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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., (© 2024. The Author(s).)

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., (Published by Oxford University Press 2021.)

Fructokinase (FRK) mediates fructose phosphorylation to regulate the carbon flow and its assignment to sink tissues. Out of five HbFRKs in the genome of the rubber tree, three (HbFRK1-3) that were highly expressed in latex (cytoplasm of laticifers) were isolated and examined. According to phylogenetic analysis and intracellular location experiment, both HbFRK2 and HbFRK3 were highly possible to be expressed in cytosol, while HbFRK1 was in plastid. As the predominant isoform in laticifers, HbFRK2 had the highest transcripts, followed by HbFRK3 and HbFRK1. In enzymatic function, HbFRK2 also showed the highest affinity for fructose. To examine the roles of FRKs in latex yield and regeneration, changes in HbFRKs were examined when latex outflow from the trees were increased through two experimental interventions. In the first approach, tapping was initiated on previously untapped trees, resulting in latex yield increasing with consecutive tapping at the initial stage before it stabilized. In the second approach, latex yield from trees that were already in regular tapping was stimulated by treatment with the ethylene-based yield stimulant, ethephon. Using either method to induce an increase in latex yield, the abundance of HbFRK2 and HbFRK3 in transcripts, was increased. This development, which was especially marked in HbFRK2, may reflect a strengthening of glycolysis to meet the carbon flux and energy demands for increased rubber biosynthesis to replace rubber lost in the increased latex yield. Our results, therefore, suggest that HbFRK2 plays a critical role in fructose catabolism to facilitate rubber regeneration in the commercially exploited rubber tree., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

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 β-actin and β-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.

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.

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.

Metallothioneins (MTs) as reactive oxygen species (ROS) scavengers play important roles in stress response and heavy metal homeostasis. In Hevea brasiliensis (the para rubber tree that is the source of commercial natural rubber) and in other trees, the functions of MTs are not well understood. Latex exudes when the rubber tree is tapped. The flow of latex and its regeneration can be enhanced by tapping, wounding and ethylene treatment, all of which produce ROS as a by-product. Here, we show the presence of four MT genes in H. brasiliensis, comprising three Type 2 (HbMT2, -2a and -2b) and one Type 3 (HbMT3L) isoforms, representing one of the smallest MT gene families among angiosperms. The four HbMTs exhibited distinct tissue expression patterns: HbMT2 and HbMT3L mainly in leaves, HbMT2a specifically in flowers and HbMT2b in diverse tissues. The expression of HbMT2b, an isoform present in latex, decreased significantly in the latex following the stress-inducing treatments of tapping, wounding and ethephon (an ethylene generator). The expressions of the leaf-abundant isoforms, HbMT2 and -3L were up-regulated following pathogenic fungus infection and high-temperature stress, but down-regulated by low-temperature stress. These reactions were consistent with multiple defense- and hormone-responsive cis-acting elements in the HbMT promoters. Nine transcription factors were shown to implicate in the high-temperature responsiveness of HbMT2 and -3L in leaves. Overexpression of HbMT2 in Escherichia coli enhanced the bacterium's tolerance to heavy metals and ROS, consistent with its predicted role as an ROS scavenger. Taken together, our results, along with other relevant studies, suggest an important role of HbMTs in latex regeneration as well as species adaptation via the regulation of ROS homeostasis.

Along with changes in morphology in the course of maturation, leaves of Hevea brasiliensis become more resistant to leaf diseases, including the South American Leaf Blight (SALB), a devastating fungal disease of this economically important tree species. To understand the underlying mechanisms of this defense, and to identify the candidate genes involved, we sequenced the Hevea leaf transcriptome at four developmental stages (I to IV) by Illumina sequencing. A total of 62.6 million high-quality reads were generated, and assembled into 98,796 unique transcripts. We identified 3,905 differentially expressed genes implicated in leaf development, 67.8% (2,651) of which were during the transition to leaf maturation. The genes involved in cyanogenic metabolism, lignin and anthocyanin biosynthesis were noteworthy for their distinct patterns of expression between developing leaves (stages I to III) and mature leaves (stage IV), and the correlation with the change in resistance to SALB and the Oidium/Colletotrichum leaf fall. The results provide a first profile of the molecular events that relate to the dynamics of leaf morphology and defense strategies during Hevea leaf development. This dataset is beneficial to devising strategies to engineer resistance to leaf diseases as well as other in-depth studies in Hevea tree.

