Downregulation of HbFPS1 affects rubber biosynthesis of Hevea brasiliensis suffering from tapping panel dryness.

Tapping panel dryness (TPD) is a century-old problem that has plagued the natural rubber production of Hevea brasiliensis. TPD may result from self-protective mechanisms of H. brasiliensis in response to stresses such as excessive hormone stimulation and mechanical wounding (bark tapping). It has been hypothesized that TPD impairs rubber biosynthesis; however, the underlying mechanisms remain poorly understood. In the present study, we firstly verified that TPD-affected rubber trees exhibited lower rubber biosynthesis activity and greater rubber molecular weight compared to healthy rubber trees. We then demonstrated that HbFPS1, a key gene of rubber biosynthesis, and its expression products were downregulated in the latex of TPD-affected rubber trees, as revealed by transcriptome sequencing and iTRAQ-based proteome analysis. We further discovered that the farnesyl diphosphate synthase HbFPS1 could be recruited to small rubber particles by HbSRPP1 through protein-protein interactions to catalyze farnesyl diphosphate (FPP) synthesis and facilitate rubber biosynthesis initiation. FPP content in the latex of TPD-affected rubber trees was significantly decreased with the downregulation of HbFPS1, ultimately resulting in abnormal development of rubber particles, decreased rubber biosynthesis activity, and increased rubber molecular weight. Upstream regulator assays indicated that a novel regulator, MYB2-like, may be an important regulator of downregulation of HbFPS1 in the latex of TPD-affected rubber trees. Our findings not only provide new directions for studying the molecular events involved in rubber biosynthesis and TPD syndrome and contribute to rubber management strategies, but also broaden our knowledge of plant isoprenoid metabolism and its regulatory networks.

HbWRKY82, a novel IIc WRKY transcription factor from Hevea brasiliensis associated with abiotic stress tolerance and leaf senescence in Arabidopsis.

WRKY group transcription factors of model plants and major crops are confirmed to play essential roles in stress responses, senescence, secondary metabolism processes and hormone signal transduction. Previous studies have identified 81 HbWRKY genes from Hevea brasiliensis (the Pará rubber tree), but the functions of HbWRKYs in response to abiotic stresses and leaf senescence are unclear. In this study, one novel group IIc WRKY transcription factor named HbWRKY82 was identified and characterized as a stress-associated WRKY in rubber tree. Transient expression and transcriptional activation analyses indicated that HbWRKY82 encoded a nuclear protein and functioned as a transcription activator. The transcription levels of HbWRKY82 were induced by exogenous Ethrel (ET) (ethylene releaser) and abscisic acid (ABA) stimulations, down-regulated in tapping panel dryness rubber trees, and also exhibits significant decrease during the progression of leaf senescence. Overexpression of HbWRKY82 in Arabidopsis improved the tolerance to dehydration and salinity, and decreased the sensitivity to exogenous ABA. Moreover, real-time quantitative PCR analysis demonstrated that HbWRKY82 regulated the transcriptional expression of several stress-responsive genes (DREB1A, ERD10, HKT1, P5CS, RD22, RD29B, SKOR), leaf senescence marker genes (EIN3, WRKY53, NAP), ROS-related genes (RbohD, CSD1, CSD2, FSD3) and hormone signaling genes (EIN3, ABF3, ABF4). Collectively, our findings suggested that HbWRKY82 might function as an important transcriptional regulator in ET- and ABA-mediated leaf senescence and abiotic stress responses, and also be involved in tapping panel dryness, latex flow and regeneration processes of rubber trees via participating in the ET and reactive oxygen species signaling pathways.

Molecular characterization and functional analysis of a novel WRKY transcription factor HbWRKY83 possibly involved in rubber production of Hevea brasiliensis.

