Jasmonate signalling in the regulation of rubber biosynthesis in laticifer cells of rubber tree, Hevea brasiliensis

Jasmonate signalling in the regulation of rubber biosynthesis in laticifer cells of rubber tree,... Abstract Rubber trees are the world’s major source of natural rubber. Rubber-containing latex is obtained from the laticifer cells of the rubber tree (Hevea brasiliensis) via regular tapping. Rubber biosynthesis is a typical isoprenoid metabolic process in the laticifer cells; however, little is known about the positive feedback regulation caused by the loss of latex that occurs through tapping. In this study, we demonstrate the crucial role of jasmonate signalling in this feedback regulation. The endogenous levels of jasmonate, the expression levels of rubber biosynthesis-related genes, and the efficiency of in vitro rubber biosynthesis were found to be significantly higher in laticifer cells of regularly tapped trees than those of virgin (i.e. untapped) trees. Application of methyl jasmonate had similar effects to latex harvesting in up-regulating the rubber biosynthesis-related genes and enhancing rubber biosynthesis. The specific jasmonate signalling module in laticifer cells was identified as COI1–JAZ3–MYC2. Its activation was associated with enhanced rubber biosynthesis via up-regulation of the expression of a farnesyl pyrophosphate synthase gene and a small rubber particle protein gene. The increase in the corresponding proteins, especially that of farnesyl pyrophosphate synthase, probably contributes to the increased efficiency of rubber biosynthesis. To our knowledge, this is the first study to reveal a jasmonate signalling pathway in the regulation of rubber biosynthesis in laticifer cells. The identification of the specific jasmonate signalling module in the laticifer cells of the rubber tree may provide a basis for genetic improvement of rubber yield potential. Hevea brasiliensis, isoprenoid metabolism, jasmonate signalling, laticifer cell, rubber biosynthesis, secondary metabolism Introduction Major progress in elucidating the jasmonate signalling pathway in model plants has been achieved. This includes the characterization of endogenous bioactive jasmonate as (&#x002B;)-7-iso-jasmonoyl-L-isoleucine (Ile-JA) (Fonseca et al., 2009b) and its receptor as COI1 (Katsir et al., 2008; Mach, 2009; Yan et al., 2009), the identification of the COI1–JAZ co-receptor (Sheard et al., 2010), COI1 as a component of an SCF (Skp/Cullin/F-box) E3 ubiquitin ligase (Devoto et al., 2002; Xu et al., 2002), its targets as jasmonate ZIM-domain (JAZ) proteins (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007), and MYC2 as a crucial transcription factor for activating many jasmonate-responsive genes (Lorenzo et al., 2004; Dombrecht et al., 2007). These studies have demonstrated the importance of the COI1–JAZ–MYC2 core module in jasmonate signalling (Fonseca et al., 2009a). Recently, Thireault et al. (2015) reported repression of jasmonate signalling by non-TIFY JAZ proteins, and other transcription factors have been shown to positively or negatively regulate jasmonate responses (Fonseca et al., 2014; Sasaki-Sekimoto et al., 2014; Zhou and Memelink, 2016). The core module COI1–JAZ–MYC2 is conserved in plants (Thines et al., 2007; Shoji et al., 2008) and mediates activation of the biosynthesis of several secondary metabolites, such as nicotine in tobacco (Shoji and Hashimoto, 2011), tanshinone in Salvia miltiorrhiza (Shi et al., 2016a), and anthocyanin in Arabidopsis (Niu et al., 2011; Qi et al., 2011; Xie et al., 2016) and apple (Chen et al., 2017). However, little is known about this core signalling module in tissues that are specific for secondary metabolite biosynthesis. The rubber tree (Hevea brasiliensis) is the major source of natural rubber worldwide. Rubber biosynthesis is a typical isoprenoid metabolic process and occurs in the laticifer cells, a specific tissue for biosynthesis and storage in the rubber tree (Hao and Wu, 2000). Rubber biosynthesis consists of the general mevalonate (MVA) pathway that converts pyruvic acid into isopentenyl pyrophosphate (IPP) and the 2C-methyl-d-erythritol 4-phosphate (MEP) pathway that converts pyruvic acid and glycerate 3-phosphate into IPP. The specific integration of IPP units into a prenyl chain occurs with the aid of initiators, such as farnesyl diphosphate (FPP) (Adiwilaga and Kush, 1996; Cornish and Siler, 1999). Critical reactions are those catalysed by hydroxymethylglutaryl-CoA reductase (HMGR), 1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), farnesyl pyrophosphate synthase (FPS), and Hevea rubber transferase (HRT), and the small rubber particle protein (SRPP) and rubber elongation factor (REF) are also important; these steps are involved in the synthesis of MVA, MEP, and FPP, and in the integration of IPP units into the prenyl chain, which determines the efficiency of rubber biosynthesis (Oh et al., 1999). The cells that are directly related to rubber production are the secondary laticifers in the bark of the trunk. The secondary laticifer is a single-cell type tissue and consists of cells that are differentiated from the fusiform initials of the vascular cambia in the rubber tree (Hao and Wu, 2000). For rubber production, latex is harvested by tapping, i.e. cutting the bark of the trunk, which is generally done every 3 d. Natural latex is the cytoplasm of laticifer cells, where the rubber is synthesized and stored, and contains numerous rubber particles. Although it is well known that latex harvesting enhances latex regeneration, including rubber biosynthesis in the laticifer cells (Paardekooper, 1989), little is known about the positive feedback regulation of biosynthesis. Ethylene appears to be a key regulator given that application of ethrel (an ethylene releaser) increases rubber yield per tapping (Kush, 1994). The available data indicate that this ethylene-induced increase is primarily due to a prolonged duration of latex flow (Coupé and Chrestin, 1989) in addition to up-regulation of genes related to sucrose allocation (Tang et al., 2010), water transportation (Tungngoen et al., 2009), glycolysis, and C3 carbon fixation (Liu et al., 2016). These effects are directly related to energy regeneration, maintenance of turgor pressure, and supply of carbon building blocks, which are required for the biosynthesis of rubber and other organic metabolites in the laticifer cells. Jasmonate signalling has also been suggested as providing an alternative means of control for the positive feedback regulation of rubber biosynthesis per se based on its pivotal roles in activating biosynthesis of secondary metabolites (Peng et al., 2009; Tian et al., 2010; Zhao et al., 2011). Although orthologs of the COI1, MYC, and JAZ genes in Arabidopsis have been cloned and characterized from laticifer cells in the rubber tree (Peng et al., 2009; Tian et al., 2010; Zhao et al., 2011; Pirrello et al., 2014; Hong et al., 2015), there is still a lack of direct evidence to show the presence of the core module of jasmonate signalling and its regulatory involvement in rubber biosynthesis. In the present study, we examined whether the COI1–JAZ–MYC2 signalling pathway is present in laticifer cells and is used to regulate rubber biosynthesis. We observed that latex harvesting raised the level of endogenous jasmonates, up-regulated most of rubber-biosynthesis genes tested, and enhanced rubber biosynthesis in the laticifer cells. Application of methyl jasmonate also had similar effects to latex harvesting. We further identified an HbCOI1–HbJAZ3–HbMYC2 module in laticifer cells and demonstrated that it mediated the latex harvesting-induced up-regulation of the rubber biosynthesis-related genes HbFPS1 and HbSRPP1. The resulting increases in the levels of HbSRPP1 and especially HbFPS1 should contribute to the increased efficiency of rubber biosynthesis. Materials and methods Plant materials and treatments Eight-year-old virgin (i.e. untapped) and tapped trees as well as the epicormic shoots described by Hao and Wu (2000) of the rubber tree (Hevea brasiliensis) clone CATAS7-33–97 were grown at the Experimental Farm of the Chinese Academy of Tropical Agricultural Sciences in Danzhou City, Hainan Province, China. The tapped trees had been 7-year-old virgins before being regularly tapped every 3 d for 1 year in a downward half spiral without ethylene stimulation. After the tapped trees have been harvested 60 times, a total of 30 virgin trees and 30 tapped trees with the same circumference were selected for determination of the content of endogenous jasmonates, the efficiency of rubber biosynthesis, and rubber biosynthesis-related gene expression in latex collected 2 d after the last tapping. Latex samples collected from batches of 10 trees were pooled into single samples, i.e. there were three biological replicates each for virgin and tapped trees. All the 30 tapped trees were then rested from tapping for 15 d, after which nine were treated with 0.07% methyl jasmonate, nine with 0.5% ethrel, and nine were designated as untreated controls. Latex samples were collected 1 d after the treatments. For determination of in vitro rubber biosynthesis, latex samples from batches of three trees for each treatment were pooled; a total of nine trees were sampled, forming three replicates. To investigate the effects of jasmonate (JA) on rubber biosynthesis-related gene expression, 120 epicormic shoots were treated with 0.07% methyl jasmonate as described by Hao and Wu (2000) and a further 120 epicormic shoots without any treatment were used as the control. Latex samples from the treated shoots were collected by cutting the stem bark of the first and second extension units (Hao and Wu, 2000; Zhang et al., 2015) at 4 h, 12 h, 1 d, and 5 d after the treatment. At each time interval, latex was collected from 30 epicormic shoots and batches of 10 samples were pooled to form three replicates. Samples from the control shoots were similarly collected and pooled. Tobacco (Nicotiana benthamiana) was sown and grown at 25 °C with a 16/8 h light/dark photoperiod in an illuminated incubator for approximately 6 weeks prior to being used for BiFC and transient expression analysis. Sequence analysis and phylogenetic tree construction The deduced amino acid sequences of the HbMYC2 and HbJAZ3 proteins were aligned with other homologous proteins using the Clustal X 2.05 program (http://www.clustal.org/clustal2/) and then edited using the Genedoc 2.7 software (http://iubio.bio.indiana.edu/soft/molbio/ibmpc/genedoc-readme.html). Phylogenetic tree construction was performed using Clustal X 2.05 and MEGA 4.0 software (https://www.megasoftware.net/mega4/). Transcriptional activation analysis in yeast cells The full-length coding region and different truncated derivatives of HbMYC2 were separately cloned into the bait vector pGBKT7 (see Supplementary Table S1) and transformed into the yeast strain Y2HGold for transcriptional activation according to the manufacturer’s protocol (Clontech, USA). The transformed strains were then streaked onto SD/–Trp or SD/–Ade–Trp–His plates, and transcriptional activation was determined according to colony growth and the blue coloration after the α-galactosidase reaction on the defective medium (SD/–Ade–Trp–His/X-α-gal). Subcellular localization and BiFC assays For determination of subcellular localization, the coding regions of HbMYC2 and HbJAZ3 were separately cloned into the pCambia1302 vector and fused with the enhanced green fluorescent protein (EGFP) gene. This recombinant plasmid and control GFP plasmids were introduced into Agrobacterium tumefaciens GV3101 and infiltrated into onion epidermal cells for fluorescence detection according to the method described by Li et al. (2016). For bimolecular fluorescence complementation (BiFC) assays in tobacco leaves and onion epidemic cells, the coding sequence of HbJAZ3 was cloned into pSPYNE(R)173 described by Waadt et al. (2008). HbMYC2 as well as HblMYC1 and HblMYC2 were cloned into pSPYCE(MR). The human lamin C gene from the pGBKT7-Lam vector was cloned and inserted into pSPYNE(R)173 for the negative control, while the murine p53 gene from the pGBKT7-53 vector was cloned and inserted into the pSPYNE(R)173 for the positive control. The SV40 large T-antigen gene was cloned from the pGADT7-T vector and inserted into pSPYCE(MR) for both the negative and positive controls. The corresponding combined plasmids were introduced into A. tumefaciens strain GV3101 and confirmed by RT-PCR with specific primers (Supplementary Table S1). The BiFC experiments were designed according to Kudla and Bock (2016) and also performed in onion epidemic cells according to the methods described previously by Li et al. (2016). The pSPYNE(R)173-HbJAZ3 and pSPYCE(MR)-HbMYC2 constructs were separately introduced into A. tumefaciens GV3101 and co-infiltrated in combination with pSPYNE(R)173-HbJAZ3 and pSPYCE(MR)-HbMYC2 at an optical density of 0.6–0.7 at 600 nm. Negative controls without HbJAZ3 or HbMYC2 were performed in the same way. After cultivation for 2–4 d, the transformed tobacco leaves were cut approximately into squares and put into a DAPI solution (10 μg ml–1; (Solarbio, Beijing, China) within 15 min and then washed with ddH2O at least twice. The fluorescence of yellow fluorescent protein (YFP) and DAPI in the transformed tobacco leaves and onion epidemic cells were recorded using confocal microscopy. Yeast one/two-hybrid assays For yeast one-hybrid assays, the HbFPS1 promoter and HbSRPP1 promoter were cloned into the pHIS2.1 vector (Clontech), generating pHIS-pHbFPS1 and pHIS-pHbSRPP1, respectively (Wang et al., 2013; Guo et al., 2014). The yeast-one library was constructed according to the manufacturer’s instructions (Clontech), as described previously (Wang et al., 2013; Guo et al., 2014). The yeast-one library strain Y187 was transfected with the bait vector pHIS-pHbFPS1 and then cultivated for 3 d at 30 °C on SD/–His/–Leu/–Trp medium supplemented with 20 mM 3-AT for the selection of transformants. For confirmation of the HbFPS1 or HbSRPP1 promoter-binding proteins, HbMYC2 was cloned