Genome-Wide Identification and Characterization of the JAZ Gene Family in Rubber Tree (Hevea brasiliensis).

Jasmonate signaling plays a vital role in the regulation of secondary laticifer differentiation and natural rubber biosynthesis in Hevea brasiliensis. Jasmonate ZIM-domain (JAZ) proteins are the master regulators of jasmonate signaling. Although several JAZs have been reported in the laticifer cells of H. brasiliensis, the genome-wide screening of HbJAZ members has not yet been explored. In the present study, 18 HbJAZs were identified based on the recent H. brasiliensis genome. Phylogenetic construction revealed that the HbJAZs were clustered into five subgroups and that members within the same subgroup shared highly conserved gene structures and protein motifs. Cis-element analysis of HbJAZ promoters suggested the presence of hormone, stress and development-related cis-elements. HbJAZ1.0, HbJAZ2.0, and HbJAZ5.0 interacted with CORONATINE INSENSITIVE1 (COI1) in the presence of coronatine (COR, a JA mimic). HbJAZ1.0, HbJAZ2.0, HbJAZ5.0, and HbJAZ12.0 could also interact with each other. Of the 18 HbJAZs, transcripts of 15 HbJAZs were present in the vascular cambium region except for that of HbJAZ7.0, HbJAZ8.0d, and HbJAZ13.0. Fourteen of the 15 HbJAZs were significantly up-regulated upon COR treatment. The transcripts of three genes that were absent from vascular cambium region were also absent from the latex. Among the 15 HbJAZs in the latex, the expression patterns of 13 HbJAZs were different between the tapping and ethrel treatments. Eight of the 14 COR-up-regulated HbJAZs in the vascular cambium region were also activated by tapping in latex. Of the eight tapping-activated HbJAZs, 5 HbJAZs were repressed by ethrel application. Based on the computational analyses and gene expression patterns described in this study, the HbJAZ5.0 and HbJAZ10.0b may be associated with laticifer differentiation while the HbJAZ8.0b is a negative regulator for natural rubber biosynthesis in H. brasiliensis.

Transcript Profiling of Hevea brasiliensis during Latex Flow.

Latex exploitation enhances latex regeneration in rubber trees. The latex exploitation-caused latex flow lasts from 10 min to a few hours, which is convenient for exploring the transcript profiling of latex metabolism-related genes at the different stages of latex flow. In the present study, the expression pattern of 62 latex metabolism-related genes involved in water transportation, carbohydrate metabolism, natural rubber biosynthesis, hormone signaling, ROS generation and scavenging, and latex coagulum across three stages of latex flow between rubber tree clones CATAS7-33-97 and CATAS8-79 were comparatively analyzed by quantitative real-time PCR. The two clones show differences in latex regeneration and have a different duration of latex flow. The results showed that the expression levels of 38 genes were significantly higher in CATAS8-79 latex than in CATAS7-33-97 during latex regeneration, while 45 genes had a notably higher expression level in CATAS8-79 latex during latex flow. Together with the activation of the MEP pathway and jasmonate pathway in CATAS8-79 latex, HbPIP1;3, HbPIP1;4, HbSUT3, HbSus3, HbHMGS1-2, HbMK should contribute to the high latex regeneration ability. The up-regulation of ethylene signaling and Hb44KD and the down-regulation of latex coagulation-related genes in CATAS8-79 latex might contribute to its longer latex flow duration. This study provides some cues for revealing the regulation of latex metabolism in rubber trees.

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Laticifers on the Basis of Latex Flow in Rubber Tree (Hevea brasiliensis Muell. Arg.).

Latex exploitation-caused latex flow is effective in enhancing latex regeneration in laticifer cells of rubber tree. It should be suitable for screening appropriate reference gene for analysis of the expression of latex regeneration-related genes by quantitative real-time PCR (qRT-PCR). In the present study, the expression stability of 23 candidate reference genes was evaluated on the basis of latex flow by using geNorm and NormFinder algorithms. Ubiquitin-protein ligase 2a (UBC2a) and ubiquitin-protein ligase 2b (UBC2b) were the two most stable genes among the selected candidate references in rubber tree clones with differential duration of latex flow. The two genes were also high-ranked in previous reference gene screening across different tissues and experimental conditions. By contrast, the transcripts of latex regeneration-related genes fluctuated significantly during latex flow. The results suggest that screening reference gene during latex flow should be an efficient and effective clue for selection of reference genes in qRT-PCR.