WRKY transcription factors play important roles in plant growth and developmental processes and various stress responses, and are also associated with jasmonic acid (JA) signaling in the regulation of secondary metabolite biosynthesis in plants. The regulatory networks mediated by WRKY proteins in the latex production of Hevea brasiliensis (the Pará rubber tree) are poorly understood. In this study, one novel WRKY gene (designated HbWRKY83) was identified from the latex of H. brasiliensis, and its functions were characterized via gene expression analysis in both the latex and HbWRKY83-overexpressing transgenic Arabidopsis. HbWRKY83 gene contains an open reading frame (ORF) of 921 bp encoding a 306-amino-acid protein which is clustered with group IIc WRKY TF. HbWRKY83 is a nuclear-localized protein with transcriptional activity. Real-time quantitative PCR analysis demonstrated that the transcription level of HbWRKY83 was up-regulated by exogenous methyl jasmonate, Ethrel (ethylene releaser) stimulation, and bark tapping (mechanical wounding). Compared with the wild-type plants, overexpression of HbWRKY83 improved the tolerance of transgenic Arabidopsis lines to drought and salt stresses by enhancing the expression levels of ethylene-insensitive3 transcription factors (EIN3s) and several stress-responsive genes, including Cu/Zn superoxide dismutases CSD1 (Cu/Zn-SOD1) and CSD2 (Cu/Zn-SOD2), related to reactive oxygen species scavenging. Additionally, these genes were also significantly up-regulated by bark tapping. In combination, these results suggest that HbWRKY83 might act as a positive regulator of rubber production by activating the expression of JA-, ethylene-, and wound-responsive genes in the laticiferous cells of rubber trees.

Acyl-CoA-binding protein family members in laticifers are possibly involved in lipid and latex metabolism of Hevea brasiliensis (the Para rubber tree).

BACKGROUND: Acyl-CoA-binding proteins (ACBPs) are mainly involved in acyl-CoA ester binding and trafficking in eukaryotic cells, and their various functions have been characterized in model plants, such as Arabidopsis thaliana (A. thaliana), Oryza sativa (rice), and other plant species. In the present study, genome-wide mining and expression analysis of ACBP genes was performed on Hevea brasiliensis (the para rubber tree), the most important latex-producing crop in the world. RESULTS: Six members of the H. brasiliensis ACBP family genes, designated HbACBP1-HbACBP6, were identified from the H. brasiliensis genome. They can be categorized into four classes with different amino acid sequences and domain structures based on the categorization of their A. thaliana counterparts. Phylogenetic analysis shows that the HbACBPs were clustered with those of other closely related species, such as Manihot esculenta, Ricinus communis, and Jatropha carcas, but were further from those of A. thaliana, a distantly related species. Expression analysis demonstrated that the HbACBP1 and HbACBP2 genes are more prominently expressed in H. brasiliensis latex, and their expression can be significantly enhanced by bark tapping (a mechanical wound) and jasmonic acid stimulation, whereas HbACBP3-HbACBP6 had almost the same expression patterns with relatively high levels in mature leaves and male flowers, but a markedly low abundance in the latex. HbACBP1 and HbACBP2 may have crucial roles in lipid and latex metabolism in laticifers, so their subcellular location was further investigated and the results indicated that HbACBP1 is a cytosol protein, whereas HbACBP2 is an endoplasmic reticulum-associated ACBP. CONCLUSIONS: In this study, the H. brasiliensis ACBP family genes were identified. Phylogenetic analyses of the HbABCPs indicate that there is a high conservation and evolutionary relationship between ACBPs in land plants. The HbACBPs are organ/tissue-specifically expressed and have different expression patterns in response to stimulation by bark tapping or ethrel/jasmonic acid. HbACBP1 and HbACBP2 are two important latex ACBPs that might be involved in the lipid and latex metabolism. The results may provide valuable information for further investigations into the biological functions of HbACBPs during latex metabolism and stress responses in H. brasiliensis.

Identification and subcellular localization analysis of two rubber elongation factor isoforms on Hevea brasiliensis rubber particles.

Rubber elongation factor (REF) is the most abundant protein found on the rubber particles or latex from Hevea brasiliensis (the Para rubber tree) and is considered to play important roles in natural rubber (cis-polyisoprene) biosynthesis. 16 BAC (benzyldimethyl-n-hexadecylammonium chloride)/SDS-PAGE separations and mass spectrometric identification had revealed that two REF isoforms shared similar amino acid sequences and common C-terminal sequences. In this study, the gene sequences encoding these two REF isoforms (one is 23.6 kDa in size with 222 amino acid residues and the other is 27.3 kDa in size with 258 amino acid residues) were obtained. Their proteins were relatively enriched by sequential extraction of the rubber particle proteins and separated by 16 BAC/SDS-PAGE. The localization of these isoforms on the surfaces of rubber particles was further verified by western blotting and immunogold electron microscopy, which demonstrated that these two REF isoforms are mainly located on the surfaces of larger rubber particles and that they bind more tightly to rubber particles than the most abundant REF and SRPP (small rubber particle protein).

