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.

Genome-Wide Identification and Expression Profile Analysis of the Phenylalanine Ammonia-Lyase Gene Family in Hevea brasiliensis.

The majority of the world's natural rubber comes from the rubber tree (Hevea brasiliensis). As a key enzyme for synthesizing phenylpropanoid compounds, phenylalanine ammonia-lyase (PAL) has a critical role in plant satisfactory growth and environmental adaptation. To clarify the characteristics of rubber tree PAL family genes, a genome-wide characterization of rubber tree PALs was conducted in this study. Eight PAL genes (HbPAL1-HbPAL8), which spread over chromosomes 3, 7, 8, 10, 12, 13, 14, 16, and 18, were found to be present in the genome of H. brasiliensis. Phylogenetic analysis classified HbPALs into groups I and II, and the group I HbPALs (HbPAL1-HbPAL6) displayed similar conserved motif compositions and gene architectures. Tissue expression patterns of HbPALs quantified by quantitative real-time PCR (qPCR) proved that distinct HbPALs exhibited varying tissue expression patterns. The HbPAL promoters contained a plethora of cis-acting elements that responded to hormones and stress, and the qPCR analysis demonstrated that abiotic stressors like cold, drought, salt, and H2O2-induced oxidative stress, as well as hormones like salicylic acid, abscisic acid, ethylene, and methyl jasmonate, controlled the expression of HbPALs. The majority of HbPALs were also regulated by powdery mildew, anthracnose, and Corynespora leaf fall disease infection. In addition, HbPAL1, HbPAL4, and HbPAL7 were significantly up-regulated in the bark of tapping panel dryness rubber trees relative to that of healthy trees. Our results provide a thorough comprehension of the characteristics of HbPAL genes and set the groundwork for further investigation of the biological functions of HbPALs in rubber trees.

A novel rubber tree PR-10 protein involved in host-defense response against the white root rot fungus Rigidoporus microporus.

BACKGROUND: White root rot disease in rubber trees, caused by the pathogenic fungi Rigidoporus microporus, is currently considered a major problem in rubber tree plantations worldwide. Only a few reports have mentioned the response of rubber trees occurring at the non-infection sites, which is crucial for the disease understanding and protecting the yield losses. RESULTS: Through a comparative proteomic study using the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique, the present study reveals some distal-responsive proteins in rubber tree leaves during the plant-fungal pathogen interaction. From a total of 12 selected differentially expressed protein spots, several defense-related proteins such as molecular chaperones and ROS-detoxifying enzymes were identified. The expression of 6 candidate proteins was investigated at the transcript level by Reverse Transcription Quantitative PCR (RT-qPCR). In silico, a highly-expressed uncharacterized protein LOC110648447 found in rubber trees was predicted to be a protein in the pathogenesis-related protein 10 (PR-10) class. In silico promoter analysis and structural-related characterization of this novel PR-10 protein suggest that it plays a potential role in defending rubber trees against R. microporus infection. The promoter contains WRKY-, MYB-, and other defense-related cis-acting elements. The structural model of the novel PR-10 protein predicted by I-TASSER showed a topology of the Bet v 1 protein family, including a conserved active site and a ligand-binding hydrophobic cavity. CONCLUSIONS: A novel protein in the PR-10 group increased sharply in rubber tree leaves during interaction with the white root rot pathogen, potentially contributing to host defense. The results of this study provide information useful for white root rot disease management of rubber trees in the future.

Agroforestry orchards support greater butterfly diversity than monoculture plantations in the tropics.