Ethrel-stimulated prolongation of latex flow in the rubber tree (Hevea brasiliensis Muell. Arg.): an Hev b 7-like protein acts as a universal antagonist of rubber particle aggregating factors from lutoids and C-serum.

Ethrel is the most effective stimuli in prolonging the latex flow that consequently increases yield per tapping. This effect is largely ascribed to the enhanced lutoid stability, which is associated with the decreased release of initiators of rubber particle (RP) aggregation from lutoid bursting. However, the increase in both the bursting index of lutoids and the duration of latex flow after applying ethrel or ethylene gas in high concentrations suggests that a new mechanism needs to be introduced. In this study, a latex allergen Hev b 7-like protein in C-serum was identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF MS). In vitro analysis showed that the protein acted as a universal antagonist of RP aggregating factors from lutoids and C-serum. Ethrel treatment obviously weakened the effect of C-serum on RP aggregation, which was closely associated with the increase in the level of the Hev b 7-like protein and the decrease in the level of the 37 kDa protein, as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting analysis and antibody neutralization. Thus, the increase of the Hev b 7-like protein level or the ratio of the Hev b 7-like protein to the 37 kDa protein in C-serum should be primarily ascribed to the ethrel-stimulated prolongation of latex flow duration.

Cloning, heterologous expression and characterization of ascorbate peroxidase (APX) gene in laticifer cells of rubber tree (Hevea brasiliensis Muell. Arg.).

Ascorbate peroxidases (APXs) are a kind of crucial enzymes for removing reactive oxygen species (ROS) in plant cell. In the present study, a full-length cDNA encoding an APX, designated HbAPX, was isolated from Hevea brasiliensis by the rapid amplification of cDNA ends (RACE) method. HbAPX was 1174-bp in length and contained a 912-bp open reading frame (ORF) encoding a putative protein of 304 amino acids. The predicted molecular mass of HbAPX was 27.6 kDa (kDa) with an isoelectric point (pI) of 6.73. The phylogenetic analysis showed that HbAPX belonged to the cytosolic subgroup and was more relative to PtAPX and MdAPX2. By using PlantCare online analysis, such cis-acting elements as W-box and MRE were detected in the promoter region of HbAPX. Overproduction of recombinant HbAPX protein either in Escherichia coli or yeast enhanced their tolerance to such abiotic stresses as Cu(2+), Zn(2+), Na(2+) and hydrogen peroxide (H2O2). Ethrel application significantly down-regulated the expression of HbAPX and inhibited the activity of HbAPX in vivo. The ethrel-caused down-regulation of HbAPX may disturb the redox homeostasis in laticifer cells of rubber tree.

Cloning and characterization of HbMT2a, a metallothionein gene from Hevea brasiliensis Muell. Arg differently responds to abiotic stress and heavy metals.

Metallothioneins (MTs) are of low molecular mass, cysteine-rich proteins. They play an important role in the detoxification of heavy metals and homeostasis of intracellular metal ions, and protecting against intracellular oxidative damages. In this study a full-length cDNA of type 2 plant metallothioneins, HbMT2a, was isolated from 25 mM Polyethyleneglycol (PEG) stressed leaves of Hevea brasiliensis by RACE. The HbMT2a was 372bp in length and had a 237bp open reading frame (ORF) encoding for a protein of 78 amino acid residues with molecular mass of 7.772 kDa. The expression of HbMT2a in the detached leaves of rubber tree clone RY7-33-97 was up-regulated by Me-JA, ABA, PEG, H2O2, Cu(2+) and Zn(2+), but down-regulated by water. The role of HbMT2a protein in protecting against metal toxicity was demonstrated in vitro. PET-28a-HbMT2-beared Escherichia coli. Differential expression of HbMT2a upon treatment with 10 °C was observed in the detached leaves of rubber tree clone 93-114 which is cold-resistant and Reken501 which is cold-sensitive. The expression patterns of HbMT2a in the two rubber tree clones may be ascribed to a change in the level of endogenous H2O2.

Comparative proteomics of primary and secondary lutoids reveals that chitinase and glucanase play a crucial combined role in rubber particle aggregation in Hevea brasiliensis.

