Molecular Mechanisms of Natural Rubber Biosynthesis.

Natural rubber (NR), principally comprising cis-1,4-polyisoprene, is an industrially important natural hydrocarbon polymer because of its unique physical properties, which render it suitable for manufacturing items such as tires. Presently, industrial NR production depends solely on latex obtained from the Pará rubber tree, Hevea brasiliensis. In latex, NR is enclosed in rubber particles, which are specialized organelles comprising a hydrophobic NR core surrounded by a lipid monolayer and membrane-bound proteins. The similarity of the basic carbon skeleton structure between NR and dolichols and polyprenols, which are found in most organisms, suggests that the NR biosynthetic pathway is related to the polyisoprenoid biosynthetic pathway and that rubber transferase, which is the key enzyme in NR biosynthesis, belongs to the cis-prenyltransferase family. Here, we review recent progress in the elucidation of molecular mechanisms underlying NR biosynthesis through the identification of the enzymes that are responsible for the formation of the NR backbone structure.

Uncovering mechanisms of rubber biosynthesis in Taraxacum koksaghyz - role of cis-prenyltransferase-like 1 protein.

The Russian dandelion Taraxacum koksaghyz synthesizes considerable amounts of high-molecular-weight rubber in its roots. The characterization of factors that participate in natural rubber biosynthesis is fundamental for the establishment of T. koksaghyz as a rubber crop. The cis-1,4-isoprene polymers are stored in rubber particles. Located at the particle surface, the rubber transferase complex, member of the cis-prenyltransferase (cisPT) enzyme family, catalyzes the elongation of the rubber chains. An active rubber transferase heteromer requires a cisPT subunit (CPT) as well as a CPT-like subunit (CPTL), of which T. koksaghyz has two homologous forms: TkCPTL1 and TkCPTL2, which potentially associate with the rubber transferase complex. Knockdown of TkCPTL1, which is predominantly expressed in latex, led to abolished poly(cis-1,4-isoprene) synthesis but unaffected dolichol content, whereas levels of triterpenes and inulin were elevated in roots. Analyses of latex from these TkCPTL1-RNAi plants revealed particles that were similar to native rubber particles regarding their particle size, phospholipid composition, and presence of small rubber particle proteins (SRPPs). We found that the particles encapsulated triterpenes in a phospholipid shell stabilized by SRPPs. Conversely, downregulating the low-expressed TkCPTL2 showed no altered phenotype, suggesting its protein function is redundant in T. koksaghyz. MS-based comparison of latex proteomes from TkCPTL1-RNAi plants and T. koksaghyz wild-types discovered putative factors that convert metabolites in biosynthetic pathways connected to isoprenoids or that synthesize components of the rubber particle shell.

Proteomic Landscape Has Revealed Small Rubber Particles Are Crucial Rubber Biosynthetic Machines for Ethylene-Stimulation in Natural Rubber Production.

Rubber particles are a specific organelle for natural rubber biosynthesis (NRB) and storage. Ethylene can significantly improve rubber latex production by increasing the generation of small rubber particles (SRPs), regulating protein accumulation, and activating many enzyme activities. We conducted a quantitative proteomics study of different SRPs upon ethylene stimulation by differential in-gel electrophoresis (DIGE) and using isobaric tags for relative and absolute quantification (iTRAQ) methods. In DIGE, 79 differentially accumulated proteins (DAPs) were determined as ethylene responsive proteins. Our results show that the abundance of many NRB-related proteins has been sharply induced upon ethylene stimulation. Among them, 23 proteins were identified as rubber elongation factor (REF) and small rubber particle protein (SRPP) family members, including 16 REF and 7 SRPP isoforms. Then, 138 unique phosphorylated peptides, containing 129 phosphorylated amino acids from the 64 REF/SRPP family members, were identified, and most serine and threonine were phosphorylated. Furthermore, we identified 226 DAPs from more than 2000 SRP proteins by iTRAQ. Integrative analysis revealed that almost all NRB-related proteins can be detected in SRPs, and many proteins are positively responsive to ethylene stimulation. These results indicate that ethylene may stimulate latex production by regulating the accumulation of some key proteins. The phosphorylation modification of REF and SRPP isoforms might be crucial for NRB, and SRP may act as a complex natural rubber biosynthetic machine.

Proteomic Landscape of the Mature Roots in a Rubber-Producing Grass Taraxacum Kok-saghyz.