into pGADT7-Rec2. The pGADT7-HbMYC2 and pHIS-pHbFPS1 or pHIS-pHbSRPP1 vector was co-transformed into the yeast strain Y187. pGADT7-Rec53 and p53-His were used as positive controls (CK&#x002B;) and p53-His as a negative control (CK–). Transformed clones were grown on SD/–His/–Leu/–Trp medium with 20 mM (for pHbFPS1) or 42.5 mM 3-AT (for pHbSRPP1) for 3 d at 30 °C. For yeast two-hybrid assays, the full-length coding region of HbMYC2 was cloned into the pGADT7 vector (Clontech) with specific primers (Supplementary Table S1). HbJAZ3 and HbCOI1 were respectively cloned into the pGADT7 and pGBKT7 vectors. HbJAZ8.0a, HbJAZ8.0c, and HbJAZ10.0a were each cloned into the pGBKT7 vector. Detection of protein–protein interactions between HbMYC2 and JAZs or JAZs and HbCOI1 was performed using SD/–His/–Leu/–Trp–Ade/X-α-gal medium (DDO/X medium, –4&#x002B;X) or SD/–His/–Leu/–Trp–Ade/X-α-gal (–4&#x002B;X) medium containing 0 μM or 50 μM coronatine (COR). Electrophoretic mobility shift assays The coding sequence of HbMYC2 was cloned into the pET-30a vector fused with the His tag. The recombinant His-HbMYC2 protein was expressed and affinity-purified from E. coli (BL21[DE3]) using Ni&#x002B; affinity resin (GE). The EMSA was performed with an Electrophoretic Mobility Shift Assay kit (Invitrogen, USA) following the manufacturer’s instructions, as described previously (Wang et al., 2013; Guo et al., 2014). Transient dual-luciferase assays The coding regions of HbMYC2 and HbJAZ3 were respectively cloned into the effector vectors pGreenII-62SK and pCAMBIA2301 (CAMBIA). The empty vector pGreenII-62SK was used as a negative control. The inhibitory effect of HbJAZ3 on the activity of HbMYC2 with regards to the transcription of HbFPS1 and HbSRPP1 was determined by transient dual-luciferase assays. The reporter vectors (pGreen II-0800-LUC containing pHbFPS1 or pHbSRPP1) and the effector vector (p35S::HbMYC2) were transferred into A. tumefaciens (strain GV3101). Overnight cultures of Agrobacterium were collected by centrifugation, resuspended in the infiltration buffer (10 mM MES, 150 mM acetosyringone, and 10 mM MgCl2), and incubated at room temperature for 4 h before infiltration. The strain containing the reporter was used alone or mixed with the strains harboring the effectors or the empty vector control. An Agrobacterium suspension in a 5-ml syringe was carefully press-infiltrated manually onto healthy leaves of 6-week-old N. benthamiana. After culture for 3 d, the infected area was harvested for total protein extraction. The supernatant of total proteins was treated with the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions, and the fluorescent values of LUC and REN were detected using a GloMaxR-Multi&#x002B;Detection System (Promega). The LUC value was normalized to that of REN. Three biological repeats were measured for each combination. qRT-PCR Latex samples from plants subjected to the treatments described above were used for quantitative real-time PCR analysis of the expression levels of relevant genes. Total RNAs were extracted using a Total plant RNA isolation kit (Tiangen, China) and reverse-transcribed using M-MLV reverse transcriptase (Fermentas, USA). Quantitative real-time PCR (qRT-PCR) was performed using the CFX384 real-time PCR system (Bio-Rad, USA) with the SYBR Prime Script RT-PCR kit (TaKaRa, Japan). Relative gene expression levels were calculated using the 18S-rRNA gene as the internal normalization control. The qRT-PCR experiments were performed with each sample measured in triplicate. Primers used for real-time PCR analysis are listed in Supplementary Table S1). JA determination The extraction and quantification of JA from the latex samples were conducted according to Tian et al. (2015) with modifications in sample preparation. Approximately 3 g of each latex sample was dripped into 20 ml of 80% methanol containing 0.01% (w/v) sodium diethyldithiocarbamate as an antioxidant. [9,10-2H2]Dihydro-JA (250 ng) was added as an internal standard for JA determination. The admixture was stirred overnight at 4 °C and then centrifuged. After the methanol phase was collected, the rubber deposit was extracted twice with 20 ml of cold (4 °C) 80% methanol. The organic phase was combined, adjusted to pH 8.0 with ammonia, condensed to the aqueous phase (approximately 5 ml) in a rotary film evaporator (Heidolph Laborota 4000 efficient, Germany), and frozen at –20 °C, followed by three rounds of melting and thawing. After centrifugation at 270 g for 20 min, 1 mg of polyvinylpolypyrrolidone was added to the collected supernatant, and it was homogenized at 4.5 m s–1 for 10 s and filtered. The filtrate was collected, adjusted to pH 2.5–3.0 with 2 M acetic acid, and then extracted three times by mixing the filtrate with 5 ml of ethyl acetate. The organic layers were combined, adjusted to pH 8 with ammonia and evaporated to dryness in a rotary film evaporator. The dried extracts were dissolved in 5 ml of 0.1 M acetic acid and passed through a C18 column (Sep-Pak® Classic C18, Part No. WAT051910, Waters Corporation, Milford, MA, USA) that had been pre-washed with 5 ml 100% methanol and pre-equilibrated with 5 ml 0.1 M acetic acid. The column was eluted with 5 ml of 17% (v/v) acid methanol (100% methanol:0.1 M acetic acid, 17:83), followed in turn by 5 ml of 60% acid methanol (100% methanol:0.1 M acetic acid, 60:40) for JA extraction. JA fractions were collected and evaporated to dryness (40 °C) after being adjusted to pH 8 with ammonia. Methyl esterification was carried out by dissolving the residue in 0.5 ml of methanol and adding 3 ml of ethereal diazomethane. The excess diazomethane was removed under a stream of oxygen-free nitrogen gas. The methylated samples were re-dissolved in ethyl acetate for analysis by GC-MS as described by Tian et al, (2015). All analyses were performed with three biological replicates. In vitro rubber biosynthesis assay The reaction system was as described by Archer et al. (1963) with modifications as follows. A sample of 25 μl fresh latex was added into a 400-μl reaction system containing 10 μM MgCl2, 10 μM DTT, 5 μM ATP, and 0.736 μM 13C-MVA (mevalonolactone-2-13C) (Sigma Aldrich, Lot# MBBB5201 V) in 0.1 M PBS buffer (pH 7.8). The reaction was carried out at 30 °C for 8 h with constant shaking (70 rpm). Thereafter, the rubber was extracted according to the method described by Rattanapittayaporn et al. (2004). A total volume of 400 μl saturated NaCl solution was added into the reaction solution and mixed thoroughly, an equal volume (800 μl) of extractant (toluene/hexane, 1:1, v/v) was added to the mixture and subjected to centrifugation at 16 000 g for 30 min. The upper organic phase was collected and mixed with equal volume of 1-butanol to dissolve out the rubber overnight. The organic phase was removed after centrifugation at 16 000 g for 30 min, and the remaining rubber was dried. Approximately 0.5 mg of dried rubber sample was embedded into a tin capsule (Elemental Microanalysis, 6 × 4 mm, BN261080) and the 13C/12C ratio was determined using a stable isotope mass spectrometer (GV Instruments, IsoPrime JB312) and an elemental analyser (Thermo, FLASH EA1112 Series). The results were expressed as values of atom percentage (APC), equal to the percentage of the content of 13C relative to the total carbon (13C &#x002B;12C) content in the rubber sample. All analyses were performed with three biological replicates. Latex collection for western blot assays Latex from each of 30 virgin rubber trees and 30 tapped trees were collected within 5 min after tapping. Then equal volumes of samples that were collected from 10 virgin trees were pooled to give a single sample for western blot assays. The samples from the tapped trees were pooled in the same manner. In this way, three biological replicates were conducted. Then an equal volume of the isotonic solution used in the in vitro rubber biosynthesis efficiency assay was added to the pooled samples and mixed gently. The pooled samples were stored on ice for subsequent use. The latex samples were centrifuged at 4 °C at 16 000 g for 10 min. The middle layer of the suspension liquid was filtered through a 0.45-μm membrane, and the filtrate was collected. SDS-PAGE and western blots were performed as described by Towbin et al. (1979) and Shi et al. (2016b) with the following modifications. The loading amounts were 20 μl and 6 μl for HbFPS1 and HbSRPP1 protein detection, respectively. FPS and SRPP proteins were recognized by specific polyclonal antibodies (1:1000 dilution for anti-FPS1, 1:2500 dilution for anti-SRPP1) and were identified using an Enhanced Chemiluminescence (ECL) Kit and a BCIP/NBT Kit (Pierce, USA), respectively. Statistical analysis Statistical analysis was carried out by ANOVA using SPSS Statistics 17.0, and statistical significance was evaluated using Student’s t-test (two-group comparisons between treated samples and control samples) as described previously (Wang et al., 2013; Guo et al., 2014; Li et al., 2016). Results Increase in the level of endogenous jasmonates induced by removal of latex, up-regulation of rubber biosynthesis genes, and enhancement of rubber biosynthesis The levels of endogenous jasmonates in the latex from virgin trees and regularly tapped trees were determined by GC-MS. Levels in the regularly tapped trees were approximately six times that of virgin trees (Fig. 1A). The levels of expression of rubber biosynthesis-related genes in the latex were also different between the virgin trees and the regularly tapped trees. Most of the genes in the MVA pathway (Fig. 1B), including HbHMGR1, HbMVK1, HbPMK1, HbMVD1, HbMVD2, HbIPPI1, HbFPS1, HbREF1, HbCPT6 (also designated as HbHRT2), HbCPT7, and HbCPT8 (HbHRT1), were significantly up-regulated in regularly tapped trees in comparison with virgin trees (Fig. 1C). Most of the up-regulated genes in the regularly tapped trees were also up-regulated in the latex from epicormic shoots at 12 h or 1 d after being treated with methyl jasmonate (Fig. 1D). HbCMK, HbMCS1/HbMDS1, and HbMCS2/HbMDS2 in the MEP pathway (Supplementary Fig. S1A) were significantly up-regulated, while HbDXS1, HbDXS2, HbDXR, HbCMS1/HbMCT1, HbCMS2/HbMCT2, and HbHDR were moderately up-regulated in the regularly tapped trees in comparison with the virgin trees (Supplementary Fig. S1B). These genes were also significantly up-regulated in epicormic shoots at 1 d after being treated with 0.07% methyl jasmonate (Supplementary Fig. S1C). Fig. 1. View largeDownload slide (A) Effect of tapping on endogenous jasmonate (MeJA) levels in laticifer cells. (B) Rubber biosynthesis-related genes of the MVA pathway that were examined. Effect of tapping of trees (C) and application of methyl jasmonate to epicormic shoots (D) on the expression of rubber biosynthesis-related genes. (E) Determination of the efficiency of in vitro rubber biosynthesis in the latex from the virgin trees, regularly tapped trees, trees rested from tapping for 15 d without any treatment (rested), rested trees treated with ethrel (ET), and rested trees treated with methyl jasmonate (JA). Data are means (±SD) of three biological replicates and are expressed as atom percentage of 13C (APC; see Methods). Significant differences were determined using Student’s t-test; *P<0.05, **P<0.01. Abbreviations: HMGR, 3-hydroxy-3-methylglutaryl CoA reductase; MK, mevalonate kinase; PMK, phosphomevalonate kinase; MVD, mevalonate diphosphate decarboxylase; IPPI, isopentenyl diphosphate isomerase; FPS, farnesyl diphosphate synthesis; CPT, cis-prenyltransferase; HRT, Hevea rubber transferase; SRPP, small rubber particle protein; REF, rubber elongation factor. Fig. 1. View largeDownload slide (A) Effect of tapping on endogenous jasmonate (MeJA) levels in laticifer cells. (B) Rubber biosynthesis-related genes of the MVA pathway that were examined. Effect of tapping of trees (C) and application of methyl jasmonate to epicormic shoots (D) on the expression of rubber biosynthesis-related genes. (E) Determination of the efficiency of in vitro rubber biosynthesis in the latex from the virgin trees, regularly tapped trees, trees rested from tapping for 15 d without any treatment (rested), rested trees treated with ethrel (ET), and rested trees treated with methyl jasmonate (JA). Data are means (±SD) of three biological replicates and are expressed as atom percentage of 13C (APC; see Methods). Significant differences were determined using Student’s t-test; *P<0.05, **P<0.01. Abbreviations: HMGR, 3-hydroxy-3-methylglutaryl CoA reductase; MK, mevalonate kinase; PMK, phosphomevalonate kinase; MVD, mevalonate diphosphate decarboxylase; IPPI, isopentenyl diphosphate isomerase; FPS, farnesyl diphosphate synthesis; CPT, cis-prenyltransferase; HRT, Hevea rubber transferase; SRPP, small rubber particle protein; REF, rubber elongation factor. The efficiency of in vitro rubber biosynthesis in the regularly tapped trees was significantly higher than that in virgin trees that had never been tapped (Fig. 1E). It decreased significantly when the regularly tapped trees were rested from tapping for 15 d (Fig. 1E). It further decreased significantly 1 d after the rested trees were treated with 0.5% ethrel (an ethylene releaser), but treatment with 0.07% methyl jasmonate instead caused a significant increase 1 d after application (Fig. 1E). Identification of the HbCOI1–HbJAZ3–MYC2 module in laticifer cells To examine jasmonate signalling in the laticifer cells of rubber trees, a gene homologous to AtJAZ3 and SmJAZ3 was cloned and characterized. It was designated as HbJAZ3 (GenBank No. KJ911911) and had preferential expression in the latex compared to the bark tissues (Supplementary Fig. S2A). The ORF of this gene was 1164 bp in length and encoded a predicted protein of 387 amino acids with a molecular weight of 41.0 kDa and a pI of 8.77. HbJAZ3 possesses a ZIM domain and a Jas domain that are conserved in the JAZ proteins from other plant species (Supplementary Fig. S3). It was clustered closely with AtJAZ3 and SmJAZ3 in the phylogenetic tree (Fig. 2A). Subcellular localization analysis showed that the green fluorescence from HbJAZ3-GFP was