Arabidopsis membrane protein AMAR1 interaction with type III effector XopAM triggers a hypersensitive response.

The efficient infection of plants by the bacteria Xanthomonas campestris pv. campestris (Xcc) depends on its type III effectors (T3Es). Although the functions of AvrE family T3Es have been reported in some bacteria, the member XopAM in Xcc has not been studied. As XopAM has low sequence similarity to reported AvrE-T3Es and different reports have shown that these T3Es have different targets in hosts, we investigated the functions of XopAM in the Xcc-plant interaction. Deletion of xopAM from Xcc reduced its virulence in cruciferous crops but increased virulence in Arabidopsis (Arabidopsis thaliana) Col-0, indicating that XopAM may perform opposite functions depending on the host species. We further found that XopAM is a lipase that may target the cytomembrane and that this activity might be enhanced by its membrane-targeted protein XOPAM-ACTIVATED RESISTANCE 1 (AMAR1) in Arabidopsis Col-0. The binding of XopAM to AMAR1 induced an intense hypersensitive response that restricted Xcc proliferation. Our results showed that the roles of XopAM in Xcc infection are not the same as those of other AvrE-T3Es, indicating that the functions of this type of T3E have differentiated during long-term bacterium‒host interactions.

Overexpression of HbGRF4 or HbGRF4-HbGIF1 Chimera Improves the Efficiency of Somatic Embryogenesis in Hevea brasiliensis.

Transgenic technology is a crucial tool for gene functional analysis and targeted genetic modification in the para rubber tree (Hevea brasiliensis). However, low efficiency of plant regeneration via somatic embryogenesis remains a bottleneck of successful genetic transformation in H. brasiliensis. Enhancing expression of GROWTH-REGULATING FACTOR 4 (GRF4)-GRF-INTERACTING FACTOR 1 (GIF1) has been reported to significantly improve shoot and embryo regeneration in multiple crops. Here, we identified endogenous HbGRF4 and HbGIF1 from the rubber clone Reyan7-33-97, the expressions of which dramatically increased along with somatic embryo (SE) production. Intriguingly, overexpression of HbGRF4 or HbGRF4-HbGIF1 markedly enhanced the efficiency of embryogenesis in two H. brasiliensis callus lines with contrasting rates of SE production. Transcriptional profiling revealed that the genes involved in jasmonic acid response were up-regulated, whereas those in ethylene biosynthesis and response as well as the S-adenosylmethionine-dependent methyltransferase activity were down-regulated in HbGRF4- and HbGRF4-HbGIF1-overexpressing H. brasiliensis embryos. These findings open up a new avenue for improving SE production in rubber tree, and help to unravel the underlying mechanisms of HbGRF4-enhanced somatic embryogenesis.

Advances in Genome Sequencing and Natural Rubber Biosynthesis in Rubber-Producing Plants.

Natural rubber (cis-1,4-polyisoprene, NR) is an important raw material utilized widely in the manufacturing of medical, agricultural, and industrial products. Rubber tree (Hevea brasiliensis) and several alternative rubber-producing plants (Taraxacum kok-saghyz, Lactuca sativa, and Parthenium argentatum) have the capability to produce high-quality NR. With the progress of genome sequencing, similar rubber biosynthesis pathways have been discovered among different rubber-producing plant species. NR is synthesized and stored in rubber particles, which are specialized organelles comprising a hydrophobic NR core surrounded by a lipid monolayer and membrane-bound proteins. The rubber transferase complex is considered to be the pivotal enzyme involved in catalyzing NR biosynthesis. However, the exact compositions of the RT complex in rubber-producing plants remain elusive and poorly understood. Here, we review the progress of genome sequencing, natural rubber biosynthesis, and the components of the RT complex in rubber-producing plants. We emphasize that identifying the detailed components of the RT complex holds great significance for exploring the mechanism of NR biosynthesis and accelerating molecular breeding in rubber-producing plants.

Single-cell transcriptomic analyses reveal cellular and molecular patterns of rubber tree response to early powdery mildew infection.