The rubber grass Taraxacum kok-saghyz (TKS) contains large amounts of natural rubber (cis-1,4-polyisoprene) in its enlarged roots and it is an alternative crop source of natural rubber. Natural rubber biosynthesis (NRB) and storage in the mature roots of TKS is a cascade process involving many genes, proteins and their cofactors. The TKS genome has just been annotated and many NRB-related genes have been determined. However, there is limited knowledge about the protein regulation mechanism for NRB in TKS roots. We identified 371 protein species from the mature roots of TKS by combining two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Meanwhile, a large-scale shotgun analysis of proteins in TKS roots at the enlargement stage was performed, and 3545 individual proteins were determined. Subsequently, all identified proteins from 2-DE gel and shotgun MS in TKS roots were subject to gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and most proteins were involved in carbon metabolic process with catalytic activity in membrane-bounded organelles, followed by proteins with binding ability, transportation and phenylpropanoid biosynthesis activities. Fifty-eight NRB-related proteins, including eight small rubber particle protein (SRPP) and two rubber elongation factor(REF) members, were identified from the TKS roots, and these proteins were involved in both mevalonate acid (MVA) and methylerythritol phosphate (MEP) pathways. To our best knowledge, it is the first high-resolution draft proteome map of the mature TKS roots. Our proteomics of TKS roots revealed both MVA and MEP pathways are important for NRB, and SRPP might be more important than REF for NRB in TKS roots. These findings would not only deepen our understanding of the TKS root proteome, but also provide new evidence on the roles of these NRB-related proteins in the mature TKS roots.

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.

Function of Hevea brasiliensis NAC1 in dehydration-induced laticifer differentiation and latex biosynthesis.

MAIN CONCLUSIONS: HbNAC1 is a transcription factor in rubber plants whose expression is induced by dehydration, leading to latex biosynthesis. Laticifer is a special tissue in Hevea brasiliensis where natural rubber is biosynthesized and accumulated. In young stems of epicormic shoots, the differentiation of secondary laticifers can be induced by wounding, which can be prevented when the wounding site is wrapped. Using this system, differentially expressed genes were screened by suppression subtractive hybridization (SSH) and macroarray analyses. This led to the identification of several dehydration-related genes that could be involved in laticifer differentiation and/or latex biosynthesis, including a NAC transcription factor (termed as HbNAC1). Tissue sections confirmed that local tissue dehydration was a key signal for laticifer differentiation. HbNAC1 was localized at the nucleus and showed strong transcriptional activity in yeast, suggesting that HbNAC1 is a transcription factor. Furthermore, HbNAC1 was found to bind to the cis-element CACG in the promoter region of the gene encoding the small rubber particle protein (SRPP). Transgenic experiments also confirmed that HbNAC1 interacted with the SRPP promoter when co-expressed, and enhanced expression of the reporter gene ß-glucuronidase occurred in planta. In addition, overexpression of HbNAC1 in tobacco plants conferred drought tolerance. Together, the data suggest that HbNAC1 might be involved in dehydration-induced laticifer differentiation and latex biosynthesis.

Novel Insights into the Organization of Laticifer Cells: A Cell Comprising a Unified Whole System.

Laticifer cells are specialized plant cells that synthesize and accumulate latex. Studies on laticifers have lagged behind in recent years, and data regarding the functional role of laticifers and their fitness benefit still remain elusive. Laticifer differentiation and its impact on plant growth and development also remain to be investigated. Here, cellular, molecular, and genetic tools were developed to examine the distribution, differentiation, ontogeny, and other characteristic features, as well as the potential developmental role of laticifer cells in the latex-bearing plant Euphorbia lathyris. The organization of the laticiferous system within the E. lathyris plant body is reported, emerging as a single elongated and branched coenocytic cell, constituting the largest cell type existing in plants. We also report the ontogeny and organization of laticifer cells in the embryo and the identification of a laticifer-associated gene expression pattern. Moreover, the identification of laticifer- and latex-deficient mutants (pil mutants) allowed for the identification of distinct loci regulating laticifer differentiation, growth, and metabolic activity. Additionally, pil mutants revealed that laticifer cells appear nonessential for plant growth and development, thus pointing toward their importance, instead, for specific ecophysiological adaptations of latex-bearing plants in natural environments.

