Metabolite profiling and transcriptomic analyses reveal an essential role of UVR8-mediated signal transduction pathway in regulating flavonoid biosynthesis in tea plants (Camellia sinensis) in response to shading

Abstract

Background: Tea is the most popular nonalcoholic beverage worldwide for its pleasant characteristics and healthful properties. Catechins, theanine and caffeine are the major natural products in tea buds and leaves that determine tea qualities such as infusion colors, tastes and fragrances, as well as their health benefits. Shading is a traditional and effective practice to modify natural product accumulation and to enhance the tea quality in tea plantation. However, the mechanism underlying the shading effects is not fully understood. This study aims to explore the regulation of flavonoid biosynthesis in Camellia sinensis under shading by using both metabolomic and transcriptional analyses.

Results: While shading enhanced chlorophyll accumulation, major catechins, including C, EC, GC and EGC, decreased significantly in tea buds throughout the whole shading period. The reduction of catechins and flavonols were consistent with the simultaneous down-regulation of biosynthetic genes and TFs associated with flavonoid biosynthesis. Of 16 genes involved in the flavonoid biosynthetic pathway, F3'H and FLS significantly decreased throughout shading while the others (PAL, CHSs, DFR, ANS, ANR and LAR, etc.) temporally decreased in early or late shading stages. Gene co-expression cluster analysis suggested that a number of photoreceptors and potential genes involved in UV-B signal transductions (UVR8_L, HY5, COP1 and RUP1/2) showed decreasing expression patterns consistent with structural genes (F3'H, FLS, ANS, ANR, LAR, DFR and CHSs) and potential TFs (MYB4, MYB12, MYB14 and MYB111) involved in flavonoid biosynthesis, when compared with genes in the UV-A/blue and red/far-red light signal transductions. The KEGG enrichment and matrix correlation analyses also attributed the regulation of catechin biosynthesis to the UVR8-mediated signal transduction pathway. Further UV-B treatment in the controlled environment confirmed UV-B induction on flavonols and EGCG accumulation in tea leaves.

Conclusions: We proposed that catechin biosynthesis in C. sinensis leaves is predominantly regulated by UV through the UVR8-mediated signal transduction pathway to MYB12/MYB4 downstream effectors, to modulate flavonoid accumulation. Our study provides new insights into our understanding of regulatory mechanisms for shading-enhanced tea quality.

Keywords: Camellia sinensis; Flavonoids; Light signal transduction; Shading; UV-B responses; UVR8.

Fig. 1

The effects of shading on chlorophylls and daily environmental parameters in shading experiment in tea plantation. a The set up of shading treatments in tea plantation. b The effects of shading on appearance of tea leaves after 14 days. c The effects of shading on chlorophylls after 14 days. d The effects of shading on daily environmental parameters among different treatments in tea plantation. The treatments are: tea plants with naturally growth (Control); tea plants with 50–60% shading treatment (S50–60%); tea plants with 80–90% shading treatment (S80–90%). PAR, photosynthetic actively radiation; Chl_a, chlorophyll a; Chl_b, chlorophyll b; ns, no significance. Data shown are the average mean ± SE of three replicates (n = 3). *Significant differences comparing the Control treatment at each time point according to one-way analysis of variance (ANOVA) test and a Fisher’s least significant difference (LSD) at the 5% significance level (*p < 0.05, **p < 0.01). Different letters (a, b, c) indicate statistical significance among treatments using one-way ANOVA and a Fisher’s LSD test at the 5% significance level

Fig. 2

The OPLS-DA analysis of flavonoids and gene expression in tea buds from shading and UV-B experiments. a The OPLS-DA analysis of flavonoids in tea buds at different time points throughout shading period between the Control and medium shading (S50–60%) treatments. b The OPLS-DA analysis of flavonoids in tea buds at different time points throughout shading period between the Control and heavy shading (S80–90%) treatments. c The OPLS-DA analysis of transcript abundance of all unigenes annotated in transcriptome datasets between the Control and S80–90% treatments. d The OPLS-DA analysis of major catechins in tea buds between the Control and UV-B treatments in the controlled environment. Treatments in the shading experiment in tea plantation are shown as above in Fig. 1. Treatments in the UV-B experiment in the controlled environment are: tea plants exposed to pure PAR (Control); tea plants exposed to PAR + UV-B radiation (UV-B). FPKM, Fragment Per Kilo base of exon model per Million mapped reads. Data shown are from the value of three replicates (n = 3). OPLS-DA analysis was conducted by SIMCA 13.0 (UMETRICS, https://umetrics.com/)

Fig. 3

The effects of shading on flavonoid accumulation in tea buds in tea plantation. a The composition of flavonoids in tea buds from the control and shading treatments at time point of 14d. b The changes of major catechins in tea buds in different treatments throughout shading period. C, catechin; EC, epicatechin; EGC, epigallocatechin; GC, gallocatechin; GCG, gallocatechin 3-O-gallate; EGCG, epigallocatechin 3-O-gallate; ECG, epicatechin 3-O-gallate; K7G, keampferol-7-O-glucoside; K3Gal, kaempferol-3-O-galactopyranoside; RT, retention time; ns, no significance. Data shown are the average mean ± SE of three replicates (n = 3). Different letters (a, b, c) indicate statistical significance among different treatments according to one-way ANOVA and a Fisher’s LSD test at the 5% level