Large-scale deforestation in the tropics, triggered by logging and subsequent agricultural monoculture has a significant adverse impact on biodiversity due to habitat degradation. Here, we measured the diversity of butterfly species in three agricultural landscapes, agroforestry orchards, oil palm, and rubber tree plantations. Butterfly species were counted at 127 sampling points over the course of a year using the point count method. We found that agroforestry orchards supported a greater number of butterfly species (74 species) compared to rubber tree (61 species) and oil palm plantations (54 species) which were dominated by generalist (73%) followed by forest specialists (27%). We found no significant difference of butterfly species composition between agroforestry orchards and rubber tree plantation, with both habitats associated with more butterfly species compared to oil palm plantations. This indicates butterflies were able to persist better in certain agricultural landscapes. GLMMs suggested that tree height, undergrowth coverage and height, and elevation determined butterfly diversity. Butterfly species richness was also influenced by season and landscape-level variables such as proximity to forest, mean NDVI, and habitat. Understanding the factors that contributed to butterfly species richness in an agroecosystem, stakeholders should consider management practices to improve biodiversity conservation such as ground vegetation management and retaining adjacent forest areas to enhance butterfly species richness. Furthermore, our findings suggest that agroforestry system should be considered to enhance biodiversity in agricultural landscapes.

Brassinosteroids Regulate the Water Deficit and Latex Yield of Rubber Trees.

Brassinolide (BR) is an important plant hormone that regulates the growth and development of plants and the formation of yield. The yield and quality of latex from Hevea brasiliensis are regulated by phytohormones. The understanding of gene network regulation mechanism of latex formation in rubber trees is still very limited. In this research, the rubber tree variety CATAS73397 was selected to analyze the relationship between BR, water deficit resistance, and latex yield. The results showed that BR improves the vitality of rubber trees under water deficit by increasing the rate of photosynthesis, reducing the seepage of osmotic regulatory substances, increasing the synthesis of energy substances, and improving the antioxidant system. Furthermore, BR increased the yield and quality of latex by reducing the plugging index and elevating the lutoid bursting index without decreasing mercaptan, sucrose, and inorganic phosphorus. This was confirmed by an increased expression of genes related to latex flow. RNA-seq analysis further indicated that DEG encoded proteins were enriched in the MAPK signaling pathway, plant hormone signal transduction and sucrose metabolism. Phytohormone content displayed significant differences, in that trans-Zeatin, ethylene, salicylic acid, kinetin, and cytokinin were induced by BR, whereas auxin, abscisic acid, and gibberellin were not. In summary, the current research lays a foundation for comprehending the molecular mechanism of latex formation in rubber trees and explores the potential candidate genes involved in natural rubber biosynthesis to provide useful information for further research in relevant areas.

Identification and Characterization of the HbPP2C Gene Family and Its Expression in Response to Biotic and Abiotic Stresses in Rubber Tree.

Plant PP2C genes are crucial for various biological processes. To elucidate the potential functions of these genes in rubber tree (Hevea brasiliensis), we conducted a comprehensive analysis of these genes using bioinformatics methods. The 60 members of the PP2C family in rubber tree were identified and categorized into 13 subfamilies. The PP2C proteins were conserved across different plant species. The results revealed that the HbPP2C genes contained multiple elements responsive to phytohormones and stresses in their promoters, suggesting their involvement in these pathways. Expression analysis indicated that 40 HbPP2C genes exhibited the highest expression levels in branches and the lowest expression in latex. Additionally, the expression of A subfamily members significantly increased in response to abscisic acid, drought, and glyphosate treatments, whereas the expression of A, B, D, and F1 subfamily members notably increased under temperature stress conditions. Furthermore, the expression of A and F1 subfamily members was significantly upregulated upon powdery mildew infection, with the expression of the HbPP2C6 gene displaying a remarkable 33-fold increase. These findings suggest that different HbPP2C subgroups may have distinct roles in the regulation of phytohormones and the response to abiotic and biotic stresses in rubber tree. This study provides a valuable reference for further investigations into the functions of the HbPP2C gene family in rubber tree.

A putative effector of the rubber-tree powdery mildew fungus has elicitor activity that can trigger plant immunity.