Comparative Proteomics of Rubber Latex Revealed Multiple Protein Species of REF/SRPP Family Respond Diversely to Ethylene Stimulation among Different Rubber Tree Clones.

Rubber elongation factor (REF) and small rubber particle protein (SRPP) are two key factors for natural rubber biosynthesis. To further understand the roles of these proteins in rubber formation, six different genes for latex abundant REF or SRPP proteins, including REF138,175,258 and SRPP117,204,243, were characterized from Hevea brasiliensis Reyan (RY) 7-33-97. Sequence analysis showed that REFs have a variable and long N-terminal, whereas SRPPs have a variable and long C-terminal beyond the REF domain, and REF258 has a ß subunit of ATPase in its N-terminal. Through two-dimensional electrophoresis (2-DE), each REF/SRPP protein was separated into multiple protein spots on 2-DE gels, indicating they have multiple protein species. The abundance of REF/SRPP proteins was compared between ethylene and control treatments or among rubber tree clones with different levels of latex productivity by analyzing 2-DE gels. The total abundance of each REF/SRPP protein decreased or changed a little upon ethylene stimulation, whereas the abundance of multiple protein species of the same REF/SRPP changed diversely. Among the three rubber tree clones, the abundance of the protein species also differed significantly. Especially, two protein species of REF175 or REF258 were ethylene-responsive only in the high latex productivity clone RY 8-79 instead of in RY 7-33-97 and PR 107. Some individual protein species were positively related to ethylene stimulation and latex productivity. These results suggested that the specific protein species could be more important than others for rubber production and post-translational modifications might play important roles in rubber biosynthesis.

Molecular mechanism of ethylene stimulation of latex yield in rubber tree (Hevea brasiliensis) revealed by de novo sequencing and transcriptome analysis.

BACKGROUND: Rubber tree (Hevea brasiliensis) is an important industrial crop cultivated in tropical areas for natural rubber production. Treatment of the bark of rubber trees with ehephon (an ethylene releaser) has been a routine measure to increase latex yield, but the molecular mechanism behind the stimulation of rubber production by ethylene still remains a puzzle. Deciphering the enigma is of great importance for improvement of rubber tree for high yield. RESULTS: De novo sequencing and assembly of the bark transciptomes of Hevea brasiliensis induced with ethephon for 8 h (E8) and 24 h (E24) were performed. 51,965,770, 52,303,714 and 53,177,976 high-quality clean reads from E8, E24 and C (control) samples were assembled into 81,335, 80,048 and 80,800 unigenes respectively, with a total of 84,425 unigenes and an average length of 1,101 bp generated. 10,216 and 9,374 differentially expressed genes (DEGs) in E8 and E24 compared with C were respectively detected. The expression of several enzymes in crucial points of regulation in glycolysis were up-regulated and DEGs were not significantly enriched in isopentenyl diphosphate (IPP) biosynthesis pathway. In addition, up-regulated genes of great regulatory importance in carbon fixation (Calvin cycle) were identified. CONCLUSIONS: The rapid acceleration of glycolytic pathway supplying precursors for the biosynthesis of IPP and natural rubber, instead of rubber biosynthesis per se, may be responsible for ethylene stimulation of latex yield in rubber tree. The elevated rate of flux throughout the Calvin cycle may account for some durability of ethylene-induced stimulation. Our finding lays the foundations for molecular diagnostic and genetic engineering for high-yielding improvement of rubber tree.

The Hevea brasiliensis XIP aquaporin subfamily: genomic, structural and functional characterizations with relevance to intensive latex harvesting.

X-Intrinsic Proteins (XIP) were recently identified in a narrow range of plants as a full clade within the aquaporins. These channels reportedly facilitate the transport of a wide range of hydrophobic solutes. The functional roles of XIP in planta remain poorly identified. In this study, we found three XIP genes (HbXIP1;1, HbXIP2;1 and HbXIP3;1) in the Hevea brasiliensis genome. Comprehensive bioinformatics, biochemical and structural analyses were used to acquire a better understanding of this AQP subfamily. Phylogenetic analysis revealed that HbXIPs clustered into two major groups, each distributed in a specific lineage of the order Malpighiales. Tissue-specific expression profiles showed that only HbXIP2;1 was expressed in all the vegetative tissues tested (leaves, stem, bark, xylem and latex), suggesting that HbXIP2;1 could take part in a wide range of cellular processes. This is particularly relevant to the rubber-producing laticiferous system, where this isoform was found to be up-regulated during tapping and ethylene treatments. Furthermore, the XIP transcriptional pattern is significantly correlated to latex production level. Structural comparison with SoPIP2;1 from Spinacia oleracea species provides new insights into the possible role of structural checkpoints by which HbXIP2;1 ensures glycerol transfer across the membrane. From these results, we discuss the physiological involvement of glycerol and HbXIP2;1 in water homeostasis and carbon stream of challenged laticifers. The characterization of HbXIP2;1 during rubber tree tapping lends new insights into molecular and physiological response processes of laticifer metabolism in the context of latex exploitation.