Lutoids are specific vacuole-based organelles within the latex-producing laticifers in rubber tree Hevea brasiliensis. Primary and secondary lutoids are found in the primary and secondary laticifers, respectively. Although both lutoid types perform similar roles in rubber particle aggregation (RPA) and latex coagulation, they vary greatly at the morphological and proteomic levels. To compare the differential proteins and determine the shared proteins of the two lutoid types, a proteomic analysis of lutoid membranes and inclusions was performed, revealing 169 proteins that were functionally classified into 14 families. Biological function analysis revealed that most of the proteins are involved in pathogen defense, chitin catabolism, and proton transport. Comparison of the gene and protein changed patterns and determination of the specific roles of several main lutoid proteins, such as glucanase, hevamine, and hevein, demonstrated that Chitinase and glucanase appeared to play crucial synergistic roles in RPA. Integrative analysis revealed a protein-based metabolic network mediating pH and ion homeostasis, defense response, and RPA in lutoids. From these findings, we developed a modified regulation model for lutoid-mediated RPA that will deepen our understanding of potential mechanisms involved in lutoid-mediated RPA and consequent latex coagulation.

Molecular and biochemical characterization of a cyanogenic ß-glucosidase in the inner bark tissues of rubber tree (Hevea brasiliensis Muell. Arg.).

Tapping causes the loss of large amounts of latex from laticifers and subsequently enhances latex regeneration, a high carbon- and nitrogen-cost activity in rubber tree. It is suggested that a 67 kDa protein associated with protein-storing cells in the inner bark tissues of rubber tree plays an important role in meeting the nitrogen demand for latex regeneration. Here, the 67 kDa protein was further characterized by a combination of cell biological, molecular biological and biochemical techniques. Immunogold labeling showed that the 67 kDa protein was specifically localized in the central vacuole of protein-storing cells. A full-length cDNA, referred to as HbVSP1, was cloned. The HbVSP1 contained a 1584 bp open reading frame encoding a protein of 527 amino acids. The putative protein HbVSP1 shared high identity with the P66 protein from rubber tree and proteins of the linamarase, and bg1A from cassava (Manihot esculenta). HbVSP1 contained the active site sequences of ß-glucosidase, TFNEP and I/VTENG. In vitro analysis showed that the 67 kDa protein exhibited the activity of both ß-glucosidase and linamarase and was thus characterized as a cyanogenic ß-glucosidase. Proteins immuno-related to the 67 kDa protein were present in leaves and lutoids of laticifers. Tapping down-regulated the expression of HbVSP1, but up-regulated the expression of genes encoding the key enzymes for rubber biosynthesis, while the effect of resting from tapping was the reverse. Taken together, the results suggest that the 67 kDa protein is a vacuole-localized cyanogenic ß-glucosidase encoded by HbVSP1 and may have a role in nitrogen storage in inner bark tissues of trunk during the leafless periods when rubber tree is rested from tapping.

Molecular characterization of a novel 14-3-3 protein gene (Hb14-3-3c) from Hevea brasiliensis.

The cDNA encoding a 14-3-3 protein, designated as Hb14-3-3c, was isolated from Hevea brasiliensis. Hb14-3-3c was 1,269 bp long containing a 795 bp open reading frame encoding a putative protein of 264 amino acids, flanked by a 146 bp 5'UTR and a 328 bp 3' UTR. The predicted molecular mass of Hb14-3-3c is 29.67 kDa, with an isoelectric point of 4.52 and the deduced protein showed high similarity to the 14-3-3 protein from other plant species. Expression analysis revealed more significant accumulation of Hb14-3-3c transcripts in latex than in leaves, buds and flowers. The transcription of Hb14-3-3c in latex was induced by jasmonate and ethephon. Overproduction of recombinant Hb14-3-3c protein gave the Escherichia coli cells more tolerance on Co(2+), Cu(2+) and Zn(2+). Through yeast two-hybrid screening, 11 interaction partners of the Hb14-3-3c, which are involved in rubber biosynthesis, stress-related responses, defence etc., were identified in rubber tree latex. Taking these data together, it is proposed that the Hb14-3-3c may participate in regulation of rubber biosynthesis. Thus, the results of this study provide novel insights into the 14-3-3 signaling related to rubber biosynthesis, stress-related responses in rubber tree.

Characterization of HbEREBP1, a wound-responsive transcription factor gene in laticifers of Hevea brasiliensis Muell. Arg.