only detected in the nuclei, which was confirmed by DAPI staining (Fig. 2B). Using yeast two-hybrid assays, we determined that yeast cells harboring HbJAZ3 and HbCOI1 could survive in the selective medium when it contained 50 μM COR (Fig. 2C, Supplementary Fig. S6), suggesting that HbJAZ3 was the target of SCFCOI1 in response to a burst of endogenous jasmonates. Fig. 2. View largeDownload slide HbJAZ3 identification. (A) A phylogenetic tree was constructed by using the neighbor-joining method with 100 replications after ClustalX2 alignment analysis of HbJAZ3 with other JAZ proteins including AtJAZ1 (NP_564075), AtJAZ2 (Q9S7M2), AtJAZ3 (AEE76017), AtJAZ4 (NP_001117450), AtJAZ5 (ANM58475), AtJAZ6 (NP_565043), AtJAZ7 (AEC08997), AtJAZ8 (NP_564349), AtJAZ9 (Q8W4J8), AtJAZ10 (NP_974775), AtJAZ11 (NP_001190007), AaJAZ1 (AJK93412), AaJAZ2 (AJK93413), AaJAZ3 (AJK93414), AaJAZ4 (AJK93415), SmJAZ1 (AGC73980), SmJAZ3 (AHK23660), SmJAZ9 (protein sequences in Shi et al., 2016a), NtJAZ1 (BAG68655), NtJAZ2 (BAG68656), NtJAZ3 (BAG68657), HbJAZ1 (ADI39634), HbJAZ2 (AIY25007), HbJAZ3 (KJ911911), HbJAZ7 (AIY2500), HbJAZ8 (AIY25009), HbJAZ9 (AIY25010), HbJAZ10 (AIY25011), and HbJAZ11 (AIY25012). The scale bar indicates the evolutionary distance. (B) Subcellular localization of HbJAZ3 in onion epidermal cells. The nuclei of the onion epidermal cells were visualized by DAPI staining. GFP, green fluorescent protein; Merge, merged image. (C) Interaction of HbCOI1 with HbJAZ3 in yeast cells in the presence of 50 µM coronatine (COR). Fig. 2. View largeDownload slide HbJAZ3 identification. (A) A phylogenetic tree was constructed by using the neighbor-joining method with 100 replications after ClustalX2 alignment analysis of HbJAZ3 with other JAZ proteins including AtJAZ1 (NP_564075), AtJAZ2 (Q9S7M2), AtJAZ3 (AEE76017), AtJAZ4 (NP_001117450), AtJAZ5 (ANM58475), AtJAZ6 (NP_565043), AtJAZ7 (AEC08997), AtJAZ8 (NP_564349), AtJAZ9 (Q8W4J8), AtJAZ10 (NP_974775), AtJAZ11 (NP_001190007), AaJAZ1 (AJK93412), AaJAZ2 (AJK93413), AaJAZ3 (AJK93414), AaJAZ4 (AJK93415), SmJAZ1 (AGC73980), SmJAZ3 (AHK23660), SmJAZ9 (protein sequences in Shi et al., 2016a), NtJAZ1 (BAG68655), NtJAZ2 (BAG68656), NtJAZ3 (BAG68657), HbJAZ1 (ADI39634), HbJAZ2 (AIY25007), HbJAZ3 (KJ911911), HbJAZ7 (AIY2500), HbJAZ8 (AIY25009), HbJAZ9 (AIY25010), HbJAZ10 (AIY25011), and HbJAZ11 (AIY25012). The scale bar indicates the evolutionary distance. (B) Subcellular localization of HbJAZ3 in onion epidermal cells. The nuclei of the onion epidermal cells were visualized by DAPI staining. GFP, green fluorescent protein; Merge, merged image. (C) Interaction of HbCOI1 with HbJAZ3 in yeast cells in the presence of 50 µM coronatine (COR). To identify the downstream partners of HbJAZ3 in jasmonate signalling, we used HbJAZ3 as bait to screen a cDNA library from latex using yeast two-hybrid assays. A positive clone was obtained after several cycles of verification on the selective medium (Fig. 3A). It contained a single plasmid harboring an inserted fragment of 2495 bp in length. The fragment contained an ORF of 2046 bp in length, a 72-bp 5′UTR region and a 377 bp 3′UTR region. The ORF encoded a MYC-type protein of 681 amino acids with a molecular weight of 74.58 kDa and a pI of 5.70. This protein shared high similarity with AtMYC2 according to blastp analysis. Therefore, we designated the fragment as HbMYC2 (GenBank No. KM507201) and it had higher expression level in the latex than that in the bark tissues (Supplementary Fig. S2B). HbMYC2 shared highly conserved motifs with the AtMYC2, NtMYC2a, SmMYC2a, and AaMYC2 transcription factors in an amino acid alignment (Supplementary Fig. S4) and was clustered into the MYC2 ortholog subgroup in the phylogenetic tree (Fig. 3B, Supplementary Fig. S5). As a potential transcription factor, the transcriptional activation of HbMYC2 and its N- and C-terminal regions were assessed in yeast. Only the full-length protein and N-terminal region of residues 1–377 could activate expression of the HIS and MEL1 genes in yeast cells, resulting in survival of the yeast cells in the selective media lacking histidine and adenine (Fig. 3C). Subcellular localization analysis showed that the green fluorescence from HbMYC2-GFP was only present in the nuclei, which was confirmed by DAPI staining (Fig. 3D). Fig. 3. View largeDownload slide HbMYC2 identification. (A) Screening of HbMYC2 by HbJAZ3 using yeast two-hybrid assays. (B) A phylogenetic tree was constructed by using the neighbor-joining method with 100 replications after ClustalX2 alignment of HbMYC2 (AJC01627) and other bHLH proteins, including NtMYC2a (ADU60100), NtMYC2b (ADU60101), SmMYC2a (AIO09733), SlMYC2 (AGZ94899), JcMYC2 (XP_012076236), RcMYC2 (XP_002519814), AtMYC2 (Q39204), AtMYC3 (Q9FIP9), AtMYC4 (O49687), AaMYC2 (AKO62850), MaMYC2a (XP_009384727), MaMYC2b (XP_009413229), TcJAMYC (ACM48567), SmMYC2b (protein sequences in Zhou et al., (2016), AtbHLH13 (Q9 LNJ5), HblMYC1 (ADK56287), and HblMYC2 (ADK91082). The scale bar indicates the evolutionary distance. (C) Activating activity of full-length HbMYC2 and its N-terminal derivative (1–377 aa) for the transcription in yeast cells. The schematic diagram represents for the constructs of the N- and C-terminal of HbMYC2 in the transcriptional activation assay in yeast. (D) Subcellular localization of HbMYC2 in onion epidermal cells. The nuclei of the onion epidermal cells were visualized by DAPI staining. GFP, green fluorescent protein; Merge, merged image. Fig. 3. View largeDownload slide HbMYC2 identification. (A) Screening of HbMYC2 by HbJAZ3 using yeast two-hybrid assays. (B) A phylogenetic tree was constructed by using the neighbor-joining method with 100 replications after ClustalX2 alignment of HbMYC2 (AJC01627) and other bHLH proteins, including NtMYC2a (ADU60100), NtMYC2b (ADU60101), SmMYC2a (AIO09733), SlMYC2 (AGZ94899), JcMYC2 (XP_012076236), RcMYC2 (XP_002519814), AtMYC2 (Q39204), AtMYC3 (Q9FIP9), AtMYC4 (O49687), AaMYC2 (AKO62850), MaMYC2a (XP_009384727), MaMYC2b (XP_009413229), TcJAMYC (ACM48567), SmMYC2b (protein sequences in Zhou et al., (2016), AtbHLH13 (Q9 LNJ5), HblMYC1 (ADK56287), and HblMYC2 (ADK91082). The scale bar indicates the evolutionary distance. (C) Activating activity of full-length HbMYC2 and its N-terminal derivative (1–377 aa) for the transcription in yeast cells. The schematic diagram represents for the constructs of the N- and C-terminal of HbMYC2 in the transcriptional activation assay in yeast. (D) Subcellular localization of HbMYC2 in onion epidermal cells. The nuclei of the onion epidermal cells were visualized by DAPI staining. GFP, green fluorescent protein; Merge, merged image. The interaction between HbJAZ3 and HbMYC2 was further confirmed by yeast two-hybrid assays. Only the yeast cells harboring HbJAZ3 and HbMYC2 and the positive control could grow and exhibit a blue color on QDO/X-α-gal plates, suggesting that HbJAZ3 interacted with HbMYC2 (Fig. 3A). The interaction was further verified in planta by BiFC assays. Tobacco cells (Fig. 4A) or onion epidermal cells (Supplementary Fig. S7) harboring HbJAZ3-cYFP and HbMYC2-nYFP exhibited a bright yellow color in the sites that were specifically stained by DAPI, similar to the YFP signal in the cells harboring the yeast positive control p53 and SV40-T. By contrast, the bright yellow color was not detected in tobacco or onion epidermal cells harboring either HbJAZ3-cYFP or nYFP, HbMYC2-nYFP or cYFP, and HbJAZ3-cYFP and HblMYC1 or HblMYC2, or the yeast negative control Lam and SV40-T, although DAPI staining of the nuclei was observed (Supplementary Fig. S7), suggesting that the bright yellow color was specific and positive. HbCOI1, HbJAZ3, and HbMYC2 were all significantly up-regulated in laticifer cells of the regularly tapped trees compared with those of the virgin trees (Fig. 4B). The expression of these three genes in the epicormic shoots was significantly up-regulated at 12 h or 1 d after being treated with 0.07% methyl jasmonate (Fig. 4C). The positive response of the three genes to JA, the interaction between HbCOI1 and HbJAZ3 in a COR-dependent manner, and between HbJAZ3 and HbMYC2 in a COR-independent manner, demonstrated the presence of a HbCOI1–HbJAZ3–MYC2 module in the laticifer cells of rubber tree. Fig. 4. View largeDownload slide Identification of the HbCOI1–HbJAZ3–HbMCY2 module. (A) Interaction of HbJAZ3 with HbMYC2 in tobacco as shown by bimolecular fluorescence complementation (BiFC) assays. The empty pairs (n-YFP and c-YFP) were used as negative controls. YFP fluorescence was detected under a confocal microscope 3 d after infiltration. Scale bar &#x003D; 20 μm. (B, C) Up-regulated expression of HbCOI1, HbJAZ3, and HbMYC2 upon tapping (B) and treatment with methyl jasmonate (C). Fig. 4. View largeDownload slide Identification of the HbCOI1–HbJAZ3–HbMCY2 module. (A) Interaction of HbJAZ3 with HbMYC2 in tobacco as shown by bimolecular fluorescence complementation (BiFC) assays. The empty pairs (n-YFP and c-YFP) were used as negative controls. YFP fluorescence was detected under a confocal microscope 3 d after infiltration. Scale bar &#x003D; 20 μm. (B, C) Up-regulated expression of HbCOI1, HbJAZ3, and HbMYC2 upon tapping (B) and treatment with methyl jasmonate (C). Effect of HbJAZ3 on the HbMYC2-activated transcription of HbFPS1 and HbSRPP1 To identify possible transcriptional factors involved in regulation of rubber biosynthesis by JA signalling, a yeast one-hybrid screening of the latex cDNA library was performed using the promoter of the key rubber biosynthesis gene HbFPS1. A fragment harbored in a positive clone was identical to HbMYC2. The interaction of the full-length HbMYC2 with the HbFPS1 promoter was revealed by the yeast one-hybrid assay (Fig. 5A). The physical binding of HbMYC2 to the HbFPS1 promoter was further verified by in vitro EMSAs. HbFPS1 promoter fragments were more and more retarded with increased amounts of the purified HbMYC2 protein (Fig. 5B). Fig. 5. View largeDownload slide Transcriptional regulation of HbFPS1 and HbSRPP1. (A) Activation of the HbFPS1 promoter in yeast by the HbMYC2 protein. (B) Demonstration of the physical binding of the HbMYC2 protein to the promoter of HbFPS1 by electrophoretic mobility shift assays (EMSA). (C) Activation of the HbSRPP1promoter in yeast by the HbMYC2 protein. (D) Demonstration of the physical binding of the HbMYC2 protein to the promoter of HbFPS1 by EMSA. (E) Schematic diagram of the constructs for a dual-luciferase assay. (F, G) Assays of the transient transcriptional activity of HbMYC2 on HbFPS1 (F) and HbSRPP1 (G) with or without the expressed HbJAZ3 protein. Data are means (±SD) of three biological replicates. Significant differences were determined using Student’s t-test; **P<0.01. Fig. 5. View largeDownload slide Transcriptional regulation of HbFPS1 and HbSRPP1. (A) Activation of the HbFPS1 promoter in yeast by the HbMYC2 protein. (B) Demonstration of the physical binding of the HbMYC2 protein to the promoter of HbFPS1 by electrophoretic mobility shift assays (EMSA). (C) Activation of the HbSRPP1promoter in yeast by the HbMYC2 protein. (D) Demonstration of the physical binding of the HbMYC2 protein to the promoter of HbFPS1 by EMSA. (E) Schematic diagram of the constructs for a dual-luciferase assay. (F, G) Assays of the transient transcriptional activity of HbMYC2 on HbFPS1 (F) and HbSRPP1 (G) with or without the expressed HbJAZ3 protein. Data are means (±SD) of three biological replicates. Significant differences were determined using Student’s t-test; **P<0.01. Given the presence of a conserved G-box (‘CANNTG’) in the HbSRPP1 promoter (Supplementary Table S2), the potential interaction of this promoter with HbMYC2 was tested by yeast one-hybrid assays. Yeast cells harboring HbMYC2 and the HbSRPP1 promoter survived on the selective medium (Fig. 5C), suggesting that the action did indeed occur. The physical binding of HbMYC2 to the HbSRPP1 promoter was verified by in vitro EMSAs. HbSRPP1 promoter fragments were increasingly retarded with increasing amounts of the purified HbMYC2 protein (Fig. 5D). A dual-luciferase assay system (Fig. 5E) was used to assess the effect of the physical binding of HbMYC2 to either the promoter of HbFPS1 or the promoter of HbSRPP1 as well as the effect of HbJAZ3. Luciferase (LUC) activity in tobacco leaves harboring HbMYC2 and Luc that was driven by either the promoter of HbFPS1 or that of HbSRPP1 was significantly higher than that in leaves harboring Luc alone, and activity was significantly decreased when HbJAZ3 was co-expressed (Fig. 5F, G). There was no obvious difference in the expression of Luc in tobacco leaves in comparison with the empty vector control when the HbJAZ3 gene was expressed alone (Fig. 5F, G). These data demonstrated that both HbFPS1 and HbSRPP1 were positively regulated by HbMYC2, while HbJAZ3 repressed the HbMYC2-activated expression of either HbFPS1 or HbSRPP1. Effect of latex harvesting on the levels of HbFPS1 and HbSRPP1 Given that the expression levels of HbFPS1 and HbSRPP1 were higher in the regularly tapped trees than that in the virgin trees (Fig. 1D), we further examined the differences in the levels of HbFPS1 and HbSRPP1. As HbSRPP1 is more abundant than HbFPS1, different amounts of rubber particle filtrates were loaded for detection of the proteins (HbFPS1 20 μl, HbSRPP1 6 μl) (Fig. 6A). Western blotting analysis revealed that levels of HbFPS1 were significantly higher in the regularly tapped trees as compared with the virgin trees (Fig. 6B, C). Although the SDS-PAGE profiles appeared to show no difference in the levels of HbSRPP1 between the regularly tapped trees and the virgin trees (Fig. 6D), western blots showed that HbSRPP1 levels were also significantly higher in the regularly tapped trees (Fig. 6E, F). These results indicated that the increase in the transcripts was positively correlated with the increase in their corresponding proteins. Fig. 6. View largeDownload slide Changes in the levels of HbFPS1 and HbSRPP1 in the latex from regularly tapped trees and virgin trees. (A, D) SDS-PAGE profiles of latex from regularly tapped trees (T) and virgin trees (V); M, protein standards. In (A) 20 μl latex was loaded per lane, and in (D) 6 μl latex was loaded per lane. (B, E) Western blot analyses with polyclonal antibodies raised against HbFPS1 (B) and HbSRPP1 (E), as indicated by the arrows. (C, F) Relative quantitative analysis of HbFPS1 (C) and HbSRPP1 (F). Data are means (±SD) of three biological replicates. Significant differences were determined using Student’s t-test; *P<0.05, **P<0.01. Fig. 6. View largeDownload slide Changes in the levels of HbFPS1 and HbSRPP1 in the latex from regularly tapped trees and virgin trees. (A, D) SDS-PAGE profiles of latex from regularly tapped trees (T) and virgin trees (V); M, protein standards. In (A) 20 μl latex was loaded per lane, and in (D) 6 μl latex was loaded per lane. (B, E) Western blot analyses with polyclonal antibodies raised against HbFPS1 (B) and HbSRPP1 (E), as indicated by the arrows. (C, F) Relative quantitative analysis of HbFPS1 (C) and HbSRPP1 (F). Data are means (±SD) of three biological replicates. Significant differences were determined using Student’s t-test; *P<0.05, **P<0.01. Effect of HbEIN3 on the HbMYC2-activated transcription of HbFPS1 and HbSRPP1 Given that in vitro rubber biosynthesis was enhanced by exogenous methyl jasmonate and inhibited by application of ethrel (Fig. 1E), the effect of a member of the EIN3 family, HbEIN3, on the HbMYC2-activated transcription of HbFPS1 and HbSRPP1 was assessed using the transient LUC activity detection system (Fig. 7A). HbEIN3 had little influence on the expression of HbFPS1 and HbSRPP1, although ET-responsive elements are present in the promoter sequence of HbFPS1 and HbSRPP1 (Supplementary Table S2). HbEIN3 also had no influence on the HbMYC2-activated expression of HbFPS1 and HbSRPP1 (Fig. 7B, C). Therefore, the mechanism for the antagonistic effect of JA and ET on rubber biosynthesis (Fig. 1E) remains to be elucidated. Fig. 7. View largeDownload slide Effect of HbEIN3 on the regulation of HbMYC2-activated HbFPS1 and HbSRPP1 expression. (A) Schematic diagrams of the constructs for a dual-luciferase assay. (B, C) Analysis of the transient transcriptional activity of HbMYC2 on HbFPS1 (B) and HbSRPP1 (C) with or without the expressed HbEIN3 protein. Data are means (±SD) of three biological replicates. Significant differences were determined using Student’s t-test; **P<0.01. Fig. 7. View largeDownload slide Effect of HbEIN3 on the regulation of HbMYC2-activated HbFPS1 and HbSRPP1 expression. (A) Schematic diagrams of the constructs for a dual-luciferase assay. (B, C) Analysis of the transient transcriptional activity of HbMYC2 on HbFPS1 (B) and HbSRPP1 (C) with or without the expressed HbEIN3 protein. Data are means (±SD) of three biological replicates. Significant differences were determined using Student’s t-test; **P<0.01. Discussion Jasmonate signalling has been extensively studied in herbaceous model plants with the aid of forward genetics (Xie et al., 1998; Chini et al., 2007; Wasternack and Song, 2017; Zhu and Napier, 2017) and a COI1–JAZ–MYC2 module has been identified that seems to be conserved among plant species (Fonseca et al., 2009a; Shoji and Hashimoto, 2011; Shen et al., 2016; Zhou et al., 2016). However, the role of jasmonate signalling in the specific tissues that produce latex in rubber trees has not yet been elucidated. The secondary laticifer in rubber trees is a single-cell type tissue that is composed of laticifer cells. There are no plasmodesmata between the cells and their surrounding phloem parenchyma cells (de Faÿ et al., 1989). Therefore, the latex that is easily obtained from them by the process of tapping is the pure cytoplasm of laticifer cells. As the specific tissue for rubber biosynthesis (Kush, 1994), the secondary laticifer is enriched in transcripts of rubber biosynthesis-related genes (Chow et al., 2007). These structural and molecular characteristics mean that the laticifer cells are a good model for investigating the transcriptional regulation of secondary metabolism. Rubber biosynthesis in laticifer cells is a typical isoprenoid metabolism and is carried out by the MVA as well as the MEP pathways (Seetang-Nun et al., 2008; Chow et al., 2012; Deng et al., 2016; Tang et al., 2016). Most of the rubber biosynthesis-related genes in these pathways were up-regulated in regularly tapped trees (Fig. 1C, Supplementary Fig. S1B), and these genes are down-regulated when trees are rested from tapping for a short time (Tian et al., 2013), and also in trees that are affected by tapping panel dryness (TPD) that are rested from tapping for a long time (Liu et al., 2015). The up-regulated expression of rubber biosynthesis-related genes could be associated with a high level of endogenous JA in the latex of regularly tapped trees, since the expression pattern of the genes resulting from exogenous methyl jasmonate was similar to that in regularly tapped trees (Fig. 1D, Supplementary Fig. S1C). The level of endogenous JA in the laticier cells of regularly tapped trees was several times higher than that of virgin trees 2 d after tapping (Fig.1A). As mechanical wounding causes a burst of endogenous JA in bark tissues that occurs within 4 h (Tian et al., 2015), the maintenance of high levels of endogenous JA in regularly tapped trees 2 d after being tapped may not be related to the mechanical wounding per se; it could mainly be ascribed to changes in the turgor pressure of laticifer cells. Available data show that changes in turgor pressure are critical for JA biosynthesis (Creelmen and Mullet, 1997), and the turgor of laticifer cells is 10 times above atmospheric pressure before tapping and significantly decreases within the latex drainage areas, even resulting in a detectable decrease in the trunk diameter after tapping (Pakianathan et al., 1989). The high levels of endogenous JA were also positively correlated with enhanced rubber biosynthesis in regularly tapped trees, because exogenous methyl jasmonate was effective in enhancing in vitro rubber biosynthesis (Fig.1E). Our results identified for the first time a jasmonate signalling module, HbCOI1–HbJAZ3–HbMYC2, in the specific laticifer tissue of rubber trees. HbJAZ3 interacted with HbCOI1 in a coronatine (COR)-dependent manner (Fig. 2C, Supplementary Fig. S6D). It also interacted with HbMYC2 in a COR-independent manner and inhibited the HbMYC2-activated transcription of HbFPS1 and HbSRPP1. The cascade of HbCOI1–HbJAZ3–HbMYC2–HbFPS1/HbSRPP1 in laticifer cells directly links jasmonates with the regulation of rubber biosynthesis. FPS has been identified as the key rate-limiting enzyme, and this is the first time that its encoding gene is identified as the regulating target for MYC2-type transcriptional factor in the conserved MVA pathway. This finding increases our understanding of jasmonate signalling in the regulation of isoprenoid metabolism in plants. The HbFPS1 and HbSRPP1 transcripts are the most abundant in the laticifer cells of rubber tree among the FPS and SRPP gene families, respectively (Guo et al., 2015a; Tang et al., 2016), suggesting that they have significant and indispensable roles in rubber synthesis. HbFPS1 is essential for incorporating two IPP molecules and one DMAPP molecule into one FPP molecule, an initial primer for initiating rubber biosynthesis (Cornish and Siler, 1999). It shares 91% similarity and 83% identity with TkFPS1 from Taraxacum kok-saghyz (Supplementary Fig. S8). Over-expression of TkFPS1 results in a maximum increase of 7.48% or a mean increase of 3.92% in rubber content in transgenic plants (Cao et al., 2016). HbSRPP1 plays a positive role in rubber biosynthesis (Oh et al., 1999; Yamashita et al., 2016; Brown et al., 2017). Its orthologs also play vital roles in rubber biosynthesis in T. kok-saghyz (Collins-Silva et al., 2012) and T. brevicorniculatum (Hillebrand et al., 2012), although silencing the homolog of HbSRPP1 has little effect on rubber biosynthesis in Lactuca sativa (Chakrabarty et al., 2015). The increased levels of HbFPS1 and HbSRPP1 (Fig. 6) may be partly ascribed the enhanced biosynthesis of rubber in the regularly tapped trees compared with the virgin trees. HbJAZ3-mediated repression of HbMYC2-activated expression of both HbFPS1 and HbSRPP1 suggests possible potential for transgenic improvement of yields from rubber tree as silencing its ortholog, SmJAZ3, resulted in accumulation of tanshinone in S. miltiorrhiza (Shi et al., 2016a). It has long been believed that rubber biosynthesis in rubber tree is positively regulated by ethylene signalling since application of ethrel significantly raises the yield per tapping. This effect is primarily due to prolonged duration of latex flow (Coupé and Chrestin, 1989) in addition to enhanced sucrose allocation (Tang et al., 2010), water transportation (Tungngoen et al., 2009), glycolysis, and C3 carbon fixation (Liu et al., 2016). By contrast, ethylene signalling does not have a major role in activating rubber biosynthesis per se as ethylene application has little effect on up-regulating the genes related to IPP biosynthesis and IPP integration into rubber (Oh et al., 1999; Zhu and Zhang, 2009; Liu et al., 2016). It has little effect on, or even decreases, the abundance of REF and SRPP proteins (Tong et al., 2017). In the present study, we provide evidence that ethylene inhibits rubber biosynthesis (Fig. 1E); however, the antagonistic effect between JA signalling and ethylene signalling on rubber biosynthesis remains to be elucidated. Although the EIN3–MYC2 complex mediates the crosstalk between ethylene signalling and JA signalling in resistance reactions in Arabidopsis (Song et al., 2014), HbEIN3 (KR013139) preferentially expressed by the latex (Yang et al., 2015) had no apparent influence on the HbMYC2-activated transcription of HbFPS1 and HbSRPP1 (Fig. 7B, C), indicating that the involved in regulation of rubber biosynthesis may not be associated with transcriptional regulation of the HbFPS1 and HbSRPP1 genes. Alternatively, the transcriptional regulation of the HRT2 gene may be associated with the crosstalk because HbEIN3 could bind to the promoter and regulate the expression of this gene (Yang et al., 2015). Although HbMYC2 could not bind the promoter of HRT2 (data not shown), the involvement of other transcription factors that may be associated with JA signalling cannot be excluded (Guo et al., 2018). Just as WD-repeat/bHLH/MYB transcriptional complexes are essential for JA-regulated anthocyanin biosynthesis in Arabidopsis (Qi et al., 2011), the rubber biosynthesis-related genes may be regulated by multiple transcriptional factors. In addition to positive regulation by HbMYC2 in the present study, HbSRPP1 is also negatively regulated by HbWRKY1 (Wang et al., 2013) and HbMADS4 (Li et al., 2016). Ethylene up-regulates HbWRKY1 expression and down-regulates HbSRPP1 expression, while methyl jasmonate significantly up-regulates HbSRPP1 and has little effect on the expression of HbWRKY1 (Wang et al., 2013). Both ethylene and methyl jasmonate up-regulate the expression of HbMADS4 (Li et al., 2016). HMGR is the key rate-limiting enzyme in the MVA pathway (Goldstein and Brown, 1990), and in laticifer cells the transcripts of HbHMGR1 are the most abundant among the five HMGR gene members (Tang et al., 2016). HbHMGR1 is positively regulated by HbCZF1, a CCCH-type zinc finger protein (Guo et al., 2015b). HbCZF1 is significantly up-regulated by methyl jasmonate, but its expression is little influenced by ethylene (Guo et al., 2015b). In summary, the dramatic changes in the turgor pressure of laticifer cells after latex extraction result in a burst of endogenous JA. The increased JA up-regulates the rubber biosynthesis-related genes HbFPS1 and HbSRPP1 by the degradation of the repressor HbJAZ3 via 26S proteasome and the release of HbMYC2. The up-regulation of HbSRPP1 and HbFPS1 results in an increase in the HbSRPP1 and especially HbFPS1 proteins. The increased levels of HbSRPP1 and HbFPS1 are partially responsible for the increased efficiency of in vitro rubber biosynthesis. In addition to HbMYC2, other transcriptional factors such as HbCZF1, HbWRKY1, and HbMADS4 may integrate into the JA signalling and mediate the biosynthesis of rubber that is activated in the laticifer cells of rubber trees as a result of latex harvesting. Supplementary data Supplementary data are available at JXB online. Fig. S1. Analysis of expression of rubber tree MEP pathway genes. Fig. S2. Relative expression of HbJAZ3 and HbMYC2 genes in the bark and latex of the laticifer cells of rubber tree. Fig. S3. HbJAZ3 protein analysis. Fig. S4. HbMYC2 protein analysis. Fig. S5. Phylogenetic tree analysis of the reported MYC-encoding genes of rubber trees. Fig. S6. Interaction of HbMYC2 or HbCOI1 with the latex-expressed JAZ-encoding genes as shown by yeast two-hybrid assays. Supplementary Fig. S7. BiFC assay in the onion epidemic cells. Supplementary Fig. S8. Amino acid comparison between HbFPS1 and TkFPS1. Supplementary Table S1. Primers used in this study. Supplementary Table S2. The predicted regulatory elements present in the promoters of HbFPS1 and HbSRPP1. Acknowledgements We thank Prof. Jorg Kudla for providing vectors for the BiFC assays. We also thank Prof. Lei Zhang for technical guidance in protein expression in E. coli. We sincerely thank Prof. Bingzhong Hao and Prof. Jinglian Wu for giving useful advice during the writing of this manuscript. We also sincerely thank PhD student Chen Liu for technical discussions and guidance for the yeast-one hybrid experiment. This work was financially supported by grants from the National Natural Science Foundation of China (30872002; 31770122), the Earmarked Fund for Modern Agro-Industry Technology Research System (CARS-34-GW1), and Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (1630022015010; 1630022018018). References Adiwilaga K , Kush A . 1996 . 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For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

Jasmonate signalling in the regulation of rubber biosynthesis in laticifer cells of rubber tree, Hevea brasiliensis

Journal of Experimental Botany , Volume Advance Article (15) – May 3, 2018