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.

Involvement of Ethylene in the Latex Metabolism and Tapping Panel Dryness of Hevea brasiliensis.

Ethephon, an ethylene releaser, is used to stimulate latex production in Hevea brasiliensis. Ethylene induces many functions in latex cells including the production of reactive oxygen species (ROS). The accumulation of ROS is responsible for the coagulation of rubber particles in latex cells, resulting in the partial or complete stoppage of latex flow. This study set out to assess biochemical and histological changes as well as changes in gene expression in latex and phloem tissues from trees grown under various harvesting systems. The Tapping Panel Dryness (TPD) susceptibility of Hevea clones was found to be related to some biochemical parameters, such as low sucrose and high inorganic phosphorus contents. A high tapping frequency and ethephon stimulation induced early TPD occurrence in a high latex metabolism clone and late occurrence in a low latex metabolism clone. TPD-affected trees had smaller number of laticifer vessels compared to healthy trees, suggesting a modification of cambial activity. The differential transcript abundance was observed for twenty-seven candidate genes related to TPD occurrence in latex and phloem tissues for ROS-scavenging, ethylene biosynthesis and signalling genes. The predicted function for some Ethylene Response Factor genes suggested that these candidate genes should play an important role in regulating susceptibility to TPD.

Phylogenetic escalation and decline of plant defense strategies.

As the basal resource in most food webs, plants have evolved myriad strategies to battle consumption by herbivores. Over the past 50 years, plant defense theories have been formulated to explain the remarkable variation in abundance, distribution, and diversity of secondary chemistry and other defensive traits. For example, classic theories of enemy-driven evolutionary dynamics have hypothesized that defensive traits escalate through the diversification process. Despite the fact that macroevolutionary patterns are an explicit part of defense theories, phylogenetic analyses have not been previously attempted to disentangle specific predictions concerning (i) investment in resistance traits, (ii) recovery after damage, and (iii) plant growth rate. We constructed a molecular phylogeny of 38 species of milkweed and tested four major predictions of defense theory using maximum-likelihood methods. We did not find support for the growth-rate hypothesis. Our key finding was a pattern of phyletic decline in the three most potent resistance traits (cardenolides, latex, and trichomes) and an escalation of regrowth ability. Our neontological approach complements more common paleontological approaches to discover directional trends in the evolution of life and points to the importance of natural enemies in the macroevolution of species. The finding of macroevolutionary escalating regowth ability and declining resistance provides a window into the ongoing coevolutionary dynamics between plants and herbivores and suggests a revision of classic plant defense theory. Where plants are primarily consumed by specialist herbivores, regrowth (or tolerance) may be favored over resistance traits during the diversification process.

Opposing roles of plant laticifer cells in the resistance to insect herbivores and fungal pathogens.

More than 12,000 plant species (ca. 10% of flowering plants) exude latex when their tissues are injured. Latex is produced and stored in specialized cells named "laticifers". Laticifers form a tubing system composed of rows of elongated cells that branch and create an internal network encompassing the entire plant. Laticifers constitute a recent evolutionary achievement in ecophysiological adaptation to specific natural environments; however, their fitness benefit to the plant still remains to be proven. The identification of Euphorbia lathyris mutants (pil mutants) deficient in laticifer cells or latex metabolism, and therefore compromised in latex production, allowed us to test the importance of laticifers in pest resistance. We provided genetic evidence indicating that laticifers represent a cellular adaptation for an essential defense strategy to fend off arthropod herbivores with different feeding habits, such as Spodoptera exigua and Tetranychus urticae. In marked contrast, we also discovered that a lack of laticifer cells causes complete resistance to the fungal pathogen Botrytis cinerea. Thereafter, a latex-derived factor required for conidia germination on the leaf surface was identified. This factor promoted disease susceptibility enhancement even in the non-latex-bearing plant Arabidopsis. We speculate on the role of laticifers in the co-evolutionary arms race between plants and their enemies.