Profiling Ethylene-Responsive Genes Expressed in the Latex of the Mature Virgin Rubber Trees Using cDNA Microarray.

Ethylene is commonly used as a latex stimulant of Hevea brasiliensis by application of ethephon (chloro-2-ethylphosphonic acid); however, the molecular mechanism by which ethylene increases latex production is not clear. To better understand the effects of ethylene stimulation on the laticiferous cells of rubber trees, a latex expressed sequence tag (EST)-based complementary DNA microarray containing 2,973 unique genes (probes) was first developed and used to analyze the gene expression changes in the latex of the mature virgin rubber trees after ethephon treatment at three different time-points: 8, 24 and 48 h. Transcript levels of 163 genes were significantly altered with fold-change values ≥ 2 or ≤ -2 (q-value < 0.05) in ethephon-treated rubber trees compared with control trees. Of the 163 genes, 92 were up-regulated and 71 down-regulated. The microarray results were further confirmed using real-time quantitative reverse transcript-PCR for 20 selected genes. The 163 ethylene-responsive genes were involved in several biological processes including organic substance metabolism, cellular metabolism, primary metabolism, biosynthetic process, cellular response to stimulus and stress. The presented data suggest that the laticifer water circulation, production and scavenging of reactive oxygen species, sugar metabolism, and assembly and depolymerization of the latex actin cytoskeleton might play important roles in ethylene-induced increase of latex production. The results may provide useful insights into understanding the molecular mechanism underlying the effect of ethylene on latex metabolism of H. brasiliensis.

Comparative proteomic analysis of latex from Hevea brasiliensis treated with Ethrel and methyl jasmonate using iTRAQ-coupled two-dimensional LC-MS/MS.

UNLABELLED: Ethrel (ET) is an effective and widely used latex yield stimulant of Hevea brasiliensis (Pará rubber tree), and jasmonate (JA) is a key inducer of laticifer differentiation in this plant. To examine variations in the latex proteome caused by these phytohormones, ET and methyl jasmonate (MeJA) were applied to Reyan 7-33-97 rubber tree clones, and comparative proteomic analyses were conducted. On the basis of a transcriptome shotgun assembly (TSA) sequence database and an iTRAQ-coupled two-dimensional LC-MS/MS approach, 1499 latex proteins belonging to 1078 clusters were identified. With a 1.5-fold cut-off value to determine up- and down-regulated proteins, a total of 101 latex proteins were determined to be regulated by ET and/or MeJA via pairwise comparisons among the three exposure durations (0 h, 6 h, and 48 h). Proteins associated with latex regeneration, including phosphoenolpyruvate carboxylase and acetyl-CoA C-acetyltransferase, and those associated with latex flow, such as chitinase and a sieve element occlusion protein, were affected by the application of ET. Chitinase and polyphenol oxidase were also found to be regulated by MeJA. The findings of this study may provide new insight into the roles of phytohormones in latex yield and the causative mechanisms of laticifer differentiation in rubber trees. SIGNIFICANCE: On the basis of a transcriptome shotgun assembly (TSA) sequence database and an iTRAQ-coupled two-dimensional LC-MS/MS approach, the most comprehensive proteome of the latex was profiled, and the ethylene-/jasmonate-responsive proteins were identified in the latex of H. brasiliensis. The findings of this study may provide new insight into the role of phytohormones in latex yield and the causative mechanisms of laticifer differentiation in rubber trees.

In-depth proteome analysis of the rubber particle of Hevea brasiliensis (para rubber tree).