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0022-0957
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1460-2431
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10.1093/jxb/ery169
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Abstract

Abstract Rubber trees are the world’s major source of natural rubber. Rubber-containing latex is obtained from the laticifer cells of the rubber tree (Hevea brasiliensis) via regular tapping. Rubber biosynthesis is a typical isoprenoid metabolic process in the laticifer cells; however, little is known about the positive feedback regulation caused by the loss of latex that occurs through tapping. In this study, we demonstrate the crucial role of jasmonate signalling in this feedback regulation. The endogenous levels of jasmonate, the expression levels of rubber biosynthesis-related genes, and the efficiency of in vitro rubber biosynthesis were found to be significantly higher in laticifer cells of regularly tapped trees than those of virgin (i.e. untapped) trees. Application of methyl jasmonate had similar effects to latex harvesting in up-regulating the rubber biosynthesis-related genes and enhancing rubber biosynthesis. The specific jasmonate signalling module in laticifer cells was identified as COI1–JAZ3–MYC2. Its activation was associated with enhanced rubber biosynthesis via up-regulation of the expression of a farnesyl pyrophosphate synthase gene and a small rubber particle protein gene. The increase in the corresponding proteins, especially that of farnesyl pyrophosphate synthase, probably contributes to the increased efficiency of rubber biosynthesis. To our knowledge, this is the first study to reveal a jasmonate signalling pathway in the regulation of rubber biosynthesis in laticifer cells. The identification of the specific jasmonate signalling module in the laticifer cells of the rubber tree may provide a basis for genetic improvement of rubber yield potential. Hevea brasiliensis, isoprenoid metabolism, jasmonate signalling, laticifer cell, rubber biosynthesis, secondary metabolism Introduction Major progress in elucidating the jasmonate signalling pathway in model plants has been achieved. This includes the characterization of endogenous bioactive jasmonate as (&#x002B;)-7-iso-jasmonoyl-L-isoleucine (Ile-JA) (Fonseca et al., 2009b) and its receptor as COI1 (Katsir et al., 2008; Mach, 2009; Yan et al., 2009), the identification of the COI1–JAZ co-receptor (Sheard et al., 2010), COI1 as a component of an SCF (Skp/Cullin/F-box) E3 ubiquitin ligase (Devoto et al., 2002; Xu et al., 2002), its targets as jasmonate ZIM-domain (JAZ) proteins (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007), and MYC2 as a crucial transcription factor for activating many jasmonate-responsive genes (Lorenzo et al., 2004; Dombrecht et al., 2007). These studies have demonstrated the importance of the COI1–JAZ–MYC2 core module in jasmonate signalling (Fonseca et al., 2009a). Recently, Thireault et al. (2015) reported repression of jasmonate signalling by non-TIFY JAZ proteins, and other transcription factors have been shown to positively or negatively regulate jasmonate responses (Fonseca et al., 2014; Sasaki-Sekimoto et al., 2014; Zhou and Memelink, 2016). The core module COI1–JAZ–MYC2 is conserved in plants (Thines et al., 2007; Shoji et al., 2008) and mediates activation of the biosynthesis of several secondary metabolites, such as nicotine in tobacco (Shoji and Hashimoto, 2011), tanshinone in Salvia miltiorrhiza (Shi et al., 2016a), and anthocyanin in Arabidopsis (Niu et al., 2011; Qi et al., 2011; Xie et al., 2016) and apple (Chen et al., 2017). However, little is known about this core signalling module in tissues that are specific for secondary metabolite biosynthesis. The rubber tree (Hevea brasiliensis) is the major source of natural rubber worldwide. Rubber biosynthesis is a typical isoprenoid metabolic process and occurs in the laticifer cells, a specific tissue for biosynthesis and storage in the rubber tree (Hao and Wu, 2000). Rubber biosynthesis consists of the general mevalonate (MVA) pathway that converts pyruvic acid into isopentenyl pyrophosphate (IPP) and the 2C-methyl-d-erythritol 4-phosphate (MEP) pathway that converts pyruvic acid and glycerate 3-phosphate into IPP. The specific integration of IPP units into a prenyl chain occurs with the aid of initiators, such as farnesyl diphosphate (FPP) (Adiwilaga and Kush, 1996; Cornish and Siler, 1999). Critical reactions are those catalysed by hydroxymethylglutaryl-CoA reductase (HMGR), 1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), farnesyl pyrophosphate synthase (FPS), and Hevea rubber transferase (HRT), and the small rubber particle protein (SRPP) and rubber elongation factor (REF) are also important; these steps are involved in the synthesis of MVA, MEP, and FPP, and in the integration of IPP units into the prenyl chain, which determines the efficiency of rubber biosynthesis (Oh et al., 1999). The cells that are directly related to rubber production are the secondary laticifers in the bark of the trunk. The secondary laticifer is a single-cell type tissue and consists of cells that are differentiated from the fusiform initials of the vascular cambia in the rubber tree (Hao and Wu, 2000). For rubber production, latex is harvested by tapping, i.e. cutting the bark of the trunk, which is generally done every 3 d. Natural latex is the cytoplasm of laticifer cells, where the rubber is synthesized and stored, and contains numerous rubber particles. Although it is well known that latex harvesting enhances latex regeneration, including rubber biosynthesis in the laticifer cells (Paardekooper, 1989), little is known about the positive feedback regulation of biosynthesis. Ethylene appears to be a key regulator given that application of ethrel (an ethylene releaser) increases rubber yield per tapping (Kush, 1994). The available data indicate that this ethylene-induced increase is primarily due to a prolonged duration of latex flow (Coupé and Chrestin, 1989) in addition to up-regulation of genes related to sucrose allocation (Tang et al., 2010), water transportation (Tungngoen et al., 2009), glycolysis, and C3 carbon fixation (Liu et al., 2016). These effects are directly related to energy regeneration, maintenance of turgor pressure, and supply of carbon building blocks, which are required for the biosynthesis of rubber and other organic metabolites in the laticifer cells. Jasmonate signalling has also been suggested as providing an alternative means of control for the positive feedback regulation of rubber biosynthesis per se based on its pivotal roles in activating biosynthesis of secondary metabolites (Peng et al., 2009; Tian et al., 2010; Zhao et al., 2011). Although orthologs of the COI1, MYC, and JAZ genes in Arabidopsis have been cloned and characterized from laticifer cells in the rubber tree (Peng et al., 2009; Tian et al., 2010; Zhao et al., 2011; Pirrello et al., 2014; Hong et al., 2015), there is still a lack of direct evidence to show the presence of the core module of jasmonate signalling and its regulatory involvement in rubber biosynthesis. In the present study, we examined whether the COI1–JAZ–MYC2 signalling pathway is present in laticifer cells and is used to regulate rubber biosynthesis. We observed that latex harvesting raised the level of endogenous jasmonates, up-regulated most of rubber-biosynthesis genes tested, and enhanced rubber biosynthesis in the laticifer cells. Application of methyl jasmonate also had similar effects to latex harvesting. We further identified an HbCOI1–HbJAZ3–HbMYC2 module in laticifer cells and demonstrated that it mediated the latex harvesting-induced up-regulation of the rubber biosynthesis-related genes HbFPS1 and HbSRPP1. The resulting increases in the levels of HbSRPP1 and especially HbFPS1 should contribute to the increased efficiency of rubber biosynthesis. Materials and methods Plant materials and treatments Eight-year-old virgin (i.e. untapped) and tapped trees as well as the epicormic shoots described by Hao and Wu (2000) of the rubber tree (Hevea brasiliensis) clone CATAS7-33–97 were grown at the Experimental Farm of the Chinese Academy of Tropical Agricultural Sciences in Danzhou City, Hainan Province, China. The tapped trees had been 7-year-old virgins before being regularly tapped every 3 d for 1 year in a downward half spiral without ethylene stimulation. After the tapped trees have been harvested 60 times, a total of 30 virgin trees and 30 tapped trees with the same circumference were selected for determination of the content of endogenous jasmonates, the efficiency of rubber biosynthesis, and rubber biosynthesis-related gene expression in latex collected 2 d after the last tapping. Latex samples collected from batches of 10 trees were pooled into single samples, i.e. there were three biological replicates each for virgin and tapped trees. All the 30 tapped trees were then rested from tapping for 15 d, after which nine were treated with 0.07% methyl jasmonate, nine with 0.5% ethrel, and nine were designated as untreated controls. Latex samples were collected 1 d after the treatments. For determination of in vitro rubber biosynthesis, latex samples from batches of three trees for each treatment were pooled; a total of nine trees were sampled, forming three replicates. To investigate the effects of jasmonate (JA) on rubber biosynthesis-related gene expression, 120 epicormic shoots were treated with 0.07% methyl jasmonate as described by Hao and Wu (2000) and a further 120 epicormic shoots without any treatment were used as the control. Latex samples from the treated shoots were collected by cutting the stem bark of the first and second extension units (Hao and Wu, 2000; Zhang et al., 2015) at 4 h, 12 h, 1 d, and 5 d after the treatment. At each time interval, latex was collected from 30 epicormic shoots and batches of 10 samples were pooled to form three replicates. Samples from the control shoots were similarly collected and pooled. Tobacco (Nicotiana benthamiana) was sown and grown at 25 °C with a 16/8 h light/dark photoperiod in an illuminated incubator for approximately 6 weeks prior to being used for BiFC and transient expression analysis. Sequence analysis and phylogenetic tree construction The deduced amino acid sequences of the HbMYC2 and HbJAZ3 proteins were aligned with other homologous proteins using the Clustal X 2.05 program (http://www.clustal.org/clustal2/) and then edited using the Genedoc 2.7 software (http://iubio.bio.indiana.edu/soft/molbio/ibmpc/genedoc-readme.html). Phylogenetic tree construction was performed using Clustal X 2.05 and MEGA 4.0 software (https://www.megasoftware.net/mega4/). Transcriptional activation analysis in yeast cells The full-length coding region and different truncated derivatives of HbMYC2 were separately cloned into the bait vector pGBKT7 (see Supplementary Table S1) and transformed into the yeast strain Y2HGold for transcriptional activation according to the manufacturer’s protocol (Clontech, USA). The transformed strains were then streaked onto SD/–Trp or SD/–Ade–Trp–His plates, and transcriptional activation was determined according to colony growth and the blue coloration after the α-galactosidase reaction on the defective medium (SD/–Ade–Trp–His/X-α-gal). Subcellular localization and BiFC assays For determination of subcellular localization, the coding regions of HbMYC2 and HbJAZ3 were separately cloned into the pCambia1302 vector and fused with the enhanced green fluorescent protein (EGFP) gene. This recombinant plasmid and control GFP plasmids were introduced into Agrobacterium tumefaciens GV3101 and infiltrated into onion epidermal cells for fluorescence detection according to the method described by Li et al. (2016). For bimolecular fluorescence complementation (BiFC) assays in tobacco leaves and onion epidemic cells, the coding sequence of HbJAZ3 was cloned into pSPYNE(R)173 described by Waadt et al. (2008). HbMYC2 as well as HblMYC1 and HblMYC2 were cloned into pSPYCE(MR). The human lamin C gene from the pGBKT7-Lam vector was cloned and inserted into pSPYNE(R)173 for the negative control, while the murine p53 gene from the pGBKT7-53 vector was cloned and inserted into the pSPYNE(R)173 for the positive control. The SV40 large T-antigen gene was cloned from the pGADT7-T vector and inserted into pSPYCE(MR) for both the negative and positive controls. The corresponding combined plasmids were introduced into A. tumefaciens strain GV3101 and confirmed by RT-PCR with specific primers (Supplementary Table S1). The BiFC experiments were designed according to Kudla and Bock (2016) and also performed in onion epidemic cells according to the methods described previously by Li et al. (2016). The pSPYNE(R)173-HbJAZ3 and pSPYCE(MR)-HbMYC2 constructs were separately introduced into A. tumefaciens GV3101 and co-infiltrated in combination with pSPYNE(R)173-HbJAZ3 and pSPYCE(MR)-HbMYC2 at an optical density of 0.6–0.7 at 600 nm. Negative controls without HbJAZ3 or HbMYC2 were performed in the same way. After cultivation for 2–4 d, the transformed tobacco leaves were cut approximately into squares and put into a DAPI solution (10 μg ml–1; (Solarbio, Beijing, China) within 15 min and then washed with ddH2O at least twice. The fluorescence of yellow fluorescent protein (YFP) and DAPI in the transformed tobacco leaves and onion epidemic cells were recorded using confocal microscopy. Yeast one/two-hybrid assays For yeast one-hybrid assays, the HbFPS1 promoter and HbSRPP1 promoter were cloned into the pHIS2.1 vector (Clontech), generating pHIS-pHbFPS1 and pHIS-pHbSRPP1, respectively (Wang et al., 2013; Guo et al., 2014). The yeast-one library was constructed according to the manufacturer’s instructions (Clontech), as described previously (Wang et al., 2013; Guo et al., 2014). The yeast-one library strain Y187 was transfected with the bait vector pHIS-pHbFPS1 and then cultivated for 3 d at 30 °C on SD/–His/–Leu/–Trp medium supplemented with 20 mM 3-AT for the selection of transformants. For