MAIN CONCLUSION: A putative powdery mildew effector can elicit defense responses including reactive oxygen species and callose accumulations in model plants Nicotiana benthamiana and Arabidopsis thaliana and host plant Hevea brasiliensis. Powdery mildew fungi cause severe diseases in many agricultural plants, such as the mildew fungus Erysiphe quercicola infecting the rubber tree (Hevea brasiliensis), causing latex yield losses. However, effectors of E. quercicola were rarely functionally characterized. In this study, we identified a highly specific candidate-secreted effector protein, EqCSEP04187, from E. quercicola. This putative effector is expressed at the late stage but not the early stage during infection. The constitutive expression of EqCSEP04187 in model plants Nicotiana benthamiana and Arabidopsis thaliana elicited defense responses, as did transient expression of EqCSEP04187 in protoplasts of H. brasiliensis. Introducing EqCSEP04187 into another H. brasiliensis-associated fungal pathogen, Colletotrichum gloeosporioides, inhibited H. brasiliensis infection, and infection by E. quercicola was decreased in the A. thaliana eds1 mutant expressing EqCSEP04187. Further analysis suggests that these reductions in infection were the consequences of EqCSEP04187 eliciting defense responses. Our study suggests that this putative effector has elicitor activity that can improve plant resistance.

Transcriptome analysis of Pará rubber tree (H. brasiliensis) seedlings under ethylene stimulation.

BACKGROUND: Natural rubber (cis-1,4-polyioprene, NR) is an indispensable industrial raw material obtained from the Pará rubber tree (H. brasiliensis). Natural rubber cannot be replaced by synthetic rubber compounds because of the superior resilience, elasticity, abrasion resistance, efficient heat dispersion, and impact resistance of NR. In NR production, latex is harvested by periodical tapping of the trunk bark. Ethylene enhances and prolongs latex flow and latex regeneration. Ethephon, which is an ethylene-releasing compound, applied to the trunk before tapping usually results in a 1.5- to 2-fold increase in latex yield. However, intense mechanical damage to bark tissues by excessive tapping and/or over-stimulation with ethephon induces severe oxidative stress in laticifer cells, which often causes tapping panel dryness (TPD) syndrome. To enhance NR production without causing TPD, an improved understanding of the molecular mechanism of the ethylene response in the Pará rubber tree is required. Therefore, we investigated gene expression in response to ethephon treatment using Pará rubber tree seedlings as a model system. RESULTS: After ethephon treatment, 3270 genes showed significant differences in expression compared with the mock treatment. Genes associated with carotenoids, flavonoids, and abscisic acid biosynthesis were significantly upregulated by ethephon treatment, which might contribute to an increase in latex flow. Genes associated with secondary cell wall formation were downregulated, which might be because of the reduced sugar supply. Given that sucrose is an important molecule for NR production, a trade-off may arise between NR production and cell wall formation for plant growth and for wound healing at the tapping panel. CONCLUSIONS: Dynamic changes in gene expression occur specifically in response to ethephon treatment. Certain genes identified may potentially contribute to latex production or TPD suppression. These data provide valuable information to understand the mechanism of ethylene stimulation, and will contribute to improved management practices and/or molecular breeding to attain higher yields of latex from Pará rubber trees.

HbLFG1, a Rubber Tree (Hevea brasiliensis) Lifeguard Protein, Can Facilitate Powdery Mildew Infection by Suppressing Plant Immunity.

Powdery mildew causes substantial losses in crop and economic plant yields worldwide. Although powdery mildew infection of rubber trees (Hevea brasiliensis), caused by the biotrophic fungus Erysiphe quercicola, severely threatens natural rubber production, little is known about the mechanism by which E. quercicola adapts to H. brasiliensis to invade the host plant. In barley and Arabidopsis thaliana, lifeguard (LFG) proteins, which have topological similarity to BAX INHIBITOR-1, are involved in host plant susceptibility to powdery mildew infection. In this study, we characterized an H. brasiliensis LFG protein (HbLFG1) with a focus on its function in regulating defense against powdery mildew. HbLFG1 gene expression was found to be upregulated during E. quercicola infection. HbLFG1 showed conserved functions in cell death inhibition and membrane localization. Expression of HbLFG1 in Nicotiana benthamiana leaves and A. thaliana Col-0 was demonstrated to significantly suppress callose deposition induced by conserved pathogen-associated molecular patterns chitin and flg22. Furthermore, we found that overexpression of HbLFG1 in H. brasiliensis mesophyll protoplasts significantly suppressed the chitin-induced burst of reactive oxygen species. Although A. thaliana Col-0 and E. quercicola displayed an incompatible interaction, Col-0 transformants overexpressing HbLFG1 were shown to be susceptible to E. quercicola. Collectively, the findings of this study provide evidence that HbLFG1 acts as a negative regulator of plant immunity that facilitates E. quercicola infection in H. brasiliensis.