As a perennial woody plant, the rubber tree (Hevea brasiliensis) must adapt to various environmental challenges through gene expression in multiple cell types. It is still unclear how genes in this species are expressed at the cellular level and the precise mechanisms by which cells respond transcriptionally to environmental stimuli, especially in the case of pathogen infection. Here, we characterized the transcriptomes in Hevea leaves during early powdery mildew infection using single-cell RNA sequencing. We identified 10 cell types and constructed the first single-cell atlas of Hevea leaves. Distinct gene expression patterns of the cell clusters were observed under powdery mildew infection, which was especially significant in the epidermal cells. Most of the genes involved in host-pathogen interactions in epidermal cells exhibited a pattern of dramatically increased expression with increasing pseudotime. Interestingly, we found that the HbCNL2 gene, encoding a nucleotide-binding leucine-rich repeat protein, positively modulated the defence of rubber leaves against powdery mildew. Overexpression of the HbCNL2 gene triggered a typical cell death phenotype in tobacco leaves and a higher level of reactive oxygen species in the protoplasts of Hevea leaves. The HbCNL2 protein was located in the cytomembrane and nucleus, and its leucine-rich repeat domain interacted with the histidine kinase-like ATPase domain of the molecular chaperone HbHSP90 in the nucleus. Collectively, our results provide the first observation of the cellular and molecular responses of Hevea leaves to biotrophic pathogen infection and can guide the identification of disease-resistance genes in this important tree species.

Structural and Functional Annotation of Transposable Elements Revealed a Potential Regulation of Genes Involved in Rubber Biosynthesis by TE-Derived siRNA Interference in Hevea brasiliensis.

The natural rubber biosynthetic pathway is well described in Hevea, although the final stages of rubber elongation are still poorly understood. Small Rubber Particle Proteins and Rubber Elongation Factors (SRPPs and REFs) are proteins with major function in rubber particle formation and stabilization. Their corresponding genes are clustered on a scaffold1222 of the reference genomic sequence of the Hevea brasiliensis genome. Apart from gene expression by transcriptomic analyses, to date, no deep analyses have been carried out for the genomic environment of SRPPs and REFs loci. By integrative analyses on transposable element annotation, small RNAs production and gene expression, we analysed their role in the control of the transcription of rubber biosynthetic genes. The first in-depth annotation of TEs (Transposable Elements) and their capacity to produce TE-derived siRNAs (small interfering RNAs) is presented, only possible in the Hevea brasiliensis clone PB 260 for which all data are available. We observed that 11% of genes are located near TEs and their presence may interfere in their transcription at both genetic and epigenetic level. We hypothesized that the genomic environment of rubber biosynthesis genes has been shaped by TE and TE-derived siRNAs with possible transcriptional interference on their gene expression. We discussed possible functionalization of TEs as enhancers and as donors of alternative transcription start sites in promoter sequences, possibly through the modelling of genetic and epigenetic landscapes.

Characterization of a vacuolar sucrose transporter, HbSUT5, from Hevea brasiliensis: involvement in latex production through regulation of intracellular sucrose transport in the bark and laticifers.

BACKGROUND: Sucrose (Suc), as the precursor molecule for rubber biosynthesis in Hevea brasiliensis, is transported via phloem-mediated long-distance transport from leaves to laticifers in trunk bark, where latex (cytoplasm of laticifers) is tapped for rubber. In our previous report, six Suc transporter (SUT) genes have been cloned in Hevea tree, among which HbSUT3 is verified to play an active role in Suc loading to the laticifers. In this study, another latex-abundant SUT isoform, HbSUT5, with expressions only inferior to HbSUT3 was characterized especially for its roles in latex production. RESULTS: Both phylogenetic analysis and subcellular localization identify HbSUT5 as a tonoplast-localized SUT protein under the SUT4-clade (=type III). Suc uptake assay in baker's yeast reveals HbSUT5 to be a typical Suc-H+ symporter, but its high affinity for Suc (Km = 2.03 mM at pH 5.5) and the similar efficiency in transporting both Suc and maltose making it a peculiar SUT under the SUT4-clade. At the transcript level, HbSUT5 is abundantly and preferentially expressed in Hevea barks. The transcripts of HbSUT5 are conspicuously decreased both in Hevea latex and bark by two yield-stimulating treatments of tapping and ethephon, the patterns of which are contrary to HbSUT3. Under the ethephon treatment, the Suc level in latex cytosol decreases significantly, but that in latex lutoids (polydispersed vacuoles) changes little, suggesting a role of the decreased HbSUT5 expression in Suc compartmentalization in the lutoids and thus enhancing the Suc sink strength in laticifers. CONCLUSIONS: Our findings provide insights into the roles of a vacuolar sucrose transporter, HbSUT5, in Suc exchange between lutoids and cytosol in rubber-producing laticifers.