confirmation of the HbFPS1 or HbSRPP1 promoter-binding proteins, HbMYC2 was cloned into pGADT7-Rec2. The pGADT7-HbMYC2 and pHIS-pHbFPS1 or pHIS-pHbSRPP1 vector was co-transformed into the yeast strain Y187. pGADT7-Rec53 and p53-His were used as positive controls (CK&#x002B;) and p53-His as a negative control (CK–). Transformed clones were grown on SD/–His/–Leu/–Trp medium with 20 mM (for pHbFPS1) or 42.5 mM 3-AT (for pHbSRPP1) for 3 d at 30 °C. For yeast two-hybrid assays, the full-length coding region of HbMYC2 was cloned into the pGADT7 vector (Clontech) with specific primers (Supplementary Table S1). HbJAZ3 and HbCOI1 were respectively cloned into the pGADT7 and pGBKT7 vectors. HbJAZ8.0a, HbJAZ8.0c, and HbJAZ10.0a were each cloned into the pGBKT7 vector. Detection of protein–protein interactions between HbMYC2 and JAZs or JAZs and HbCOI1 was performed using SD/–His/–Leu/–Trp–Ade/X-α-gal medium (DDO/X medium, –4&#x002B;X) or SD/–His/–Leu/–Trp–Ade/X-α-gal (–4&#x002B;X) medium containing 0 μM or 50 μM coronatine (COR). Electrophoretic mobility shift assays The coding sequence of HbMYC2 was cloned into the pET-30a vector fused with the His tag. The recombinant His-HbMYC2 protein was expressed and affinity-purified from E. coli (BL21[DE3]) using Ni&#x002B; affinity resin (GE). The EMSA was performed with an Electrophoretic Mobility Shift Assay kit (Invitrogen, USA) following the manufacturer’s instructions, as described previously (Wang et al., 2013; Guo et al., 2014). Transient dual-luciferase assays The coding regions of HbMYC2 and HbJAZ3 were respectively cloned into the effector vectors pGreenII-62SK and pCAMBIA2301 (CAMBIA). The empty vector pGreenII-62SK was used as a negative control. The inhibitory effect of HbJAZ3 on the activity of HbMYC2 with regards to the transcription of HbFPS1 and HbSRPP1 was determined by transient dual-luciferase assays. The reporter vectors (pGreen II-0800-LUC containing pHbFPS1 or pHbSRPP1) and the effector vector (p35S::HbMYC2) were transferred into A. tumefaciens (strain GV3101). Overnight cultures of Agrobacterium were collected by centrifugation, resuspended in the infiltration buffer (10 mM MES, 150 mM acetosyringone, and 10 mM MgCl2), and incubated at room temperature for 4 h before infiltration. The strain containing the reporter was used alone or mixed with the strains harboring the effectors or the empty vector control. An Agrobacterium suspension in a 5-ml syringe was carefully press-infiltrated manually onto healthy leaves of 6-week-old N. benthamiana. After culture for 3 d, the infected area was harvested for total protein extraction. The supernatant of total proteins was treated with the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions, and the fluorescent values of LUC and REN were detected using a GloMaxR-Multi&#x002B;Detection System (Promega). The LUC value was normalized to that of REN. Three biological repeats were measured for each combination. qRT-PCR Latex samples from plants subjected to the treatments described above were used for quantitative real-time PCR analysis of the expression levels of relevant genes. Total RNAs were extracted using a Total plant RNA isolation kit (Tiangen, China) and reverse-transcribed using M-MLV reverse transcriptase (Fermentas, USA). Quantitative real-time PCR (qRT-PCR) was performed using the CFX384 real-time PCR system (Bio-Rad, USA) with the SYBR Prime Script RT-PCR kit (TaKaRa, Japan). Relative gene expression levels were calculated using the 18S-rRNA gene as the internal normalization control. The qRT-PCR experiments were performed with each sample measured in triplicate. Primers used for real-time PCR analysis are listed in Supplementary Table S1). JA determination The extraction and quantification of JA from the latex samples were conducted according to Tian et al. (2015) with modifications in sample preparation. Approximately 3 g of each latex sample was dripped into 20 ml of 80% methanol containing 0.01% (w/v) sodium diethyldithiocarbamate as an antioxidant. [9,10-2H2]Dihydro-JA (250 ng) was added as an internal standard for JA determination. The admixture was stirred overnight at 4 °C and then centrifuged. After the methanol phase was collected, the rubber deposit was extracted twice with 20 ml of cold (4 °C) 80% methanol. The organic phase was combined, adjusted to pH 8.0 with ammonia, condensed to the aqueous phase (approximately 5 ml) in a rotary film evaporator (Heidolph Laborota 4000 efficient, Germany), and frozen at –20 °C, followed by three rounds of melting and thawing. After centrifugation at 270 g for 20 min, 1 mg of polyvinylpolypyrrolidone was added to the collected supernatant, and it was homogenized at 4.5 m s–1 for 10 s and filtered. The filtrate was collected, adjusted to pH 2.5–3.0 with 2 M acetic acid, and then extracted three times by mixing the filtrate with 5 ml of ethyl acetate. The organic layers were combined, adjusted to pH 8 with ammonia and evaporated to dryness in a rotary film evaporator. The dried extracts were dissolved in 5 ml of 0.1 M acetic acid and passed through a C18 column (Sep-Pak® Classic C18, Part No. WAT051910, Waters Corporation, Milford, MA, USA) that had been pre-washed with 5 ml 100% methanol and pre-equilibrated with 5 ml 0.1 M acetic acid. The column was eluted with 5 ml of 17% (v/v) acid methanol (100% methanol:0.1 M acetic acid, 17:83), followed in turn by 5 ml of 60% acid methanol (100% methanol:0.1 M acetic acid, 60:40) for JA extraction. JA fractions were collected and evaporated to dryness (40 °C) after being adjusted to pH 8 with ammonia. Methyl esterification was carried out by dissolving the residue in 0.5 ml of methanol and adding 3 ml of ethereal diazomethane. The excess diazomethane was removed under a stream of oxygen-free nitrogen gas. The methylated samples were re-dissolved in ethyl acetate for analysis by GC-MS as described by Tian et al, (2015). All analyses were performed with three biological replicates. In vitro rubber biosynthesis assay The reaction system was as described by Archer et al. (1963) with modifications as follows. A sample of 25 μl fresh latex was added into a 400-μl reaction system containing 10 μM MgCl2, 10 μM DTT, 5 μM ATP, and 0.736 μM 13C-MVA (mevalonolactone-2-13C) (Sigma Aldrich, Lot# MBBB5201 V) in 0.1 M PBS buffer (pH 7.8). The reaction was carried out at 30 °C for 8 h with constant shaking (70 rpm). Thereafter, the rubber was extracted according to the method described by Rattanapittayaporn et al. (2004). A total volume of 400 μl saturated NaCl solution was added into the reaction solution and mixed thoroughly, an equal volume (800 μl) of extractant (toluene/hexane, 1:1, v/v) was added to the mixture and subjected to centrifugation at 16 000 g for 30 min. The upper organic phase was collected and mixed with equal volume of 1-butanol to dissolve out the rubber overnight. The organic phase was removed after centrifugation at 16 000 g for 30 min, and the remaining rubber was dried. Approximately 0.5 mg of dried rubber sample was embedded into a tin capsule (Elemental Microanalysis, 6 × 4 mm, BN261080) and the 13C/12C ratio was determined using a stable isotope mass spectrometer (GV Instruments, IsoPrime JB312) and an elemental analyser (Thermo, FLASH EA1112 Series). The results were expressed as values of atom percentage (APC), equal to the percentage of the content of 13C relative to the total carbon (13C &#x002B;12C) content in the rubber sample. All analyses were performed with three biological replicates. Latex collection for western blot assays Latex from each of 30 virgin rubber trees and 30 tapped trees were collected within 5 min after tapping. Then equal volumes of samples that were collected from 10 virgin trees were pooled to give a single sample for western blot assays. The samples from the tapped trees were pooled in the same manner. In this way, three biological replicates were conducted. Then an equal volume of the isotonic solution used in the in vitro rubber biosynthesis efficiency assay was added to the pooled samples and mixed gently. The pooled samples were stored on ice for subsequent use. The latex samples were centrifuged at 4 °C at 16 000 g for 10 min. The middle layer of the suspension liquid was filtered through a 0.45-μm membrane, and the filtrate was collected. SDS-PAGE and western blots were performed as described by Towbin et al. (1979) and Shi et al. (2016b) with the following modifications. The loading amounts were 20 μl and 6 μl for HbFPS1 and HbSRPP1 protein detection, respectively. FPS and SRPP proteins were recognized by specific polyclonal antibodies (1:1000 dilution for anti-FPS1, 1:2500 dilution for anti-SRPP1) and were identified using an Enhanced Chemiluminescence (ECL) Kit and a BCIP/NBT Kit (Pierce, USA), respectively. Statistical analysis Statistical analysis was carried out by ANOVA using SPSS Statistics 17.0, and statistical significance was evaluated using Student’s t-test (two-group comparisons between treated samples and control samples) as described previously (Wang et al., 2013; Guo et al., 2014; Li et al., 2016). Results Increase in the level of endogenous jasmonates induced by removal of latex, up-regulation of rubber biosynthesis genes, and enhancement of rubber biosynthesis The levels of endogenous jasmonates in the latex from virgin trees and regularly tapped trees were determined by GC-MS. Levels in the regularly tapped trees were approximately six times that of virgin trees (Fig. 1A). The levels of expression of rubber biosynthesis-related genes in the latex were also different between the virgin trees and the regularly tapped trees. Most of the genes in the MVA pathway (Fig. 1B), including HbHMGR1, HbMVK1, HbPMK1, HbMVD1, HbMVD2, HbIPPI1, HbFPS1, HbREF1, HbCPT6 (also designated as HbHRT2), HbCPT7, and HbCPT8 (HbHRT1), were significantly up-regulated in regularly tapped trees in comparison with virgin trees (Fig. 1C). Most of the up-regulated genes in the regularly tapped trees were also up-regulated in the latex from epicormic shoots at 12 h or 1 d after being treated with methyl jasmonate (Fig. 1D). HbCMK, HbMCS1/HbMDS1, and HbMCS2/HbMDS2 in the MEP pathway (Supplementary Fig. S1A) were significantly up-regulated, while HbDXS1, HbDXS2, HbDXR, HbCMS1/HbMCT1, HbCMS2/HbMCT2, and HbHDR were moderately up-regulated in the regularly tapped trees in comparison with the virgin trees (Supplementary Fig. S1B). These genes were also significantly up-regulated in epicormic shoots at 1 d after being treated with 0.07% methyl jasmonate (Supplementary Fig. S1C). Fig. 1. View largeDownload slide (A) Effect of tapping on endogenous jasmonate (MeJA) levels in laticifer cells. (B) Rubber biosynthesis-related genes of the MVA pathway that were examined. Effect of tapping of trees (C) and application of methyl jasmonate to epicormic shoots (D) on the expression of rubber biosynthesis-related genes. (E) Determination of the efficiency of in vitro rubber biosynthesis in the latex from the virgin trees, regularly tapped trees, trees rested from tapping for 15 d without any treatment (rested), rested trees treated with ethrel (ET), and rested trees treated with methyl jasmonate (JA). Data are means (±SD) of three biological replicates and are expressed as atom percentage of 13C (APC; see Methods). Significant differences were determined using Student’s t-test; *P<0.05, **P<0.01. Abbreviations: HMGR, 3-hydroxy-3-methylglutaryl CoA reductase; MK, mevalonate kinase; PMK, phosphomevalonate kinase; MVD, mevalonate diphosphate decarboxylase; IPPI, isopentenyl diphosphate isomerase; FPS, farnesyl diphosphate synthesis; CPT, cis-prenyltransferase; HRT, Hevea rubber transferase; SRPP, small rubber particle protein; REF, rubber elongation factor. Fig. 1. View largeDownload slide (A) Effect of tapping on endogenous jasmonate (MeJA) levels in laticifer cells. (B) Rubber biosynthesis-related genes of the MVA pathway that were examined. Effect of tapping of trees (C) and application of methyl jasmonate to epicormic shoots (D) on the expression of rubber biosynthesis-related genes. (E) Determination of the efficiency of in vitro rubber biosynthesis in the latex from the virgin trees, regularly tapped trees, trees rested from tapping for 15 d without any treatment (rested), rested trees treated with ethrel (ET), and rested trees treated with methyl jasmonate (JA). Data are means (±SD) of three biological replicates and are expressed as atom percentage of 13C (APC; see Methods). Significant differences were determined using Student’s t-test; *P<0.05, **P<0.01. Abbreviations: HMGR, 3-hydroxy-3-methylglutaryl CoA reductase; MK, mevalonate kinase; PMK, phosphomevalonate kinase; MVD, mevalonate diphosphate decarboxylase; IPPI, isopentenyl diphosphate isomerase; FPS, farnesyl diphosphate synthesis; CPT, cis-prenyltransferase; HRT, Hevea rubber transferase; SRPP, small rubber particle protein; REF, rubber elongation factor. The efficiency of in vitro rubber biosynthesis in the regularly tapped trees was significantly higher than that in virgin trees that had never been tapped (Fig. 1E). It decreased significantly when the regularly tapped trees were rested from tapping for 15 d (Fig. 1E). It further decreased significantly 1 d after the rested trees were treated with 0.5% ethrel (an ethylene releaser), but treatment with 0.07% methyl jasmonate instead caused a significant increase 1 d after application (Fig. 1E). Identification of the HbCOI1–HbJAZ3–MYC2 module in laticifer cells To examine jasmonate signalling in the laticifer cells of rubber trees, a gene homologous to AtJAZ3 and SmJAZ3 was cloned and characterized. It was designated as HbJAZ3 (GenBank No. KJ911911) and had preferential expression in the latex compared to the bark tissues (Supplementary Fig. S2A). The ORF of this gene was 1164 bp in length and encoded a predicted protein of 387 amino acids with a molecular weight of 41.0 kDa and a pI of 8.77. HbJAZ3 possesses a ZIM domain and a Jas domain that are conserved in the JAZ proteins from other plant species (Supplementary Fig. S3). It was clustered closely with AtJAZ3 and SmJAZ3 in the phylogenetic tree (Fig. 2A). Subcellular