AP2/ERF transcription factors play an important role in regulation of the cross-talk between ethylene and jasmonate signaling pathways mediating defense responses of plants to biotic and abiotic stresses. In this study, an AP2/ERF transcription factor gene was isolated and characterized from laticifers of rubber tree by using RACE and real time PCR. The full length cDNA, referred to as HbEREBP1, was 1,095 bp in length and contained a 732 bp open reading frame encoding a putative protein of 243 amino acid residues. The molecular mass of the putative protein is 26.4 kDa with a pI of 9.46. The deduced amino acid sequence had a specific domain of AP2 superfamily and an ethylene-responsive element binding factor-associated amphiphilic repression motif, sharing 42.4, 39.1, and 38.0% identity with that of AtERF11, AtERF4, and AtERF8 in Arabidopsis, respectively. HbEREBP1 expression was down-regulated by tapping and mechanical wounding in the laticifers of adult trees. It was also down-regulated at early stage while up-regulated at late stage upon treatment with exogenous ethephon or methyl jasmonate, which was reverse to the case of defense genes in laticifers of epicormic shoots of rubber tree. Our results suggest that HbEREBP1 may be a negative regulator of defense genes in laticifers.

MYC genes with differential responses to tapping, mechanical wounding, ethrel and methyl jasmonate in laticifers of rubber tree (Hevea brasiliensis Muell. Arg.).

MYC2 transcription factor is a key component of the core module COI1-JAZ-MYC2 of jasmonate signaling in Arabidopsis, but the MYC transcription factor (s) associated with jasmonate signaling in jasmonate-responsive laticifer cells remains to be identified. Two full-length cDNAs, designated HblMYC1 and HblMYC2, were isolated from laticifer cells in Hevea brasiliensis by the method of RACE. HblMYC1 contained 1431bp ORF encoding a putative protein of 476 amino acids while HblMYC2 contained 1428bp ORF encoding a putative protein of 475 amino acids. Bioinformatic analysis showed that the putative proteins, HblMYC1 and HblMYC2, possessed a bHLH domain and were most related to the MYC2 among the selected 27 MYC members with identified functions in Arabidopsis. In addition to the presence of cis-regulatory elements involving jasmonate responsiveness in the promoter regions of HblMYC1 and HblMYC2, the abscisic acid-, salicylic acid- and gibberellin-responsive elements were found in the promoter region of HblMYC1. Transcripts of HblMYC1 and HblMYC2 were most abundant in latex, relatively low in male flowers and nearly undetected in bark tissues and roots by real-time RT-PCR analysis. Regular tapping, mechanical wounding, and ethrel remarkably up-regulated HblMYC1 expression, but had little effect on the expression of HblMYC2 in laticifer cells. Successive tapping, however, significantly down-regulated the expression of HblMYC2 while up-regulating the expression of HblMYC1. The HblMYC2 expression took a mutual ebb and flow relationship with the HblMYC1 expression upon treatment with methyl jasmonate. Characterization of HblMYC1 and HblMYC2 will contribute to the understanding of jasmonate signaling in laticifiers, a kind of specialized tissue for natural rubber biosynthesis in Hevea brasiliensis.

Molecular characterization of a thioredoxin h gene (HbTRX1) from Hevea brasiliensis showing differential expression in latex between self-rooting juvenile clones and donor clones.

The cDNA code of thioredoxin h, designated as HbTRX1, was isolated from Hevea brasiliensis by rapid amplification of cDNA ends. HbTRX1 contained a 542-bp open reading frame encoding 123 amino acids. The deduced HbTRX1 protein showing high identity to thioredoxin h of other plant species was predicted to possess the conserved catalytic site WCXPC. Semiquantitative reverse transcription-polymerase chain reaction analysis revealed that HbTRX1 was constitutively expressed in all tested tissues. HbTRX1 transcripts accumulated at relatively low levels in the flower, somatic embryo, and leaves, while HbTRX1 transcripts accumulated at relatively high levels in the callus and latex. The HbTRX1 transcript was expressed at different levels, with higher levels in self-rooting juvenile clones than in their donor clones. HbTRX1 was expressed in Escherichia coli, and its activity was demonstrated using the dithiothreitol-dependent insulin assay. This work provides a basis for studying the biological function of thioredoxin h in rubber tree.

Three MADS-box genes of Hevea brasiliensis expressed during somatic embryogenesis and in the laticifer cells.

Three MADS-box genes, designated HbMADS1, HbMADS2 and HbMADS3, were isolated from Hevea brasiliensis. HbMADS1, HbMADS2 and HbMADS3 encode polypetides consisting of 245, 217 and 239 amino acids, respectively, containing conserved MADS-box motifs at N-terminus. Transcription pattern analysis revealed that three MADS-box genes had highly transcription in the laticifer cells. The transcriptions of HbMADS1and HbMADS3 were induced in the laticifer cells by jamonic acid, while HbMADS2 was not induction by jamonic acid. Ethephone is not effective in inducing their expression. The three genes were differentially expressed during somatic embryogenesis of rubber tree. Characterization of HbMADSs will attribute to understand their possible function in rubber tree.