SPL7 and SPL8 represent a novel flowering regulation mechanism in switchgrass.

The aging pathway in flowering regulation is controlled mainly by microRNA156 (miR156). Studies in Arabidopsis thaliana reveal that nine miR156-targeted SQUAMOSA PROMOTER BINDING-LIKE (SPL) genes are involved in the control of flowering. However, the roles of SPLs in flowering remain elusive in grasses. Inflorescence development in switchgrass was characterized using scanning electron microscopy (SEM). Microarray, quantitative reverse transcription polymerase chain reaction (qRT-PCR), chromatin immunoprecipitation (ChIP)-PCR and EMSA were used to identify regulators of phase transition and flowering. Gene function was characterized by downregulation and overexpression of the target genes. Overexpression of SPL7 and SPL8 promotes flowering, whereas downregulation of individual genes moderately delays flowering. Simultaneous downregulation of SPL7/SPL8 results in extremely delayed or nonflowering plants. Furthermore, downregulation of both genes leads to a vegetative-to-reproductive reversion in the inflorescence, a phenomenon that has not been reported in any other grasses. Detailed analyses demonstrate that SPL7 and SPL8 induce phase transition and flowering in grasses by directly upregulating SEPALLATA3 (SEP3) and MADS32. Thus, the SPL7/8 pathway represents a novel regulatory mechanism in grasses that is largely different from that in Arabidopsis. Additionally, genetic modification of SPL7 and SPL8 results in much taller plants with significantly increased biomass yield and sugar release.

The miR156-SPL4 module predominantly regulates aerial axillary bud formation and controls shoot architecture.

Grasses possess basal and aerial axillary buds. Previous studies have largely focused on basal bud (tiller) formation but scarcely touched on aerial buds, which may lead to aerial branch development. Genotypes with and without aerial buds were identified in switchgrass (Panicum virgatum), a dedicated bioenergy crop. Bud development was characterized using scanning electron microscopy. Microarray, RNA-seq and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to identify regulators of bud formation. Gene function was characterized by down-regulation and overexpression. Overexpression of miR156 induced aerial bud formation in switchgrass. Various analyses revealed that SQUAMOSA PROMOTER BINDING PROTEIN LIKE4 (SPL4), one of the miR156 targets, directly regulated aerial axillary bud initiation. Down-regulation of SPL4 promoted aerial bud formation and increased basal buds, while overexpression of SPL4 seriously suppressed bud formation and tillering. RNA-seq and RT-qPCR identified potential downstream genes of SPL4. Unlike all previously reported genes acting as activators of basal bud initiation, SPL4 acts as a suppressor for the formation of both aerial and basal buds. The miR156-SPL4 module predominantly regulates aerial bud initiation and partially controls basal bud formation. Genetic manipulation of SPL4 led to altered plant architecture with increased branching, enhanced regrowth after cutting and improved biomass yield.

HbNIN2, a cytosolic alkaline/neutral-invertase, is responsible for sucrose catabolism in rubber-producing laticifers of Hevea brasiliensis (para rubber tree).