localization analysis showed that the green fluorescence from HbJAZ3-GFP was only detected in the nuclei, which was confirmed by DAPI staining (Fig. 2B). Using yeast two-hybrid assays, we determined that yeast cells harboring HbJAZ3 and HbCOI1 could survive in the selective medium when it contained 50 μM COR (Fig. 2C, Supplementary Fig. S6), suggesting that HbJAZ3 was the target of SCFCOI1 in response to a burst of endogenous jasmonates. Fig. 2. View largeDownload slide HbJAZ3 identification. (A) A phylogenetic tree was constructed by using the neighbor-joining method with 100 replications after ClustalX2 alignment analysis of HbJAZ3 with other JAZ proteins including AtJAZ1 (NP_564075), AtJAZ2 (Q9S7M2), AtJAZ3 (AEE76017), AtJAZ4 (NP_001117450), AtJAZ5 (ANM58475), AtJAZ6 (NP_565043), AtJAZ7 (AEC08997), AtJAZ8 (NP_564349), AtJAZ9 (Q8W4J8), AtJAZ10 (NP_974775), AtJAZ11 (NP_001190007), AaJAZ1 (AJK93412), AaJAZ2 (AJK93413), AaJAZ3 (AJK93414), AaJAZ4 (AJK93415), SmJAZ1 (AGC73980), SmJAZ3 (AHK23660), SmJAZ9 (protein sequences in Shi et al., 2016a), NtJAZ1 (BAG68655), NtJAZ2 (BAG68656), NtJAZ3 (BAG68657), HbJAZ1 (ADI39634), HbJAZ2 (AIY25007), HbJAZ3 (KJ911911), HbJAZ7 (AIY2500), HbJAZ8 (AIY25009), HbJAZ9 (AIY25010), HbJAZ10 (AIY25011), and HbJAZ11 (AIY25012). The scale bar indicates the evolutionary distance. (B) Subcellular localization of HbJAZ3 in onion epidermal cells. The nuclei of the onion epidermal cells were visualized by DAPI staining. GFP, green fluorescent protein; Merge, merged image. (C) Interaction of HbCOI1 with HbJAZ3 in yeast cells in the presence of 50 µM coronatine (COR). Fig. 2. View largeDownload slide HbJAZ3 identification. (A) A phylogenetic tree was constructed by using the neighbor-joining method with 100 replications after ClustalX2 alignment analysis of HbJAZ3 with other JAZ proteins including AtJAZ1 (NP_564075), AtJAZ2 (Q9S7M2), AtJAZ3 (AEE76017), AtJAZ4 (NP_001117450), AtJAZ5 (ANM58475), AtJAZ6 (NP_565043), AtJAZ7 (AEC08997), AtJAZ8 (NP_564349), AtJAZ9 (Q8W4J8), AtJAZ10 (NP_974775), AtJAZ11 (NP_001190007), AaJAZ1 (AJK93412), AaJAZ2 (AJK93413), AaJAZ3 (AJK93414), AaJAZ4 (AJK93415), SmJAZ1 (AGC73980), SmJAZ3 (AHK23660), SmJAZ9 (protein sequences in Shi et al., 2016a), NtJAZ1 (BAG68655), NtJAZ2 (BAG68656), NtJAZ3 (BAG68657), HbJAZ1 (ADI39634), HbJAZ2 (AIY25007), HbJAZ3 (KJ911911), HbJAZ7 (AIY2500), HbJAZ8 (AIY25009), HbJAZ9 (AIY25010), HbJAZ10 (AIY25011), and HbJAZ11 (AIY25012). The scale bar indicates the evolutionary distance. (B) Subcellular localization of HbJAZ3 in onion epidermal cells. The nuclei of the onion epidermal cells were visualized by DAPI staining. GFP, green fluorescent protein; Merge, merged image. (C) Interaction of HbCOI1 with HbJAZ3 in yeast cells in the presence of 50 µM coronatine (COR). To identify the downstream partners of HbJAZ3 in jasmonate signalling, we used HbJAZ3 as bait to screen a cDNA library from latex using yeast two-hybrid assays. A positive clone was obtained after several cycles of verification on the selective medium (Fig. 3A). It contained a single plasmid harboring an inserted fragment of 2495 bp in length. The fragment contained an ORF of 2046 bp in length, a 72-bp 5′UTR region and a 377 bp 3′UTR region. The ORF encoded a MYC-type protein of 681 amino acids with a molecular weight of 74.58 kDa and a pI of 5.70. This protein shared high similarity with AtMYC2 according to blastp analysis. Therefore, we designated the fragment as HbMYC2 (GenBank No. KM507201) and it had higher expression level in the latex than that in the bark tissues (Supplementary Fig. S2B). HbMYC2 shared highly conserved motifs with the AtMYC2, NtMYC2a, SmMYC2a, and AaMYC2 transcription factors in an amino acid alignment (Supplementary Fig. S4) and was clustered into the MYC2 ortholog subgroup in the phylogenetic tree (Fig. 3B, Supplementary Fig. S5). As a potential transcription factor, the transcriptional activation of HbMYC2 and its N- and C-terminal regions were assessed in yeast. Only the full-length protein and N-terminal region of residues 1–377 could activate expression of the HIS and MEL1 genes in yeast cells, resulting in survival of the yeast cells in the selective media lacking histidine and adenine (Fig. 3C). Subcellular localization analysis showed that the green fluorescence from HbMYC2-GFP was only present in the nuclei, which was confirmed by DAPI staining (Fig. 3D). Fig. 3. View largeDownload slide HbMYC2 identification. (A) Screening of HbMYC2 by HbJAZ3 using yeast two-hybrid assays. (B) A phylogenetic tree was constructed by using the neighbor-joining method with 100 replications after ClustalX2 alignment of HbMYC2 (AJC01627) and other bHLH proteins, including NtMYC2a (ADU60100), NtMYC2b (ADU60101), SmMYC2a (AIO09733), SlMYC2 (AGZ94899), JcMYC2 (XP_012076236), RcMYC2 (XP_002519814), AtMYC2 (Q39204), AtMYC3 (Q9FIP9), AtMYC4 (O49687), AaMYC2 (AKO62850), MaMYC2a (XP_009384727), MaMYC2b (XP_009413229), TcJAMYC (ACM48567), SmMYC2b (protein sequences in Zhou et al., (2016), AtbHLH13 (Q9 LNJ5), HblMYC1 (ADK56287), and HblMYC2 (ADK91082). The scale bar indicates the evolutionary distance. (C) Activating activity of full-length HbMYC2 and its N-terminal derivative (1–377 aa) for the transcription in yeast cells. The schematic diagram represents for the constructs of the N- and C-terminal of HbMYC2 in the transcriptional activation assay in yeast. (D) Subcellular localization of HbMYC2 in onion epidermal cells. The nuclei of the onion epidermal cells were visualized by DAPI staining. GFP, green fluorescent protein; Merge, merged image. Fig. 3. View largeDownload slide HbMYC2 identification. (A) Screening of HbMYC2 by HbJAZ3 using yeast two-hybrid assays. (B) A phylogenetic tree was constructed by using the neighbor-joining method with 100 replications after ClustalX2 alignment of HbMYC2 (AJC01627) and other bHLH proteins, including NtMYC2a (ADU60100), NtMYC2b (ADU60101), SmMYC2a (AIO09733), SlMYC2 (AGZ94899), JcMYC2 (XP_012076236), RcMYC2 (XP_002519814), AtMYC2 (Q39204), AtMYC3 (Q9FIP9), AtMYC4 (O49687), AaMYC2 (AKO62850), MaMYC2a (XP_009384727), MaMYC2b (XP_009413229), TcJAMYC (ACM48567), SmMYC2b (protein sequences in Zhou et al., (2016), AtbHLH13 (Q9 LNJ5), HblMYC1 (ADK56287), and HblMYC2 (ADK91082). The scale bar indicates the evolutionary distance. (C) Activating activity of full-length HbMYC2 and its N-terminal derivative (1–377 aa) for the transcription in yeast cells. The schematic diagram represents for the constructs of the N- and C-terminal of HbMYC2 in the transcriptional activation assay in yeast. (D) Subcellular localization of HbMYC2 in onion epidermal cells. The nuclei of the onion epidermal cells were visualized by DAPI staining. GFP, green fluorescent protein; Merge, merged image. The interaction between HbJAZ3 and HbMYC2 was further confirmed by yeast two-hybrid assays. Only the yeast cells harboring HbJAZ3 and HbMYC2 and the positive control could grow and exhibit a blue color on QDO/X-α-gal plates, suggesting that HbJAZ3 interacted with HbMYC2 (Fig. 3A). The interaction was further verified in planta by BiFC assays. Tobacco cells (Fig. 4A) or onion epidermal cells (Supplementary Fig. S7) harboring HbJAZ3-cYFP and HbMYC2-nYFP exhibited a bright yellow color in the sites that were specifically stained by DAPI, similar to the YFP signal in the cells harboring the yeast positive control p53 and SV40-T. By contrast, the bright yellow color was not detected in tobacco or onion epidermal cells harboring either HbJAZ3-cYFP or nYFP, HbMYC2-nYFP or cYFP, and HbJAZ3-cYFP and HblMYC1 or HblMYC2, or the yeast negative control Lam and SV40-T, although DAPI staining of the nuclei was observed (Supplementary Fig. S7), suggesting that the bright yellow color was specific and positive. HbCOI1, HbJAZ3, and HbMYC2 were all significantly up-regulated in laticifer cells of the regularly tapped trees compared with those of the virgin trees (Fig. 4B). The expression of these three genes in the epicormic shoots was significantly up-regulated at 12 h or 1 d after being treated with 0.07% methyl jasmonate (Fig. 4C). The positive response of the three genes to JA, the interaction between HbCOI1 and HbJAZ3 in a COR-dependent manner, and between HbJAZ3 and HbMYC2 in a COR-independent manner, demonstrated the presence of a HbCOI1–HbJAZ3–MYC2 module in the laticifer cells of rubber tree. Fig. 4. View largeDownload slide Identification of the HbCOI1–HbJAZ3–HbMCY2 module. (A) Interaction of HbJAZ3 with HbMYC2 in tobacco as shown by bimolecular fluorescence complementation (BiFC) assays. The empty pairs (n-YFP and c-YFP) were used as negative controls. YFP fluorescence was detected under a confocal microscope 3 d after infiltration. Scale bar &#x003D; 20 μm. (B, C) Up-regulated expression of HbCOI1, HbJAZ3, and HbMYC2 upon tapping (B) and treatment with methyl jasmonate (C). Fig. 4. View largeDownload slide Identification of the HbCOI1–HbJAZ3–HbMCY2 module. (A) Interaction of HbJAZ3 with HbMYC2 in tobacco as shown by bimolecular fluorescence complementation (BiFC) assays. The empty pairs (n-YFP and c-YFP) were used as negative controls. YFP fluorescence was detected under a confocal microscope 3 d after infiltration. Scale bar &#x003D; 20 μm. (B, C) Up-regulated expression of HbCOI1, HbJAZ3, and HbMYC2 upon tapping (B) and treatment with methyl jasmonate (C). Effect of HbJAZ3 on the HbMYC2-activated transcription of HbFPS1 and HbSRPP1 To identify possible transcriptional factors involved in regulation of rubber biosynthesis by JA signalling, a yeast one-hybrid screening of the latex cDNA library was performed using the promoter of the key rubber biosynthesis gene HbFPS1. A fragment harbored in a positive clone was identical to HbMYC2. The interaction of the full-length HbMYC2 with the HbFPS1 promoter was revealed by the yeast one-hybrid assay (Fig. 5A). The physical binding of HbMYC2 to the HbFPS1 promoter was further verified by in vitro EMSAs. HbFPS1 promoter fragments were more and more retarded with increased amounts of the purified HbMYC2 protein (Fig. 5B). Fig. 5. View largeDownload slide Transcriptional regulation of HbFPS1 and HbSRPP1. (A) Activation of the HbFPS1 promoter in yeast by the HbMYC2 protein. (B) Demonstration of the physical binding of the HbMYC2 protein to the promoter of HbFPS1 by electrophoretic mobility shift assays (EMSA). (C) Activation of the HbSRPP1promoter in yeast by the HbMYC2 protein. (D) Demonstration of the physical binding of the HbMYC2 protein to the promoter of HbFPS1 by EMSA. (E) Schematic diagram of the constructs for a dual-luciferase assay. (F, G) Assays of the transient transcriptional activity of HbMYC2 on HbFPS1 (F) and HbSRPP1 (G) with or without the expressed HbJAZ3 protein. Data are means (±SD) of three biological replicates. Significant differences were determined using Student’s t-test; **P<0.01. Fig. 5. View largeDownload slide Transcriptional regulation of HbFPS1 and HbSRPP1. (A) Activation of the HbFPS1 promoter in yeast by the HbMYC2 protein. (B) Demonstration of the physical binding of the HbMYC2 protein to the promoter of HbFPS1 by electrophoretic mobility shift assays (EMSA). (C) Activation of the HbSRPP1promoter in yeast by the HbMYC2 protein. (D) Demonstration of the physical binding of the HbMYC2 protein to the promoter of HbFPS1 by EMSA. (E) Schematic diagram of the constructs for a dual-luciferase assay. (F, G) Assays of the transient transcriptional activity of HbMYC2 on HbFPS1 (F) and HbSRPP1 (G) with or without the expressed HbJAZ3 protein. Data are means (±SD) of three biological replicates. Significant differences were determined using Student’s t-test; **P<0.01. Given the presence of a conserved G-box (‘CANNTG’) in the HbSRPP1 promoter (Supplementary Table S2), the potential interaction of this promoter with HbMYC2 was tested by yeast one-hybrid assays. Yeast cells harboring HbMYC2 and the HbSRPP1 promoter survived on the selective medium (Fig. 5C), suggesting that the action did indeed occur. The physical binding of HbMYC2 to the HbSRPP1 promoter was verified by in vitro EMSAs. HbSRPP1 promoter fragments were increasingly retarded with increasing amounts of the purified HbMYC2 protein (Fig. 5D). A dual-luciferase assay system (Fig. 5E) was used to assess the effect of the physical binding of HbMYC2 to either the promoter of HbFPS1 or the promoter of HbSRPP1 as well as the effect of HbJAZ3. Luciferase (LUC) activity in tobacco leaves harboring HbMYC2 and Luc that was driven by either the promoter of HbFPS1 or that of HbSRPP1 was significantly higher than that in leaves harboring Luc alone, and activity was significantly decreased when HbJAZ3 was co-expressed (Fig. 5F, G). There was no obvious difference in the expression of Luc in tobacco leaves in comparison with the empty vector control when the HbJAZ3 gene was expressed alone (Fig. 5F, G). These data demonstrated that both HbFPS1 and HbSRPP1 were positively regulated by HbMYC2, while HbJAZ3 repressed the HbMYC2-activated expression of either HbFPS1 or HbSRPP1. Effect of latex harvesting on the levels of HbFPS1 and HbSRPP1 Given that the expression levels of HbFPS1 and HbSRPP1 were higher in the regularly tapped trees than that in the virgin trees (Fig. 1D), we further examined the differences in the levels of HbFPS1 and HbSRPP1. As HbSRPP1 is more abundant than HbFPS1, different amounts of rubber particle filtrates were loaded for detection of the proteins (HbFPS1 20 μl, HbSRPP1 6 μl) (Fig. 6A). Western blotting analysis revealed that levels of HbFPS1 were significantly higher in the regularly tapped trees as compared with the virgin trees (Fig. 6B, C). Although the SDS-PAGE profiles appeared to show no difference in the levels of HbSRPP1 between the regularly tapped trees and the virgin trees (Fig. 6D), western blots showed that HbSRPP1 levels were also significantly higher in the regularly tapped trees (Fig. 6E, F). These results indicated that the increase in the transcripts was positively correlated with the increase in their corresponding proteins. Fig. 6. View largeDownload slide Changes in the levels of HbFPS1 and HbSRPP1 in the latex from regularly tapped trees and virgin