aurantia by mediating flavonoid glycosides biosynthesis

Abstract

Tea plant [Camellia sinensis (L.) O. Kuntze] is a globally important crop but is severely threatened by Toxoptera aurantia infestations, which impact yield and safety. However, the response of tea plants to aphid feeding remains largely unexplored. This study investigates the feeding behavior of T. aurantia on different cultivars and identifies 'Huangjinya' and 'Qiancha 1' as susceptible and resistant cultivars, respectively. Transcriptome analysis revealed that CsUGT89A2 was significantly upregulated in response to T. aurantia infestation. In vitro biochemical assays demonstrated that CsUGT89A2 encodes a flavonoid 7-glycosyltransferase that catalyzes the conversion of flavonoids and UDP-glucose into flavonoid 7-O-glucosides. In vivo, silencing CsUGT89A2 significantly reduced flavonoid glycoside accumulation. To further clarify the role of CsUGT89A2 in tea plant resistance to T. aurantia, we used tobacco and tea flowers to evaluate aphid feeding and reproduction under chemical treatment, gene silencing, and gene overexpression conditions. Statistical analysis showed that, compared with flavonoids, the application of flavonoid 7-O-glycosides significantly reduced T. aurantia reproductive capacity. Furthermore, compared with the control, overexpression of CsUGT89A2 significantly reduced the reproductive ability of aphids, while its silencing increased reproductive rates. Overall, our findings demonstrate that CsUGT89A2 mediates flavonoid glycosylation and enhances insect resistance in tea plants by increasing flavonoid glycoside levels, offering new insights into the role of flavonoid glycosides in the insect resistance of C. sinensis.

Figure 1

Evaluation and transcriptomic analysis of T. aurantia feeding preferences. (A, B) Field observations of T. aurantia damage to tea leaves in tea gardens. (C) Feeding preference experiment of T. aurantia among eight tea cultivars. (D) Population dynamics of T. aurantia across eight tea cultivars. Values represent the mean ± standard deviation, and different letters above the bars indicate significant differences (P < 0.05), determined by one-way ANOVA. (E) Heatmap showing the expression profiles of differentially expressed genes across six groups. (F) Number of upregulated and downregulated genes across four pairwise comparisons. (G) Venn diagrams showing the numbers of DEGs under T. aruantii infestation at different time points.

Figure 2

Transcriptional response of tea plants to T. aurantia feeding. (A–D) Top 15 significantly enriched GO terms among DEGs in each pairwise comparison. Complete GO term names are provided in Table S2. (E–H) Top 15 enriched KEGG pathways among DEGs. (I) Venn diagram showing overlap of DEGs involved in the phenylpropanoid biosynthetic pathway. (J) Functional annotation of upregulated and downregulated genes within the phenylpropanoid pathway.

Figure 3

Stress-responsive expression and phylogenetic analysis of CsUGT89A2. (A) Schematic illustrating 23 distinct stress treatments applied to tea plants, including biotic and abiotic stressors. (B) Heatmap showing transcript levels of CsUGT89A2 under the 23 stress conditions. (C) Phylogenetic tree of CsUGT89A2 and functionally characterized UGTs from Camellia and related species. GenBank accession numbers used in the phylogenetic analysis are listed in Table S3.

Figure 4

HPLC analysis of recombinant CsUGT89A2 enzymatic activity. (A) HPLC chromatogram showing products derived from UDP-glucose (UDP-Glu) conversion by recombinant CsUGT89A2. (B , C) Substrate specificity analysis of recombinant CsUGT89A2. Panels B and C show results for flavonoid aglycones and sugar donors, respectively. (D) Proposed biosynthetic role of CsUGT89A2 in catalyzing flavonoid 7-O-glycoside formation.