In Hevea brasiliensis, an alkaline/neutral invertase (A/N-Inv) is responsible for sucrose catabolism in latex (essentially the cytoplasm of rubber-producing laticifers, the source of natural rubber) and implicated in rubber yield. However, neither the gene encoding this enzyme nor its molecular and biochemical properties have been well documented. Three Hevea A/N-Inv genes, namely HbNIN1, 2 and 3, were first cloned and characterized in planta and in Escherichia coli. Cellular localizations of HbNIN2 mRNA and protein were probed. From latex, active A/N-Inv proteins were purified, identified, and explored for enzymatic properties. HbNIN2 was identified as the major A/N-Inv gene functioning in latex based on its functionality in E. coli, its latex-predominant expression, the conspicuous localization of its mRNA and protein in the laticifers, and its expressional correlation with rubber yield. An active A/N-Inv protein was partially purified from latex, and determined as HbNIN2. The enhancement of HbNIN2 enzymatic activity by pyridoxal is peculiar to A/N-Invs in other plants. We conclude that HbNIN2, a cytosolic A/N-Inv, is responsible for sucrose catabolism in rubber laticifers. The results contribute to the studies of sucrose catabolism in plants as a whole and natural rubber synthesis in particular.

Transcriptional and post-transcriptional regulation of the jasmonate signalling pathway in response to abiotic and harvesting stress in Hevea brasiliensis.

BACKGROUND: Latex harvesting in Hevea brasiliensis amounts to strong abiotic stress that can cause a halt in production in the most susceptible clones. Although the role of jasmonic acid has been suggested in laticifer differentiation, its role in latex production and in the response to harvesting stress has received very little attention. Only a few key genes acting in the COI-JAZ-MYC module have been isolated and studied at transcriptional level. RESULTS: Use of a reference transcriptome obtained on rubber clone PB 260 covering a large number of tissues under different environmental conditions enabled us to identify 24 contigs implicated in the jasmonate signalling pathway in the rubber tree. An analysis of their expression profile by qPCR, combined with hierarchical clustering, suggested that the jasmonate signalling pathway is highly activated in laticifer cells and, more particularly, in the response to harvesting stress. By comparison with their genomic sequences, the existence of regulation by alternative splicing was discovered for JAZ transcripts in response to harvesting stress. Lastly, positive transcriptional regulation of the HbJAZ_1405 gene by MYC was demonstrated. CONCLUSION: This study led to the identification of all actors of jasmonate signalling pathway and revealed a specific gene expression pattern in latex cells. In-depth analysis of this regulation showed alternative splicing that has been previously shown in Arabidopsis. Interestingly, genotypic variation was observed in Hevea clones with contrasting latex metabolism. This result suggests an involvement of jasmonate signalling pathway in latex production. The data suggest that specific variability of the JA pathway may have some major consequences for resistance to stress. The data support the hypothesis that a better understanding of transcriptional regulations of jasmonate pathway during harvesting stress, along with the use of genotypic diversity in response to such stress, can be used to improve resistance to stress and rubber production in Hevea.

Pan-genome and phylogenomic analyses highlight Hevea species delineation and rubber trait evolution.

The para rubber tree (Hevea brasiliensis) is the world's sole commercial source of natural rubber, a vital industrial raw material. However, the narrow genetic diversity of this crop poses challenges for rubber breeding. Here, we generate high-quality de novo genome assemblies for three H. brasiliensis cultivars, two H. brasiliensis wild accessions, and three other Hevea species (H. nitida, H. pauciflora, and H. benthamiana). Through analyzing genomes of 94 Hevea accessions, we identify five distinct lineages that do not align with their previous species delineations. We discover multiple accessions with hybrid origins between these lineages, indicating incomplete reproductive isolation between them. Only two out of four wild lineages have been introduced to commercial rubber cultivars. Furthermore, we reveal that the rubber production traits emerged following the development of a large REF/SRPP gene cluster and its functional specialization in rubber-producing laticifers within this genus. These findings would enhance rubber breeding and benefit research communities.

Regulation of MIR genes in response to abiotic stress in Hevea brasiliensis.