Nanopore/Illumina Hybrid Genome Sequence Resource for Corynespora cassiicola Strain XJ Infecting Rubber Tree in China.

Corynespora cassiicola is a ubiquitous pathogenic fungus that can infect a broad range of plant hosts. Corynespora leaf fall, caused by C. cassiicola, is one of the major diseases on rubber tree in China. This disease is having an increasing affect on natural rubber production worldwide. In this study, by combining the Nanopore and Illumina sequencing technologies, we present the chromosome-scale genome sequence of the rubber tree-sampled C. cassiicola strain XJ collected in the subtropical region of China. The assembly consists of 23 scaffolds (N50 = 4.62 Mb) with an estimated genome size of 44.42 Mb (only 166 non-ATCG bases) and 16,108 protein-coding genes. The genome will provide a valuable resource for further research on the pathogenesis and comparative genomics of C. cassiicola on rubber tree and other hosts.

Quantitative Detection and Monitoring of Colletotrichum siamense in Rubber Trees Using Real-Time PCR.

Colletotrichum siamense is one of the most important pathogens of rubber trees in Asia. The proper detection and quantification of C. siamense populations in rubber trees are of importance for monitoring the epidemics of the disease. In this study, we developed an internal transcribed spacer-based real-time PCR method to efficiently detect C. siamense infecting rubber trees, which reliably detected as little as 100 fg of genomic DNA, 100 copies of target DNA, and 20 conidia. The real-time PCR protocol recognized all C. siamense isolates collected from three provinces in China, whereas no amplification was observed with the rubber tree and its other pathogens. Detection and quantification of C. siamense were performed in artificially and naturally infected rubber leaves. We could still detect C. siamense in plant mixes, of which only 0.0001% of the tissue was infected. An accumulation of C. siamense DNA was observed during the whole infection process at all three leaf phenological stages, suggesting that the real-time PCR method can be used to monitor C. siamense development in rubber trees. Finally, the method allowed the detection of C. siamense in naturally infected and symptomless leaves of rubber trees in the fields. Compared with earlier detection methods, the real-time PCR method is more specific and more sensitive, and it will be of great use for studies aiming to gain a better understanding of the epidemiology of Colletotrichum leaf disease, as well as the prediction of disease risk and proposals to control it.

Identification and evaluation of suitable reference genes for gene expression analysis in rubber tree leaf.

Gene expression profiles are increasingly applied to investigate molecular mechanism for which, normalization with suitable reference genes is critical. Previously we have reported several suitable reference genes for laticifer samples from rubber tree, however, little is known in leaf. The main objective of this current study was to identify some stable expression reference genes at various developmental stages of leaf, as well as during abiotic (high and low temperature extremes) and biotic stresses (pathogen stress). Gene expression profilings identified the ubiquitin-proteasome system as excellent potential as reference genes for rubber tree leaf. Among a total of 30 tested genes investigated, 24 new candidate (including 11 genes involved in the ubiquitin-proteasome system), 4 previously identified and 2 specific genes, were further evaluated using quantitative real-time PCR. Our results indicated that the new candidate genes had better expression stability comparing with others. For instance, an ubiquitin conjugating enzyme (RG0099) and three ubiquitin-protein ligases (RG0928, RG2190 and RG0118) expressed stably in all samples, and were confirmed to be suitable reference genes for rubber tree leaf under four different conditions. Finally, we suggest that using more than one reference gene may be appropriate in gene expression studies when employing different software to normalize gene expression data. Our findings have significant implications for the reliability of data obtained from genomics studies in rubber tree and perhaps in other 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.

Lipid Composition of Latex and Rubber Particles in Hevea brasiliensis and Taraxacum kok-saghyz.