Figure 5

Optimization of catalytic conditions and kinetic analysis of recombinant CsUGT89A2. (A) Optimal reaction temperature (20–65°C) for the glycosylation activity of CsUGT89A2 on four different substrates, measured at pH 8.0. (B) Optimal pH range (4.0–11.0) for the glycosylation activity of CsUGT89A2 on four different substrates, measured at 40°C. (C) Kinetic parameters of recombinant CsUGT89A2, measured at pH 8.0 and 40°C. Data are presented as means ± standard errors from three replicates.

Figure 6

Impact of heterologous CsUGT89A2 expression and flavonoid treatment on aphids in tobacco (Nicotiana tabacum). (A) Schematic diagram of tobacco infection with M. persicae following various treatments. (B) Relative expression levels of CsUGT89A2 under different experimental conditions. (C, F) Effects of CsUGT89A2 overexpression versus the empty vector on adult and nymph M. persicae populations. (D, G) Effects of CsUGT89A2 overexpression versus the empty vector with exogenous flavonoid aglycone treatment on adult and nymph M. persicae populations. (E, H) Effects of flavonoid aglycones versus the corresponding flavonoid glycosides on adult and nymph M. persicae populations. Dashed lines represent different individual replicates; solid lines show means ± standard errors of four replicates. Statistical differences were determined using two-way ANOVA in SPSS 27.0 (*P < 0.05, **P < 0.01, and ***P <0.001). “av” denotes the average of replicates.

Figure 7

Metabolic changes in tea leaves following CsUGT89A2 gene silencing. (A) Changes in NBT staining intensity in tea leaves after gene silencing. (B) Quantification of NBT-stained areas. (C) Relative expression levels of CsUGT89A2 post silencing. (D) PCA comparing AsODN-CsUGT89A2 and sODN treatments. (E, F) KEGG pathway enrichment analysis of DEGs from AsODN-CsUGT89A2 versus sODN. (G) Differential flavonoid metabolism profiles between AsODN-CsUGT89A2 and sODN. Data are presented as means ± standard errors from three biological replicates.

Figure 8

Effects of exogenous flavonoid application on T. aurantia. (A) Schematic representation of T. aurantia treatments using flavonoid aglycones and glycosides. (B) Population dynamics of adult T. aurantia over 17 days following treatment. (C) Population dynamics of nymphs over the same period. Colored dashed lines indicate biological replicates; solid lines show the means ± standard errors of four replicates. Two-way ANOVA was performed in SPSS 27.0 to determine significant differences between the treatment and control groups (*P < 0.05, **P < 0.01, and ***P < 0.001).“F” refers to the flavonoid aglycone mixture, whereas “FG” denotes the flavonoid glycoside mixture. “av” denotes the average of replicates.

Figure 9

Effects of CsUGT89A2 gene silencing on T. aurantia behavior and tea flower metabolites. (A) Schematic of aphid inoculation following AsODN or sODN treatment. (B) Relative expression levels of CsUGT89A2 in silenced versus control tea flowers. (C) Relative peak areas of key flavonoids following gene silencing. (D) Relative peak areas of apigenin 7-O-glucoside, kaempferol 7-O-glucoside, luteolin 7-O-glucoside, and quercetin 7-O-glucoside after gene silencing. (E) Population trend of adult T. aurantia over 17 days following AsODN-CsUGT89A2 and sODN treatment. (F) Population trend of T. aurantia nymphs over the same period. (B–D) Data represent means ± standard errors of three biological replicates, with one-way ANOVA used to determine significant differences between groups. (E, F) Dashed lines indicate biological replicates (n = 4); solid lines denote means ± standard errors. Two-way ANOVA was employed to determine significant differences between the treatment and control groups (*P < 0.05, **P < 0.01, and ***P < 0.001).“av” denotes the average of replicates.

Figure 10

Proposed model of T. aurantia–induced CsUGT89A2 activation and its role in antiherbivore defense. Feeding by T. aurantia induces sustained expression of CsUGT89A2, which facilitates position-specific 7-O-glycosylation of flavonoid scaffolds, enhancing the tea plant’s defense against subsequent T. aurantia attacks.