Fig. 4

Functional distribution of the DEGs in candidate pathways in response to shading at time points of 4h and 8h. The enrichment score indicates intensiveness of DEGs (fold change ≥2) in a certain pathway (Enrichment score = (m/n): (M/N); m, the number of DEGs mapped to a certain pathway; n, the number of all DEGs annotated in transcriptome datasets; M, the number of unigenes mapped to a certain pathway; N, the number of all unigenes annotated in transcriptome datasets). A large enrichment score denotes a high degree of intensiveness. The p value (ranging from 0~1) was calculated using hypergeometric test through Bonferroni Correction and less p value means greater intensiveness. Gene number means number of DEGs mapped to a certain pathway according to KEGG database. S 4h and S 8h indicate time points at 4h and 8h throughout shading period

Fig. 5

The shading effects on transcript abundance of potential genes and TFs involved in flavonoid biosynthesis and light signal transductions in tea buds. a The shading effects on transcript abundance of genes and potential TFs involved in the flavonoid biosynthetic pathway. b The shading effects on transcript abundance of potential genes and TFs involved in the UVR8-mediated UV-B, UV-A/blue light and red/far-red light signal transduction pathways, respectively. c Cluster analysis of expression of all potential genes and TFs in response to shading treatment. The heatmaps are constructed from the competitive expression of genes (log₂ FPKM_S80–90%/FPKM_control) from the transcriptome datasets. S 4h, S 8h, S 2d, S 8d and S 14d indicate time points at 4h, 8h, 2d, 8d and 14d throughout shading period. The triangle with colors represents potential genes involved in the flavonoid biosynthetic pathway (functional enzymes, yellow; TFs, green) and different light transduction pathways (UV-B radiation, purple; UV-A/blue light, blue; red/far-red light, red; genes and TFs involved in three light signal transduction, black). Data shown are the average mean of three biological replicates (n = 3). *Significant differences comparing the Control treatment at each time point according to one-way ANOVA and a Fisher’s LSD test at the 5% level (*p < 0.05, **p < 0.01; fold change ≥2)

Fig. 6

The matrix correlation of transcript abundance of potential genes and TFs involved in flavonoid biosynthesis and different light signal transduction pathways in response to shading treatment. The heatmap was conducted from the FPKM profiles of genes and TFs from transcriptome dataset in tea buds at five time points throughout shading treatments. Correlation factor indicates the correlation of transcriptional expression of two genes (− 1~0, expression of genes are negatively correlated; 0, expression of genes are not correlated; 0~1, expression of genes are positively correlated). Data were conducted from three biological replicates (n = 3), analysed by the Speaman test in SPSS 13.0 software (IBM SPSS Software, https://www.ibm.com/analytics/data-science/predictive-analytics/spss-statistical-software) and visualized by the “pheatmap” package implemented in R (https://cran.r-project.org/web/packages/pheatmap/index.html)

Fig. 7

The shading effects on gene expression in tea buds analysed by both RNA-seq and qRT-PCR. R, relevance factor of gene expression between RNA-Seq and qRT-PCR data by the double factor correlation test in SPSS 13.0 software. C4h, C8h, C2d and C14d indicate samples collected at 4h, 8h, 2d and 14d in the Control treatment. S4h, S8h, S2d and S14d indicate samples collected at 4h, 8h, 2d and 14d in the shading treatment. Data shown are the average mean ± SE of three replicates (n = 3). *Significant differences comparing the Control treatment at each time point according to one-way ANOVA and a Fisher’s LSD test at the 5% level (*p < 0.05, **p < 0.01). Different letters indicate statistical significance among time points for the Control (a, b, c, d) and Shading (e, f, g, h) treatments in qRT-PCR using one-way ANOVA and a Fisher’s LSD test at the 5% significance level

Fig. 8

Working model for flavonoid biosynthesis in tea plants regulated by the UVR8-mediated signal transduction pathway in response to shading conditions. The full names of genes and TFs are shown in abbreviation

Fig. 9

The effects of UV-B on major catechins and gene expression in UV-B experiment carried out in the controlled environment. a The concentration of major catechins in tea sample (bud with one developing leaf) from both the control and UV-B treatments in the controlled environment analysed by HPLC. b The expression of annotated unigenes in tea buds from both the Control and UV-B treatments in the controlled environment analysed by qRT-PCR. The full name of catechins and description of treatments in the controlled environment UV-B experiment are shown as above in Fig. 2. C1d, C3d, C7d and C14d indicate samples collected at 1d, 3d, 7d and 14d in the Control treatment. U1d, U3d, U7d and U14d indicate samples collected at 1d, 3d, 7d and 14d in the UV-B treatment. Data shown are the average mean ± SE of three replicates (n = 3). ns, no significance. *Significant differences comparing the Control treatment at each time point according to one-way ANOVA and a Fisher’s LSD test at the 5% level (*p < 0.05, **p < 0.01). Different letters indicate statistical significance among time points for the Control (a, b, c, d) and UV-B (e, f, g, h) treatments using one-way ANOVA and a Fisher’s LSD test at the 5% significance level