Natural rubber is usually synthesized in the rubber particles present in the latex of rubber-producing plants such as the Pará rubber tree (Hevea brasiliensis) and rubber dandelion (Taraxacum kok-saghyz). Since the detailed lipid compositions of fresh latex and rubber particles of the plants are poorly known, the present study reports detailed compound lipid composition, focusing on phospholipids and galactolipids in the latex and rubber particles of the plants. In the fresh latex and rubber particles of both plants, phospholipids were much more dominant (85-99%) compared to galactolipids. Among the nine classes of phospholipids, phosphatidylcholines (PCs) were most abundant, at ~80%, in both plants. Among PCs, PC (36:4) and PC (34:2) were most abundant in the rubber tree and rubber dandelion, respectively. Two classes of galactolipids, monogalactosyl diacylglycerol and digalactosyl diacylglycerol, were detected as 12% and 1%, respectively, of total compound lipids in rubber tree, whereas their percentages in the rubber dandelion were negligible (< 1%). Overall, the compound lipid composition differed only slightly between the fresh latex and the rubber particles of both rubber plants. These results provide fundamental data on the lipid composition of rubber particles in two rubber-producing plants, which can serve as a basis for artificial rubber particle production in the future.

Reference gene validation in Eotetranychus sexmaculatus (Acari: Tetranychidae) feeding on mite-susceptible and mite-resistant rubber tree germplasms.

Reliable reference genes are quite important in calculating gene transcript levels by using real-time quantitative reverse transcription-PCR (RT-qPCR). Eotetranychus sexmaculatus is known as a dangerous mite causing significant yield reduction of rubber tree latex; however, selection of appropriate reference genes for validation of target gene expression in E. sexmaculatus has not been conducted yet. In the present study, nine candidate reference genes were analyzed for their expression stability in different life stages of E. sexmaculatus by using common algorithms including comparative ΔCq method, geNorm, NormFinder and BestKeeper. In addition, a comprehensive analysis software (RefFinder) was used to assign an overall final rank for each candidate gene. The results showed that ß-actin and ß-TUB were the best two reference genes and were subjected to evaluate expression of two protective enzyme genes (EsCu/ZnSOD and EsCAT1) in E. sexmaculatus. We found that the expression of EsCu/ZnSOD and EsCAT1 in E. sexmaculatus feeding on mite-resistant rubber tree germplasm was significantly lower compared with those feeding on mite-susceptible germplasm. These results will facilitate research in revealing molecular mechanisms underlying rubber tree resistance to the spider mite.

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.

The ICE-like transcription factor HbICE2 is involved in jasmonate-regulated cold tolerance in the rubber tree (Hevea brasiliensis).

KEY MESSAGE: An ICE-like transcription factor mediates jasmonate-regulated cold tolerance in the rubber tree (Hevea brasiliensis), and confers cold tolerance in transgenic Arabidopsis. The rubber tree (Hevea brasiliensis) is susceptible to low temperatures, and understanding the mechanisms regulating cold stress is of great potential value for enhancing tolerance to this environmental variable. In this study, we find that treatment with exogenous methyl jasmonate (MeJA) could significantly enhance Hevea brasiliensis cold tolerance. In addition, yeast two-hybrid and bimolecular fluorescence complementation (BiFC) experiments show that JASMONATE ZIM-DOMAIN(JAZ) proteins, HbJAZ1 and HbJAZ12, key repressors of JA signaling pathway, interact with HbICE2, a novel ICE (Inducer of CBF Expression)-like protein. HbICE2 was nuclear-localised and bound to the MYC recognition (MYCR) sequence. The transcriptional activation activity of HbICE2 in yeast cells was dependent on the N-terminus, and overexpression of HbICE2 in Arabidopsis resulted in elevated tolerance to chilling stress. Furthermore, dual-luciferase transient assay reveals that HbJAZ1 and HbJAZ12 proteins inhibit the transcriptional function of HbICE2. The expression of C-repeat-binding factor (CBF) signalling pathway genes including HbCBF1, HbCBF2 and HbCOR47 were up-regulated by MeJA. Taken together, our data suggest that the new ICE-like transcription factor HbICE2 is involved in jasmonate-regulated cold tolerance in Hevea brasiliensis.