Molecular characterization of HbCDPK1, an ethephon-induced calcium-dependent protein kinase gene of Hevea brasiliensis.

Calcium-dependent protein kinases (CDPKs), as major primary Ca(2+) sensors, have been implicated in the regulation of stress and developmental signals in plants. In this study, a novel CDPK gene, designated HbCDPK1, was isolated from Hevea brasiliensis. The HbCDPK1 cDNA had 2,400 bp with an open reading frame of 1,671 bp encoding 556 amino acids, and the deduced HbCDPK1 protein contained four characteristic domains identified in CDPKs, showing a high level of sequence similarity to CDPKs from other plants. Expression analysis revealed more significant accumulation of the transcripts of HbCDPK1 in latex than in the leaves, bark, and roots in H. brasiliensis. In addition, transcription of HbCDPK1 was strongly induced by mechanical wounding, jasmonic acid (JA), and ethephon. Recombinant HbCDPK1 was expressed in E. coli, and its activity was assayed. The assay indicated that HbCDPK1 had the kinase and Ca(2+)-binding activity in vitro as a calcium-dependent protein. The potential roles of the HbCDPK1 are discussed as to latex production and rubber biosynthesis.

Cloning and molecular characterization of HbCOI1 from Hevea brasiliensis.

The full-length cDNA encoding a coronatine insensitive-1 (COI1) protein, designated HbCOI1, was isolated for the first time from Hevea brasiliensis by the rapid amplification of cDNA ends (RACE) method. HbCOI1 contained a 2,187 bp open reading frame encoding 597 amino acids. The deduced HbCOI1 protein, which showed high identity to COI1 protein of other plant species, was predicted to possess F-box and LRRs domains. The promoter region of HbCOI1 was isolated by the PCR-based DNA walking method. TATA box and other core configurations were found in the promoter. Several sequences similar to the eukaryotic cis regulatory element were found in the 5'-untranslated region (UTR) proximal 5' flanking sequence of HbCOI1. Southern blot analysis indicated that the HbCOI1 is present as a single copy in Hevea brasiliensis. Transcription pattern analysis revealed that HbCOI1 had high transcription in laticifer, low in barks and leaf. Transcription of HbCOI1 in latex was induced by jasmonate and tapping.

Cloning and characterization of a novel cysteine protease gene (HbCP1) from Hevea brasiliensis.

The full-length cDNA encoding a cysteine protease,designated HbCP1, was isolated for the first time from Hevea brasiliensis by the rapid amplification of cDNA ends (RACE) method. HbCP1 contained a 1371 bp open reading frame encoding 457 amino acids.The deduced HbCP1 protein,which showed high identity to cysteine proteases of other plant species,was predicted to possess a putative repeat in toxin (RTX) domain at the N-terminal and a granulin (GRAN) domain at the C-terminal.Southern blot analysis indicated that the HbCP1 gene is present as a single copy in the rubber tree.Transcription pattern analysis revealed that HbCP1 had high transcription in laticifer,and low transcription in bark and leaf.The transcription of HbCP1 in latex was induced by ethylene and tapping.Cloning of the HbCP1 gene will enable us to further understand the molecular characterization of cysteine protease and its possible function in the rubber tree.

Cloning and molecular characterization of a RING zinc-finger gene of Hevea brasiliensis.

Using the information about the sequence from a differentially expressed clone (designated as HbSSH10) encodes a protein specifying a cysteine-rich sequence containing a putative "RING finger" or "C3HC4" consensus motif that was cloned recently by the subtractive hybridization between latex and leaves from rubber tree (Hevea brasiliensis). A full-length cDNA encoding C3HC4 type zinc-finger protein was isolated and characterized from rubber tree. Sequence analysis revealed that the ORFs of HbRZF encode 156 amino acid residues with a total predicted molecular mass of 17.2 kD, HbRZF protein having a putative "RING finger" segment (amino acid residues 100-144). The deduced amino acid sequences of HbRZF showed high identities of 48%, 52% and 50% to those of the ring zinc protein from Poncirus trifoliata, Arabidopsis thaliana, Thellungiella halophila. The result of Northern blot analysis indicated that the transcripts of the HbRZF were expressed more in the latex than in the leaves, whereas little expression was detected in roots and flowers. The transcription of HbRZF was induced by jasmonic acid, whereas ethylene had little effect.