Increasing demand for natural rubber (NR) calls for an increase in latex yield and also an extension of rubber plantations in marginal zones. Both harvesting and abiotic stresses lead to tapping panel dryness through the production of reactive oxygen species. Many microRNAs regulated during abiotic stress modulate growth and development. The objective of this paper was to study the regulation of microRNAs in response to different types of abiotic stress and hormone treatments in Hevea. Regulation of MIR genes differs depending on the tissue and abiotic stress applied. A negative co-regulation between HbMIR398b with its chloroplastic HbCuZnSOD target messenger is observed in response to salinity. The involvement of MIR gene regulation during latex harvesting and tapping panel dryness (TPD) occurrence is further discussed.

Identification of miRNAs and their targets in two Taraxacum species with contrasting rubber-producing ability.

MicroRNAs (miRNAs) are widely involved in various aspects of plant growth and development. However, how miRNAs and their targets regulate natural rubber metabolism remains unclear in the rubber-producing dandelions, which are being developed as alternative commercial sources of natural rubber. Here, we combined small RNA sequencing, degradome sequencing, target gene prediction, and mRNA sequencing to identify miRNAs and their targets in two dandelion species, the high rubber-yielding Taraxacum kok-saghyz (Tk) and the low rubber-yielding T. spadiceum (Ts). A total of 142 miRNAs, including 108 known and 34 novel ones, were discovered, with 53 identified as differentially expressed (DE) between the latex of Tk and Ts. Degradome sequencing identified 145 targets corresponding to 74 miRNAs. TAPIR and psRNATarget, respectively, predicted 165 and 164 non-redundant targets for the 53 aforementioned DE miRNAs. Gene ontology (GO) enrichment analysis indicated the DE miRNAs and their targets might affect natural rubber production via regulating macromolecular biosynthesis and metabolism in latex. Four critical types of regulatory modules, including miR172-AP2/ERF, miR164-NAC, miR160-ARF, and miRN19-protein kinase, were identified and their interaction networks were constructed, indicating a potential involvement in natural rubber production. The findings and the large miRNA dataset presented here are beneficial to further deciphering the roles of miRNAs in the biosynthesis of natural rubber and medicinal metabolites in dandelion.

Genome-wide analysis of the SWEET genes in Taraxacum kok-saghyz Rodin: An insight into two latex-abundant isoforms.

Taraxacum kok-saghyz Rodin (Tk) is a promising alternative rubber-producing grass. However, low biomass and rubber-producing capability limit its commercial application. As a carbon source transporter in plants, sugar will eventually be exported transporters (SWEETs) have been reported to play pivotal roles in diverse physiological events in the context of carbon assimilate transport and utilization. Theoretically, SWEETs would participate in Tk growth, development and response to environmental cues with relation to the accumulation of rubber and biomass, both of which rely on the input of carbon assimilates. Here, we identified 22 TkSWEETs through homology searching of the Tk genomes and bioinformatics analyses. RNA-seq and qRT-PCR analysis revealed these TkSWEETs to have overlapping yet distinct tissue expression patterns. Two TkSWEET isofroms, TkSWEET1 and TkSWEET12 expressed substantially in the latex, the cytoplasm of rubber-producing laticifers as well as the rubber source. As revealed by the transient expression analysis using Tk mesophyll protoplasts, both TkSWEET1 and TkSWEET12 were located in the plasma membrane. Heterologous expressions of the two TkSWEETs in a yeast mutant revealed that only TkSWEET1 exhibited apparent sugar transport activities, with a preference for monosaccharides. Interestingly, TkSWEET12, the latex-predominant TkSWEET isoform, seemed to have evolved from a tandem duplication event that results in a cluster of six TkSWEET genes with the TkSWEET12 therein, suggesting its specialized roles in the laticifers.

The rubber tree RALF peptide hormone and its receptor protein kinase FER implicates in rubber production.