Structure-function studies of ultrahigh molecular weight isoprenes provide key insights into their biosynthesis.

Some plant trans-1,4-prenyltransferases (TPTs) produce ultrahigh molecular weight trans-1,4-polyisoprene (TPI) with a molecular weight of over 1.0 million. Although plant-derived TPI has been utilized in various industries, its biosynthesis and physiological function(s) are unclear. Here, we identified three novel Eucommia ulmoides TPT isoforms-EuTPT1, 3, and 5, which synthesized TPI in vitro without other components. Crystal structure analysis of EuTPT3 revealed a dimeric architecture with a central hydrophobic tunnel. Mutation of Cys94 and Ala95 on the central hydrophobic tunnel no longer synthesizd TPI, indicating that Cys94 and Ala95 were essential for forming the dimeric architecture of ultralong-chain TPTs and TPI biosynthesis. A spatiotemporal analysis of the physiological function of TPI in E. ulmoides suggested that it is involved in seed development and maturation. Thus, our analysis provides functional and mechanistic insights into TPI biosynthesis and uncovers biological roles of TPI in plants.

The sucrose transporter HbSUT3 plays an active role in sucrose loading to laticifer and rubber productivity in exploited trees of Hevea brasiliensis (para rubber tree).

Efficient sucrose loading in rubber-producing cells (laticifer cells) is essential for retaining rubber productivity in Hevea brasiliensis, but the molecular mechanisms underlying the regulation of this process remain unknown. Here, we functionally characterized a putative Hevea SUT member, HbSUT3, mainly in samples from regularly exploited trees. When expressed in yeast, HbSUT3 encodes a functional sucrose transporter that exhibits high sucrose affinity with a K(m) value of 1.24 mm at pH 4.0, and possesses features typical of sucrose/H(+) symporters. In planta, when compared to the expression of other Hevea SUT genes, HbSUT3 was found to be the predominant member expressed in the rubber-containing cytoplasm (latex) of laticifers. The comparison of HbSUT3 expression among twelve Hevea tissues demonstrates a relatively tissue-specific pattern, i.e. expression primarily in the latex and in female flowers. HbSUT3 expression is induced by the latex stimulator Ethrel (an ethylene generator), and relates to its yield-stimulating effect. Tapping (the act of rubber harvesting) markedly increased the expression of HbSUT3, whereas wounding alone had little effect. Moreover, the expression of HbSUT3 was found to be positively correlated with latex yield. Taken together, our results provide evidence favouring the involvement of HbSUT3 in sucrose loading into laticifers and in rubber productivity.

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.

Got milk? The secret life of laticifers.

Laticifers are specialized cells that occur in over 20 plant families in several unrelated angiosperm orders. Although laticifers are likely to be of polyphyletic origin, their occurrence is considered a morphological indicator of relatedness among species. The classification of laticifers is based on developmental patterns and overall morphology. The cytoplasmic latex exuded in response to damage often includes specialized metabolites, such as cardenolides, alkaloids and natural rubber. Laticifers provide an effective location to store defense metabolites, although not all latex-bearing plants accumulate bioactive natural products. Ecophysiological studies have shown that latex and its associated metabolites are vital for the defense of plants against insects. The anatomy, development and physiology of laticifers are discussed with a focus on evolutionary and ecological perspectives.

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.

Ball milling pretreatment facilitating α-amylase hydrolysis for production of starch-based bio-latex with high performance.

Starch based bio-latex has been widely researched in the coating paper area for the purpose of partial replacement of petroleum-based binders. In this paper, a green and facile ball milling pretreatment was proposed to modify the starch granules before α-amylase hydrolysis by breaking up their crystalline structure, thus improving the accessibility and susceptibility of amylase into starch structure. It was found that the improved hydrolysis process after 8 h ball milling can generate suitable degree of polymerization of polysaccharides or oligosaccharides, which further facilitated the following H2O2 oxidation and SHMP crosslinking processes. In addition, a mechanism was also demonstrated to illustrate the improvement induced by ball milling pretreatment. The prepared bio-latex with crosslinking-structure performed excellent adhesive properties when substituted 25 % of petroleum-based latex during paper coating application, which showed great potential in improving the economic, cost, and environment benefits of traditional production of coated paper.