CsPHRs-CsJAZ3 incorporates phosphate signaling and jasmonate pathway to regulate catechin biosynthesis in Camellia sinensis

Abstract

Catechins constitute abundant metabolites in tea and have potential health benefits and high economic value. Intensive study has shown that the biosynthesis of tea catechins is regulated by environmental factors and hormonal signals. However, little is known about the coordination of phosphate (Pi) signaling and the jasmonic acid (JA) pathway on biosynthesis of tea catechins. We found that Pi deficiency caused changes in the content of catechins and modulated the expression levels of genes involved in catechin biosynthesis. Herein, we identified two transcription factors of phosphate signaling in tea, named CsPHR1 and CsPHR2, respectively. Both regulated catechin biosynthesis by activating the transcription of CsANR1 and CsMYB5c. We further demonstrated CsSPX1, a Pi pathway repressor, suppressing the activation by CsPHR1/2 of CsANR1 and CsMYB5c. JA, one of the endogenous plant hormones, has been reported to be involved in the regulation of secondary metabolism. Our work demonstrated that the JA signaling repressor CsJAZ3 negatively regulated catechin biosynthesis via physical interaction with CsPHR1 and CsPHR2. Thus, the CsPHRs-CsJAZ3 module bridges the nutrition and hormone signals, contributing to targeted cultivation of high-quality tea cultivars with high fertilizer efficiency.

Figure 1

Phosphate starvation induced catechin biosynthetic gene expression and production accumulation. A–C Relative transcript levels of genes. Young shoots were harvested after Pi-sufficient (+P) or Pi starvation (−P ) treatment for 21 days and used for RT–qPCR. A Relative transcript levels of two phosphate transporter genes. B Relative transcript levels of key genes in flavonoid biosynthesis. C Relative transcript levels of the CsMYB5 subfamily, which plays key roles in regulating flavonoid biosynthesis. Values are means ± standard deviation (n = 6). D Accumulation of catechin compounds with Pi starvation (−P ) for 60 days. Values are means ± standard deviation (n = 6). ****P < 0.0001, ***P < 0.001, **P < 0.01; Student’s t-test compared with samples under +P condition. DW, dry weight.

Figure 2

CsPHRs were involved in catechin biosynthesis. A Transcriptional autoactivation of CsPHR1 and CsPHR2 in yeast grown on SD/−Leu/−Trp and SD/−Ade/−Leu/−Trp/−His nutrition-deficient medium. Co-expression of BK-AtPHR1 and pGADT7 (AD) served as positive control, and BK-Lam and AD served as negative control. B, C Anthocyanin phenotyping (scale bar = 1 mm) and AtF3′H expression level in Col-0, phr1, CsPHR1/phr1, and CsPHR2/phr1 seedlings grown in the Pi-sufficient (+P) or Pi starvation (−P) conditions for 8 days; transcript levels were analyzed by RT–qPCR. Values are means ± standard deviation of three biological replicates. Significant differences were analyzed by ANOVA and Tukey’s multiple comparisons test. D Knockdown of CsPHRs with antisense oligonucleotide (asODN) solution for 24 h and sense oligonucleotide (ODN) solution as control. E RT–qPCR validated the silencing effect of CsPHRs and genes that may be regulated by CsPHRs. GAPDH was used as an internal control. Values represent means ± standard error of the mean (n = 6). ^****P < 0.0001, ^***P < 0.001, ^**P < 0.01; Student’s t-test compared with ODN-treated samples. F, G Accumulation of total catechins (F) and catechin compounds (G) in tea leaves treated with asODN or ODN solution. Values represent means ± standard deviation (n = 4). ^****P < 0.0001; ^***P < 0.001, ^**P < 0.01, ^*P < 0.05; Student’s t-test compared with ODN-treated samples.

