The 14-3-3 protein HbGF14a interacts with a RING zinc finger protein to regulate expression of the rubber transferase gene in Hevea brasiliensis.

Hevea brasiliensis is a key commercial source of natural rubber (cis 1,4-polyisoprene). In H. brasiliensis, rubber transferase is responsible for cis-1,4-polymerization of isoprene units from isopentenyl diphosphate and thus affects the yield of rubber. Little is known about the regulatory mechanisms of the rubber transferase gene at a molecular level. In this study we show that the 5'UTR intron of the promoter of the rubber transferase gene (HRT2) suppresses the expression of HRT2. A H. brasiliensis RING zinc finger protein (designated as HbRZFP1) was able to interact specifically with the HRT2 promoter to down-regulate its transcription in vivo. A 14-3-3 protein (named as HbGF14a) was identified as interacting with HbRZFP1, both in yeast and in planta. Transient co-expression of HbGF14a and HbRZFP1-encoding cDNAs resulted in HbRZFP1-mediated HRT2 transcription inhibition being relieved. HbGF14a repressed the protein-DNA binding of HbRZFP1 with the HRT2 promoter in yeast. We propose a regulatory mechanism by which the binding of HbGF14a to HbRZFP1 interferes with the interaction of HbRZFP1 with the HRT2 promoter, thereby repressing the protein-DNA binding between them. This study provides new insights into the role of HbGF14a in mediating expression of the rubber transferase gene in Hevea brasiliensis.

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

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.

Genome-wide identification and characterization of WRKY gene family in Hevea brasiliensis.

WRKY proteins constitute a large family of transcription factors. In this study, we identified 81 WRKY genes (named HbWRKY1 to HbWRKY81) in the latest rubber tree genome. Tissue-specific expression profiles showed that 74 HbWRKYs were expressed in at least one of the tissues and the other 7 genes showed very low expression in all tissues tested, which suggested that HbWRKYs took part in many cellular processes. The responses of 20 selected HbWRKYs to jasmonic acid (JA) and ethylene (ET) were analyzed in the latex. 17 HbWRKYs responded to at least one treatment, which included 15 HbWRKYs responding to JA treatment, 15 HbWRKYs to ET, which suggested that these HbWRKYs were regulated by JA and ET. We also observed that HbWRKY3, 14, and 55 bind HbSRPP promoter and activate the transcription in yeast. This study suggests that HbWRKY proteins maybe involved in the transcriptional regulation of nature rubber biosynthesis.

Genomic insight into domestication of rubber tree.

Understanding the genetic basis of rubber tree (Hevea brasiliensis) domestication is crucial for further improving natural rubber production to meet its increasing demand worldwide. Here we provide a high-quality H. brasiliensis genome assembly (1.58 Gb, contig N50 of 11.21 megabases), present a map of genome variations by resequencing 335 accessions and reveal domestication-related molecular signals and a major domestication trait, the higher number of laticifer rings. We further show that HbPSK5, encoding the small-peptide hormone phytosulfokine (PSK), is a key domestication gene and closely correlated with the major domestication trait. The transcriptional activation of HbPSK5 by myelocytomatosis (MYC) members links PSK signaling to jasmonates in regulating the laticifer differentiation in rubber tree. Heterologous overexpression of HbPSK5 in Russian dandelion (Taraxacum kok-saghyz) can increase rubber content by promoting laticifer formation. Our results provide an insight into target genes for improving rubber tree and accelerating the domestication of other rubber-producing plants.

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.

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.

HbWRKY27, a group IIe WRKY transcription factor, positively regulates HbFPS1 expression in Hevea brasiliensis.

Farnesyl pyrophosphate synthase (FPS) is a key enzyme that catalyzes the formation of farnesyl pyrophosphate, the main initiator for rubber chain initiation in Hevea brasiliensis Muell. Arg. The transcriptional regulatory mechanisms of the FPS gene still not well understood. Here, a WRKY transcription factor designated HbWRKY27 was obtained by screening the latex cDNA library applied the HbFPS1 promoter as bait. HbWRKY27 interacted with the HbFPS1 promoter was further identified by individual Y1H and EMSA assays. HbWRKY27 belongs to group IIe WRKY subfamily which contains a typical WRKY domain and C-X5-CX23-HXH motif. HbWRKY27 was localized to the nucleus. HbWRKY27 predominantly accumulated in latex. HbWRKY27 was up-regulated in latex by ethrel, salicylic acid, abscisic acid, and methyl jasmonate treatment. Transient expression of HbWRKY27 led to increasing the activity of the HbFPS1 promoter in tobacco plant, suggesting that HbWRKY27 positively regulates the HbFPS1 expression. Taken together, an upstream transcription factor of the key natural rubber biosynthesis gene HbFPS1 was identified and this study will provide novel transcriptional regulatory mechanisms of the FPS gene in Hevea brasiliensis.

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.

Identification and characterization of the MADS-box genes highly expressed in the laticifer cells of Hevea brasiliensis.

MADS-box transcription factors possess many functions in plant reproduction and development. However, few MADS-box genes related to secondary metabolites regulation have been identified. In Hevea brasiliensis, natural rubber is a representative cis-polyisoprenoids in secondary metabolism which occurs in the rubber laticifer cells, the molecular regulation basis of natural rubber biosynthesis is not clear. Here, a total of 24 MADS-box genes including 4 type I MADS-box genes and 20 type II MADS-box genes were identified in the transcriptome of rubber tree latex. The phylogenetic analysis was performed to clarify the evolutionary relationships of all the 24 rubber tree MADS-box proteins with MADS-box transcription factors from Arabidopsis thaliana and Oryza sativa. Four type I MADS-box genes were subdivided into Mα (3 genes) and Mß (1 gene). Twenty type II MADS-box genes were subclassified into MIKC* (8 genes) and MIKCc (12 genes). Eight MADS-box genes (HblMADS3, 5, 6, 7, 9, 13, 23, 24) were predominant expression in laticifers. ABA up-regulated the expression of HblMADS9, and the expression of HblMADS3, HblMADS5, HblMADS24 were up-regulated by MeJA. The function of HblMADS24 was elucidated. HblMADS24 bound HbFPS1 promoter in yeast and HblMADS24 activated HbFPS1 promoter in tobacco plants. Moreover, we proposed that HblMADS24 is a transcription activator of HbFPS1 which taking part in natural rubber biosynthesis.

Identification and characterization of the abscisic acid (ABA) receptor gene family and its expression in response to hormones in the rubber tree.

Abscisic acid (ABA) is an essential phytohormone involved in diverse physiological processes. Although genome-wide analyses of the ABA receptor PYR/PYL/RCAR (PYL) protein/gene family have been performed in certain plant species, little is known about the ABA receptor protein/gene family in the rubber tree (Hevea brasiliensis). In this study, we identified 14 ABA receptor PYL proteins/genes (designated HbPYL1 through HbPYL14) in the most recent rubber tree genome. A phylogenetic tree was constructed, which demonstrated that HbPYLs can be divided into three subfamilies that correlate well with the corresponding Arabidopsis subfamilies. Eight HbPYLs are highly expressed in laticifers. Five of the eight genes are simultaneously regulated by ABA, jasmonic acid (JA) and ethylene (ET). The identification and characterization of HbPYLs should enable us to further understand the role of ABA signal in the rubber tree.