trees. (A, D) SDS-PAGE profiles of latex from regularly tapped trees (T) and virgin trees (V); M, protein standards. In (A) 20 μl latex was loaded per lane, and in (D) 6 μl latex was loaded per lane. (B, E) Western blot analyses with polyclonal antibodies raised against HbFPS1 (B) and HbSRPP1 (E), as indicated by the arrows. (C, F) Relative quantitative analysis of HbFPS1 (C) and HbSRPP1 (F). Data are means (±SD) of three biological replicates. Significant differences were determined using Student’s t-test; *P<0.05, **P<0.01. Fig. 6. View largeDownload slide Changes in the levels of HbFPS1 and HbSRPP1 in the latex from regularly tapped trees and virgin trees. (A, D) SDS-PAGE profiles of latex from regularly tapped trees (T) and virgin trees (V); M, protein standards. In (A) 20 μl latex was loaded per lane, and in (D) 6 μl latex was loaded per lane. (B, E) Western blot analyses with polyclonal antibodies raised against HbFPS1 (B) and HbSRPP1 (E), as indicated by the arrows. (C, F) Relative quantitative analysis of HbFPS1 (C) and HbSRPP1 (F). Data are means (±SD) of three biological replicates. Significant differences were determined using Student’s t-test; *P<0.05, **P<0.01. Effect of HbEIN3 on the HbMYC2-activated transcription of HbFPS1 and HbSRPP1 Given that in vitro rubber biosynthesis was enhanced by exogenous methyl jasmonate and inhibited by application of ethrel (Fig. 1E), the effect of a member of the EIN3 family, HbEIN3, on the HbMYC2-activated transcription of HbFPS1 and HbSRPP1 was assessed using the transient LUC activity detection system (Fig. 7A). HbEIN3 had little influence on the expression of HbFPS1 and HbSRPP1, although ET-responsive elements are present in the promoter sequence of HbFPS1 and HbSRPP1 (Supplementary Table S2). HbEIN3 also had no influence on the HbMYC2-activated expression of HbFPS1 and HbSRPP1 (Fig. 7B, C). Therefore, the mechanism for the antagonistic effect of JA and ET on rubber biosynthesis (Fig. 1E) remains to be elucidated. Fig. 7. View largeDownload slide Effect of HbEIN3 on the regulation of HbMYC2-activated HbFPS1 and HbSRPP1 expression. (A) Schematic diagrams of the constructs for a dual-luciferase assay. (B, C) Analysis of the transient transcriptional activity of HbMYC2 on HbFPS1 (B) and HbSRPP1 (C) with or without the expressed HbEIN3 protein. Data are means (±SD) of three biological replicates. Significant differences were determined using Student’s t-test; **P<0.01. Fig. 7. View largeDownload slide Effect of HbEIN3 on the regulation of HbMYC2-activated HbFPS1 and HbSRPP1 expression. (A) Schematic diagrams of the constructs for a dual-luciferase assay. (B, C) Analysis of the transient transcriptional activity of HbMYC2 on HbFPS1 (B) and HbSRPP1 (C) with or without the expressed HbEIN3 protein. Data are means (±SD) of three biological replicates. Significant differences were determined using Student’s t-test; **P<0.01. Discussion Jasmonate signalling has been extensively studied in herbaceous model plants with the aid of forward genetics (Xie et al., 1998; Chini et al., 2007; Wasternack and Song, 2017; Zhu and Napier, 2017) and a COI1–JAZ–MYC2 module has been identified that seems to be conserved among plant species (Fonseca et al., 2009a; Shoji and Hashimoto, 2011; Shen et al., 2016; Zhou et al., 2016). However, the role of jasmonate signalling in the specific tissues that produce latex in rubber trees has not yet been elucidated. The secondary laticifer in rubber trees is a single-cell type tissue that is composed of laticifer cells. There are no plasmodesmata between the cells and their surrounding phloem parenchyma cells (de Faÿ et al., 1989). Therefore, the latex that is easily obtained from them by the process of tapping is the pure cytoplasm of laticifer cells. As the specific tissue for rubber biosynthesis (Kush, 1994), the secondary laticifer is enriched in transcripts of rubber biosynthesis-related genes (Chow et al., 2007). These structural and molecular characteristics mean that the laticifer cells are a good model for investigating the transcriptional regulation of secondary metabolism. Rubber biosynthesis in laticifer cells is a typical isoprenoid metabolism and is carried out by the MVA as well as the MEP pathways (Seetang-Nun et al., 2008; Chow et al., 2012; Deng et al., 2016; Tang et al., 2016). Most of the rubber biosynthesis-related genes in these pathways were up-regulated in regularly tapped trees (Fig. 1C, Supplementary Fig. S1B), and these genes are down-regulated when trees are rested from tapping for a short time (Tian et al., 2013), and also in trees that are affected by tapping panel dryness (TPD) that are rested from tapping for a long time (Liu et al., 2015). The up-regulated expression of rubber biosynthesis-related genes could be associated with a high level of endogenous JA in the latex of regularly tapped trees, since the expression pattern of the genes resulting from exogenous methyl jasmonate was similar to that in regularly tapped trees (Fig. 1D, Supplementary Fig. S1C). The level of endogenous JA in the laticier cells of regularly tapped trees was several times higher than that of virgin trees 2 d after tapping (Fig.1A). As mechanical wounding causes a burst of endogenous JA in bark tissues that occurs within 4 h (Tian et al., 2015), the maintenance of high levels of endogenous JA in regularly tapped trees 2 d after being tapped may not be related to the mechanical wounding per se; it could mainly be ascribed to changes in the turgor pressure of laticifer cells. Available data show that changes in turgor pressure are critical for JA biosynthesis (Creelmen and Mullet, 1997), and the turgor of laticifer cells is 10 times above atmospheric pressure before tapping and significantly decreases within the latex drainage areas, even resulting in a detectable decrease in the trunk diameter after tapping (Pakianathan et al., 1989). The high levels of endogenous JA were also positively correlated with enhanced rubber biosynthesis in regularly tapped trees, because exogenous methyl jasmonate was effective in enhancing in vitro rubber biosynthesis (Fig.1E). Our results identified for the first time a jasmonate signalling module, HbCOI1–HbJAZ3–HbMYC2, in the specific laticifer tissue of rubber trees. HbJAZ3 interacted with HbCOI1 in a coronatine (COR)-dependent manner (Fig. 2C, Supplementary Fig. S6D). It also interacted with HbMYC2 in a COR-independent manner and inhibited the HbMYC2-activated transcription of HbFPS1 and HbSRPP1. The cascade of HbCOI1–HbJAZ3–HbMYC2–HbFPS1/HbSRPP1 in laticifer cells directly links jasmonates with the regulation of rubber biosynthesis. FPS has been identified as the key rate-limiting enzyme, and this is the first time that its encoding gene is identified as the regulating target for MYC2-type transcriptional factor in the conserved MVA pathway. This finding increases our understanding of jasmonate signalling in the regulation of isoprenoid metabolism in plants. The HbFPS1 and HbSRPP1 transcripts are the most abundant in the laticifer cells of rubber tree among the FPS and SRPP gene families, respectively (Guo et al., 2015a; Tang et al., 2016), suggesting that they have significant and indispensable roles in rubber synthesis. HbFPS1 is essential for incorporating two IPP molecules and one DMAPP molecule into one FPP molecule, an initial primer for initiating rubber biosynthesis (Cornish and Siler, 1999). It shares 91% similarity and 83% identity with TkFPS1 from Taraxacum kok-saghyz (Supplementary Fig. S8). Over-expression of TkFPS1 results in a maximum increase of 7.48% or a mean increase of 3.92% in rubber content in transgenic plants (Cao et al., 2016). HbSRPP1 plays a positive role in rubber biosynthesis (Oh et al., 1999; Yamashita et al., 2016; Brown et al., 2017). Its orthologs also play vital roles in rubber biosynthesis in T. kok-saghyz (Collins-Silva et al., 2012) and T. brevicorniculatum (Hillebrand et al., 2012), although silencing the homolog of HbSRPP1 has little effect on rubber biosynthesis in Lactuca sativa (Chakrabarty et al., 2015). The increased levels of HbFPS1 and HbSRPP1 (Fig. 6) may be partly ascribed the enhanced biosynthesis of rubber in the regularly tapped trees compared with the virgin trees. HbJAZ3-mediated repression of HbMYC2-activated expression of both HbFPS1 and HbSRPP1 suggests possible potential for transgenic improvement of yields from rubber tree as silencing its ortholog, SmJAZ3, resulted in accumulation of tanshinone in S. miltiorrhiza (Shi et al., 2016a). It has long been believed that rubber biosynthesis in rubber tree is positively regulated by ethylene signalling since application of ethrel significantly raises the yield per tapping. This effect is primarily due to prolonged duration of latex flow (Coupé and Chrestin, 1989) in addition to enhanced sucrose allocation (Tang et al., 2010), water transportation (Tungngoen et al., 2009), glycolysis, and C3 carbon fixation (Liu et al., 2016). By contrast, ethylene signalling does not have a major role in activating rubber biosynthesis per se as ethylene application has little effect on up-regulating the genes related to IPP biosynthesis and IPP integration into rubber (Oh et al., 1999; Zhu and Zhang, 2009; Liu et al., 2016). It has little effect on, or even decreases, the abundance of REF and SRPP proteins (Tong et al., 2017). In the present study, we provide evidence that ethylene inhibits rubber biosynthesis (Fig. 1E); however, the antagonistic effect between JA signalling and ethylene signalling on rubber biosynthesis remains to be elucidated. Although the EIN3–MYC2 complex mediates the crosstalk between ethylene signalling and JA signalling in resistance reactions in Arabidopsis (Song et al., 2014), HbEIN3 (KR013139) preferentially expressed by the latex (Yang et al., 2015) had no apparent influence on the HbMYC2-activated transcription of HbFPS1 and HbSRPP1 (Fig. 7B, C), indicating that the involved in regulation of rubber biosynthesis may not be associated with transcriptional regulation of the HbFPS1 and HbSRPP1 genes. Alternatively, the transcriptional regulation of the HRT2 gene may be associated with the crosstalk because HbEIN3 could bind to the promoter and regulate the expression of this gene (Yang et al., 2015). Although HbMYC2 could not bind the promoter of HRT2 (data not shown), the involvement of other transcription factors that may be associated with JA signalling cannot be excluded (Guo et al., 2018). Just as WD-repeat/bHLH/MYB transcriptional complexes are essential for JA-regulated anthocyanin biosynthesis in Arabidopsis (Qi et al., 2011), the rubber biosynthesis-related genes may be regulated by multiple transcriptional factors. In addition to positive regulation by HbMYC2 in the present study, HbSRPP1 is also negatively regulated by HbWRKY1 (Wang et al., 2013) and HbMADS4 (Li et al., 2016). Ethylene up-regulates HbWRKY1 expression and down-regulates HbSRPP1 expression, while methyl jasmonate significantly up-regulates HbSRPP1 and has little effect on the expression of HbWRKY1 (Wang et al., 2013). Both ethylene and methyl jasmonate up-regulate the expression of HbMADS4 (Li et al., 2016). HMGR is the key rate-limiting enzyme in the MVA pathway (Goldstein and Brown, 1990), and in laticifer cells the transcripts of HbHMGR1 are the most abundant among the five HMGR gene members (Tang et al., 2016). HbHMGR1 is positively regulated by HbCZF1, a CCCH-type zinc finger protein (Guo et al., 2015b). HbCZF1 is significantly up-regulated by methyl jasmonate, but its expression is little influenced by ethylene (Guo et al., 2015b). In summary, the dramatic changes in the turgor pressure of laticifer cells after latex extraction result in a burst of endogenous JA. The increased JA up-regulates the rubber biosynthesis-related genes HbFPS1 and HbSRPP1 by the degradation of the repressor HbJAZ3 via 26S proteasome and the release of HbMYC2. The up-regulation of HbSRPP1 and HbFPS1 results in an increase in the HbSRPP1 and especially HbFPS1 proteins. The increased levels of HbSRPP1 and HbFPS1 are partially responsible for the increased efficiency of in vitro rubber biosynthesis. In addition to HbMYC2, other transcriptional factors such as HbCZF1, HbWRKY1, and HbMADS4 may integrate into the JA signalling and mediate the biosynthesis of rubber that is activated in the laticifer cells of rubber trees as a result of latex harvesting. Supplementary data Supplementary data are available at JXB online. Fig. S1. Analysis of expression of rubber tree MEP pathway genes. Fig. S2. Relative expression of HbJAZ3 and HbMYC2 genes in the bark and latex of the laticifer cells of rubber tree. Fig. S3. HbJAZ3 protein analysis. Fig. S4. HbMYC2 protein analysis. Fig. S5. Phylogenetic tree analysis of the reported MYC-encoding genes of rubber trees. Fig. S6. Interaction of HbMYC2 or HbCOI1 with the latex-expressed JAZ-encoding genes as shown by yeast two-hybrid assays. Supplementary Fig. S7. BiFC assay in the onion epidemic cells. Supplementary Fig. S8. Amino acid comparison between HbFPS1 and TkFPS1. Supplementary Table S1. Primers used in this study. Supplementary Table S2. The predicted regulatory elements present in the promoters of HbFPS1 and HbSRPP1. Acknowledgements We thank Prof. Jorg Kudla for providing vectors for the BiFC assays. We also thank Prof. Lei Zhang for technical guidance in protein expression in E. coli. We sincerely thank Prof. Bingzhong Hao and Prof. Jinglian Wu for giving useful advice during the writing of this manuscript. We also sincerely thank PhD student Chen Liu for technical discussions and guidance for the yeast-one hybrid experiment. This work was financially supported by grants from the National Natural Science Foundation of China (30872002; 31770122), the Earmarked Fund for Modern Agro-Industry Technology Research System (CARS-34-GW1), and Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (1630022015010; 1630022018018). References Adiwilaga K , Kush A . 1996 . 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Journal of Experimental BotanyOxford University Press

Published: May 3, 2018

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