Figure 3

CsSPX1 suppresses the function of CsPHRs in binding the P1BS element of CsANR1 and CsMYB5c. A Diagram of the wild-type P1BS element and four base-mutated P1BSs (mP1BSs) in the CsANR1 promoter. B Y1H analysis of CsPHRs binding the promoter of CsANR1. Empty pGADT7 vector was used as negative control. Yeast cells containing different plasmid combinations were grown on the selective medium SD−Leu with 300 μm aureobasidin A (AbA). C EMSA results indicated that CsPHR proteins bind to the CsANR1 promoter. Arrows show CsPHRs-bound or free DNA. The competitive protein–DNA binding assay was performed with an increasing amount of unlabeled DNA probe (1- and 100-fold). D Analysis of CsSPX1 and CsPHR interaction by BiFC. Confocal images of N. benthamiana epidermal cells expressing different construct combinations as indicated. Scale bar = 20 μm. E  In vitro semi-pull-down assays showing the interaction between CsSPX1 and CsPHRs. F Schematic diagram of the luciferase reporter system. G, H Transactivation assays in N. benthamiana leaves. G Luciferase intensity was imaged 48 h after infiltration. H LUC/REN activity obtained from co-transfection with the indicated reporter constructs or empty effector constructs, Values are means ± standard deviation of three biological replicates. Different letters (a and b) indicate significant difference (ANOVA, Tukey’s multiple comparisons test, P < 0.05).

Figure 4

Function of CsJAZ3-CsPHRs transcriptional regulatory module in tea catechin biosynthesis. A Analysis of CsJAZ3 and CsPHRs interaction by BiFC. Confocal images of N. benthamiana epidermal cells expressing different construct combinations as indicated. Scale bar = 20 μm. B  In vitro semi-pull-down assays showing the interaction between CsJAZ3 and CsPHRs. C, D LUC/REN activity obtained from co-transfection with the indicated reporter constructs or empty effector constructs. Values are means ± standard deviation of three biological replicates. Different letters (a–d) indicate significant difference (ANOVA, Tukey’s multiple comparisons test, P < 0.05). E, F EMSA showed that the CsJAZ3–CsPHR2 interaction attenuated the DNA binding activity of CsPHR2 to the P1BS motif of CsANR1 (C) and CsMYB5c (D) in the promoter. The triangle shows the rise in pCold-CsJAZ3 protein concentration from 50 to 150 ng. Signs - and + indicate the absence and presence of the corresponding proteins, respectively.

Figure 5

CsPHRs integrate Pi and JA signaling to regulate catechin biosynthesis. A RT–qPCR validated the silencing effect of CsPHRs and genes that may be regulated by CsPHRs. GAPDH was used as an internal control. B Relative inducement of catechins in tea leaves treated with ODN solution and MeJA. C Relative inducement of catechins in tea leaves treated with asODN solution and MeJA. D, E Representative western blot showing the accumulation of CsJAZ3-Flag in N. benthamiana leaves treated with 25 μM MG-132 or in the presence or absence of 100 μM MeJA. Protein levels were normalized against actin levels. F Transactivation assays. Luciferase (LUC)/Renilla (REN) activity obtained from co-transfection with an empty effector (SK) and the indicated reporter (CsPHR2) construct. Leaves were infiltrated with MG132 (10 μΜ) at least 12 h prior to harvesting. Exogenous MeJA (100 μM) was rubbed onto N. benthamiana leaves and fluorescence was measured after 4 h. G Accumulation of total catechins in young shoots with Pi starvation and MeJA treatment. Data are means ± standard error, and different letters indicate significant differences at P < 0.05 tested by ANOVA.

Figure 6

A model for CsPHRs-mediated catechin biosynthesis in tea. In the phosphate-sufficient condition, CsSPX1 interacts with CsPHRs to inhibit the transcriptional activity of CsPHRs on CsANR1 and CsMYB5c, and thus negatively regulates Pi-mediated catechin accumulation. In the presence of MeJA, degradation of CsJAZ3 via the 26S-proteasome pathway releases CsPHRs from physical interaction to activate Pi-mediated catechin accumulation. When phosphate levels are low and MeJA is present, CsPHRs are liberated from CsSPX1 binding and CsJAZ3 degradation, which promotes the production of additional tea catechins.