RAPID ALKALINIZATION FACTORs (RALFs), which are secreted peptides serving as extracellular signals transduced to the inside of the cell, interact with the receptor-like kinase FERONIA (FER) and participates in various biological pathways. Here, we identified 23 RALF and 2 FER genes in Hevea brasiliensis (para rubber tree), and characterized their expression patterns in different tissues, across the process of leaf development, and in response to the rubber yield-stimulating treatments of tapping and ethylene. Four Hevea latex (the cytoplasm of rubber-producing laticifers)-abundant RALF isoforms, HbRALF19, HbRALF3, HbRALF22, and HbRALF16 were listed with descending expression levels. Of the four HbRALFs, expressions of HbRALF3 were markedly regulated in an opposite way by the treatments of tapping (depression) and ethylene (stimulation). All of the four latex-abundant RALFs specifically interacted with the extracellular domain of HbFER1. Transgenic Arabidopsis plants overexpressing these HbRALFs displayed phenotypes similar to those reported for AtRALFs, such as shorter roots, smaller plant architecture, and delayed flowering. The application of HbRALF3 and HbRALF19 recombinant proteins significantly reduced the pH of Hevea latex, an important factor regulating latex metabolism. An in vitro rubber biosynthesis assay in a mixture of latex cytosol (C-serum) revealed a positive role of HbFER1 in rubber biosynthesis. Taken together, these data provide evidence for the participation of the HbRALF-FER module in rubber production.

Trehalose 6-Phosphate/SnRK1 Signaling Participates in Harvesting-Stimulated Rubber Production in the Hevea Tree.

Trehalose 6-phosphate (T6P), the intermediate of trehalose biosynthesis and a signaling molecule, affects crop yield via targeting sucrose allocation and utilization. As there have been no reports of T6P signaling affecting secondary metabolism in a crop plant, the rubber tree Hevea brasiliensis serves as an ideal model in this regard. Sucrose metabolism critically influences the productivity of natural rubber, a secondary metabolite of industrial importance. Here, we report on the characterization of the T6P synthase (TPS) gene family and the T6P/SNF1-related protein kinase1 (T6P/SnRK1) signaling components in Hevea laticifers under tapping (rubber harvesting), an agronomic manipulation that itself stimulates rubber production. A total of fourteen TPS genes were identified, among which a class II TPS gene, HbTPS5, seemed to have evolved with a function specialized in laticifers. T6P and trehalose increased when the trees were tapped, this being consistent with the observed enhanced activities of TPS and T6P phosphatase (TPP) and expression of an active TPS-encoding gene, HbTPS1. On the other hand, SnRK1 activities decreased, suggesting the inhibition of elevated T6P on SnRK1. Expression profiles of the SnRK1 marker genes coincided with elevated T6P and depressed SnRK1. Interestingly, HbTPS5 expression decreased significantly with the onset of tapping, suggesting a regulatory function in the T6P pathway associated with latex production in laticifers. In brief, transcriptional, enzymatic, and metabolic evidence supports the participation of T6P/SnRK1 signaling in rubber formation, thus providing a possible avenue to increasing the yield of a valuable secondary metabolite by targeting T6P in specific cells.

The Calcium Sensor Calcineurin B-Like Proteins -Calcineurin B-Like Interacting Protein Kinases Is Involved in Leaf Development and Stress Responses Related to Latex Flow in Hevea brasiliensis.

Latex flow in Hevea brasiliensis (the Para rubber tree), the sole commercial source of natural rubber (cis-1,4-polyisoprene, NR), renders it uniquely suited for the study of plant stress responses. Calcineurin B-like interacting protein kinases (CIPK) serving as calcium-sensor protein kinases react with calcineurin B-like proteins (CBL) to play crucial roles in hormone signaling transduction and response to abiotic stress in plant developmental processes. However, little is known about their functions in Hevea. In this study, a total of twelve CBL (HbCBL) and thirty CIPK (HbCIPK) genes were identified from the Hevea genome. Structure and phylogenetic analysis assigned these CIPKs to five groups and CBLs to four groups, and mapped onto fourteen of the eighteen Hevea chromosomes. RNA-seq and qPCR analysis showed that the expressions of HbCBL and HbCIPK genes varied in the seven Hevea tissues examined, i.e., latex (cytoplasm of rubber-producing laticifers), bark, leaf, root, seed, female flower, and male flower. The expressions of two HbCBL and sixteen HbCIPK genes showed upward trends during leaf development. Following ethylene yield stimulation and the latex tapping treatment, both practices invoking stress, the expression levels of most latex-expressed genes were significantly altered. Yeast two-hybrid test revealed interactions for multiple combinations of HbCBLs and HbCIPKs with substantial gene expression in latex or other Hevea tissues. However, all the HbCBL-HbCIPK complexes examined did not recruit HbSOS1 or AtSOS1 to form functional salt tolerance SOS pathway in yeast cells. Taken together, the results suggested a role of the Hevea CBL-CIPK network as a point of convergence for several different signaling pathways in growth, development, and stress responses in relation to latex production.