The rubber particle is a special organelle in which natural rubber is synthesised and stored in the laticifers of Hevea brasiliensis. To better understand the biological functions of rubber particles and to identify the candidate rubber biosynthesis-related proteins, a comprehensive proteome analysis was performed on H. brasiliensis rubber particles using shotgun tandem mass spectrometry profiling approaches-resulting in a thorough report on the rubber particle proteins. A total of 186 rubber particle proteins were identified, with a range in relative molecular mass of 3.9-194.2 kDa and in isoelectric point values of 4.0-11.2. The rubber particle proteins were analysed for gene ontology and could be categorised into eight major groups according to their functions: including rubber biosynthesis, stress- or defence-related responses, protein processing and folding, signal transduction and cellular transport. In addition to well-known rubber biosynthesis-related proteins such as rubber elongation factor (REF), small rubber particle protein (SRPP) and cis-prenyl transferase (CPT), many proteins were firstly identified to be on the rubber particles, including cyclophilin, phospholipase D, cytochrome P450, small GTP-binding protein, clathrin, eukaryotic translation initiation factor, annexin, ABC transporter, translationally controlled tumour protein, ubiquitin-conjugating enzymes, and several homologues of REF, SRPP and CPT. A procedure of multiple reaction monitoring was established for further protein validation. This comprehensive proteome data of rubber particles would facilitate investigation into molecular mechanisms of biogenesis, self-homeostasis and rubber biosynthesis of the rubber particle, and might serve as valuable biomarkers in molecular breeding studies of H. brasiliensis and other alternative rubber-producing species.

Proteome analysis of the large and the small rubber particles of Hevea brasiliensis using 2D-DIGE.

The rubber particle is a specialized organelle in which natural rubber is synthesised and stored in the laticifers of Hevea brasiliensis (para rubber tree). It has been demonstrated that the small rubber particles (SRPs) has higher rubber biosynthesis ratio than the large rubber particles (LRPs), but the underlying molecular mechanism still remains unknown. In this study, LRPs and SRPs were firstly separated from the fresh latex using differential centrifugation, and two-dimensional difference in-gel electrophoresis (2D-DIGE) combined with MALDI-TOF/TOF was then applied to investigate the proteomic alterations associated with the changed rubber biosynthesis capacity between LRPs and SRPs. A total of 53 spots corresponding to 22 gene products, were significantly altered with the |ratio|≥2.0 and T value ≤0.05, among which 15 proteins were up-regulated and 7 were down-regulated in the SRPs compared with the LRPs. The 15 up-regulated proteins in the SRPs included small rubber particle protein (SRPP), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS), phospholipase D alpha (PLD α), ethylene response factor 2, eukaryotic translation initiation factor 5A isoform IV (eIF 5A-4), 70-kDa heat shock cognate protein (HSC 70), several unknown proteins, etc., whereas the 7 up-regulated proteins in the LRPs were rubber elongation factor (REF, 19.6kDa), ASR-like protein 1, REF-like stress-related protein 1, a putative phosphoglyceride transfer family protein, ß-1,3-glucanase, a putative retroelement, and a hypothetical protein. Since several proteins related to rubber biosynthesis were differentially expressed between LRPs and SRPs, the comparative proteome data may provide useful insights into understanding the mechanism involved in rubber biosynthesis and latex coagulation in H. brasiliensis.

Cloning and expression analysis of a G-protein α subunit--Gαo in the rice water weevil Lissorhoptrus oryzophilus Kuschel.

The open reading frame (ORF) encoding a novel G protein α subunit, Lo Gα(o), was cloned from the parthenogenetic rice water weevil, Lissorhoptrus oryzophilus Kuschel (Coleoptera: Curculionidae). The Lo Gα(o) ORF encodes a protein of 354 amino acid residues. The deduced protein sequence shares high homology with Gα(o) from other species. The expression patterns of Lo Gα(o) in various adult tissues were indicated by real-time quantitative PCR and Western blot. The results showed that Lo Gα(o) mRNA was expressed at similar levels in tissues except relative high levels in the antennae of adult, and Lo Gα(o) protein of an apparent molecular mass of about 40 kDa was expressed in various tissues of the adult. Immunocytochemical localization showed that Lo Gα(o) was mainly expressed in the dendrites of the trichoid sensilla in the antenna of the weevil. The tissue and cellular localization of Lo Gα(o) suggests that Lo Gα(o) may take a part in signal transduction of olfactory/gustatory.