Opposite physiological effects upon jasmonic acid and brassinosteroid treatment on laticifer proliferation and co-occurrence of differential expression of genes involved in vascular development in rubber tree.

During growth of woody plant-trunk, the secondary meristem functions in giving rise the xylem and phloem. Rubber tree (Hevea brasiliensis Muell. Arg.), in addition, contains laticifers (latex producing vessels) in the vicinity of phloem. Insights into regulatory mechanisms of gene networks underlying laticifer proliferation in rubber tree has remained very limited. The candidate vascular development-related genes were selected to investigate for expression profile in phloem and xylem tissues of high latex yield- and high wood yield-clones of rubber tree. The differential gene expression between the mature branch-xylem and -phloem tissues was clearly observed. The cis-regulatory motif analysis revealed the existent of putative jasmonic acid (JA)- and brassinosteroid (BR)-responsive regulatory motifs in promoter regions of these genes, and consequently the effect of exogenous application of JA, BR or their respective signaling inhibitors, on the formation of laticifers in rubber tree was demonstrated. Interestingly, the laticifer numbers were significantly increased in JA-treatment, correlated with up-regulation of phloem development-related genes in both rubber tree clones. On the contrary, the laticifers were decreased in BR-treatment accompanying by up-regulation of xylem development-related genes, especially in high wood yield-rubber tree clone. BR-inhibitor treatment also enhanced laticifer numbers, while JA-inhibitor suppressed laticifer differentiation. Taken together, this study unveils the molecular interplay between JA/BR on vascular development in rubber tree and how this impacts the appearance of laticifers in this plant. This process is vital for a better understanding on laticifer differentiation and its impact in the manipulation of wood and latex yield in rubber tree improvement program.

Genome-wide association mapping of latex yield and girth in Amazonian accessions of Hevea brasiliensis grown in a suboptimal climate zone.

Latex yield and growth are the key complex traits in commercial rubber production. The present study is the first to report genome-wide association mapping of latex yield and girth, for 170 Amazonian accessions grown in a suboptimal area characterized by limited rainfall and a lengthy dry season. Targeted sequence enrichment to capture gene transcripts generated 14,155 high quality filtered single nucleotide polymorphisms (SNPs) of which 94.3% resided in coding regions. The rapid decay of linkage disequilibrium over physical and genetic distance found in the accessions was comparable to those previously reported for several outcrossing species. A mixed linear model detected three significant SNPs in three candidate genes involved in plant adaptation to drought stress, individually explaining 12.7-15.7% of the phenotypic variance. The SNPs identified in the study will help to extend understanding, and to support genetic improvement of rubber trees grown in drought-affected regions.

Salicylic Acid Induces Resistance in Rubber Tree against Phytophthora palmivora.

Induced resistance by elicitors is considered to be an eco-friendly strategy to stimulate plant defense against pathogen attack. In this study, we elucidated the effect of salicylic acid (SA) on induced resistance in rubber tree against Phytophthora palmivora and evaluated the possible defense mechanisms that were involved. For SA pretreatment, rubber tree exhibited a significant reduction in disease severity by 41%. Consistent with the occurrence of induced resistance, the pronounced increase in H2O2 level, catalase (CAT) and peroxidase (POD) activities were observed. For defense reactions, exogenous SA promoted the increases of H2O2, CAT, POD and phenylalanine ammonia lyase (PAL) activities, including lignin, endogenous SA and scopoletin (Scp) contents. However, SA had different effects on the activity of each CAT isoform in the particular rubber tree organs. Besides, three partial cDNAs encoding CAT (HbCAT1, HbCAT2 and HbCAT3) and a partial cDNA encoding PAL (HbPAL) were isolated from rubber tree. Moreover, the expressions of HbCAT1, HbPAL and HbPR1 were induced by SA. Our findings suggested that, upon SA priming, the elevated H2O2, CAT, POD and PAL activities, lignin, endogenous SA and Scp contents, including the up-regulated HbCAT1, HbPAL and HbPR1 expressions could potentiate the resistance in rubber tree against P. palmivora.