Transcript and Protein Profiling Provides Insights Into the Molecular Mechanisms of Harvesting-Induced Latex Production in Rubber Tree.

Natural rubber, an important industrial raw material with wide applications, is harvested in the form of latex (cytoplasm of rubber-producing laticifers) from Hevea brasiliensis (para rubber tree) by the way of tapping. Conspicuous stimulation on latex production is observed for the first few tappings conducted on virgin (untapped before) or resting (tapped before but no tapping for a period) rubber trees. To understand the underlying mechanisms, an integrative analysis of the latex transcriptome and proteome was conducted on virgin or resting Hevea trees for the first five tappings. A total of 505 non-redundant differentially expressed (DE) transcript-derived fragments (TDFs) were identified by silver-staining cDNA-AFLP, with 217 exhibiting patterns of upregulated, 180 downregulated and 108 irregularly-regulated. Meanwhile, 117 two dimensional gel electrophoresis DE-protein spots were isolated and subjected to mass spectrometry analysis, with 89 and 57 being successfully identified by MALDI-TOF and MALDI-TOF/TOF, respectively. About 72.5% DE-TDFs and 76.1% DE-proteins were functionally annotated and categorized. Noteworthily, most of the DE-TDFs implicated in sugar transport and metabolism as well as rubber biosynthesis were upregulated by the tapping treatment. The importance of sugar metabolism in harvesting-induced latex production was reinforced by the identification of abundant relevant DE-protein spots. About 83.8% of the randomly selected DE-TDFs were validated for expression patterns by semi-quantitative RT-PCR, and an 89.7% consistency for the 29 latex regeneration-related DE-TDFs examined by quantitative RT-PCR analysis. In brief, our results reveal extensive physiological and molecular changes in Hevea laticifers incurred by the tapping treatment, and the vast number of DE genes and proteins identified here contribute to unraveling the gene regulatory network of tapping-stimulated latex production.

Ethylene response factors regulate expression of HbSUT3, the sucrose influx carrier in laticifers of Hevea brasiliensis.

Natural rubber is an important industrial raw material and is commercially produced by rubber trees (Hevea brasiliensis). The sucrose transporter HbSUT3 plays an essential role in rubber production. Its expression in latex (cytoplasm of rubber-producing laticifers) is induced by bark treatment with Ethrel, an ethylene releaser, and the inducing effect correlates well with Ethrel-stimulated rubber yield increase. However, the mechanisms of ethylene induction on HbSUT3 expression are not known. Here, five Ethylene Response Factor (ERF) genes were identified from the cDNA library of Hevea latex by yeast one-hybrid screening with the promoter of HbSUT3 gene as bait. As revealed in a tobacco (Nicotiana tabacum) protoplast transient expression system, these HbERFs were mainly localized in the nucleus and four of them exhibited apparent transactivation activity. Of the five HbERF genes, HbERF-IXc4 was the most frequently screened in yeast one-hybrid, accounting for 65% of the ERF clones obtained. Moreover, among the five HbERFs, HbERF-IXc4 showed the strongest transactivation capacity when expressed in tobacco protoplast, the highest transcript abundance in latex and a close expressional correlation with its target gene, HbSUT3, in response to the Ethrel treatment. Taken together, our results indicate that ERFs, especially HbERF-IXc4, are critically involved in the activation of HbSUT3 expression in latex after Ethrel treatment on Hevea bark, and thus the stimulated latex yield.