The Para rubber tree (Hevea brasiliensis) is an economically important tropical tree species that produces natural rubber, an essential industrial raw material. Here we present a high-quality genome assembly of this species (1.37 Gb, scaffold N50 = 1.28 Mb) that covers 93.8% of the genome (1.47 Gb) and harbours 43,792 predicted protein-coding genes. A striking expansion of the REF/SRPP (rubber elongation factor/small rubber particle protein) gene family and its divergence into several laticifer-specific isoforms seem crucial for rubber biosynthesis. The REF/SRPP family has isoforms with sizes similar to or larger than SRPP1 (204 amino acids) in 17 other plants examined, but no isoforms with similar sizes to REF1 (138 amino acids), the predominant molecular variant. A pivotal point in Hevea evolution was the emergence of REF1, which is located on the surface of large rubber particles that account for 93% of rubber in the latex (despite constituting only 6% of total rubber particles, large and small). The stringent control of ethylene synthesis under active ethylene signalling and response in laticifers resolves a longstanding mystery of ethylene stimulation in rubber production. Our study, which includes the re-sequencing of five other Hevea cultivars and extensive RNA-seq data, provides a valuable resource for functional genomics and tools for breeding elite Hevea cultivars.

In rubber tree, latex regeneration is one of the decisive factors influencing the rubber yield, although its molecular regulation is not well known. Quantitative real-time PCR (qPCR) is a popular and powerful tool used to understand the molecular mechanisms of latex regeneration. However, the suitable reference genes required for qPCR are not available to investigate the expressions of target genes during latex regeneration. In this study, 20 candidate reference genes were selected and evaluated for their expression stability across the samples during the process of latex regeneration. All reference genes showed a relatively wide range of the threshold cycle values, and their stability was validated by four different algorithms (comparative delta Ct method, Bestkeeper, NormFinder and GeNorm). Three softwares (comparative delta Ct method, NormFinder and GeNorm) exported similar results that identify UBC4, ADF, UBC2a, eIF2 and ADF4 as the top five suitable references, and 18S as the least suitable one. The application of the screened references would improve accuracy and reliability of gene expression analysis in latex regeneration experiments., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In plants, ethanolic fermentation occurs not only under anaerobic conditions but also under aerobic conditions, and involves carbohydrate and energy metabolism. Pyruvate decarboxylase (PDC) is the first and the key enzyme of ethanolic fermentation, which branches off the main glycolytic pathway at pyruvate. Here, four PDC genes were isolated and identified in a rubber tree, and the protein sequences they encode are very similar. The expression patterns of HbPDC4 correlated well with tapping-simulated rubber productivity in virgin rubber trees, indicating it plays an important role in regulating glycometabolism during latex regeneration. HbPDC1, HbPDC2 and HbPDC3 had striking expressional responses in leaves and bark to drought, low temperature and high temperature stresses, indicating that the HbPDC genes are involve in self-protection and defense in response to various abiotic and biotic stresses during rubber tree growth and development. To understand ethanolic fermentation in rubber trees, it will be necessary to perform an in-depth study of the regulatory pathways controlling the HbPDCs in the future., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Sucrose synthase (Sus, EC 2.4.1.13) is widely recognized as a key enzyme in sucrose metabolism in plants. However, nothing is known about this gene family in Hevea brasiliensis (para rubber tree). Here, we identified six Sus genes in H. brasiliensis that comprise the entire Sus family in this species. Analysis of the gene structure and phylogeny of the Sus genes demonstrates evolutionary conservation in the Sus families across Hevea and other plant species. The expression of Sus genes was investigated via Solexa sequencing and quantitative PCR in various tissues, at various phases of leaf development, and under abiotic stresses and ethylene treatment. The Sus genes exhibited distinct but partially redundant expression profiles. Each tissue has one abundant Sus isoform, with HbSus3, 4 and 5 being the predominant isoforms in latex (cytoplasm of rubber-producing laticifers), bark and root, respectively. HbSus1 and 6 were barely expressed in any tissue examined. In mature leaves (source), all HbSus genes were expressed at low levels, but HbSus3 and 4 were abundantly expressed in immature leaves (sink). Low temperature and drought treatments conspicuously induced HbSus5 expression in root and leaf, suggesting a role in stress responses. HbSus2 and 3 transcripts were decreased by ethylene treatment, consistent with the reduced sucrose-synthesizing activity of Sus enzymes in the latex in response to ethylene stimulation. Our results are beneficial to further determination of functions for the Sus genes in Hevea trees, especially roles in regulating latex regeneration., (© 2013 FEBS.)

ROP (Rho of plants) proteins are plant-specific Rho-type small GTPases which play important roles in cellular processes and stress responses. This study explores the characteristics and possible functions of ROPs that are expressed primarily in laticifers of the rubber tree (Hevea brasiliensis). The work serves as a preliminary step to determining their involvement in latex flow and regeneration, laticifers formation and tapping panel dryness (TPD, a physiological disorder in rubber trees that result in the stoppage of latex flow). In this connection, we (i) identified five HbROPs (HbROP1-HbROP5) by searching latex transcripts database and the genome databases, (ii) characterized molecular and phylogenic aspects of the HbROPs and examined the cis-regulatory elements in their promoter regions; (iii) analyzed by Real-time Quantitative PCR (QPCR) the tissue specificity of the HbROPs and their expression patterns in response to tapping, bark wounding and growth regulator treatments. All five HbROP genes were strongly expressed in the latex, with HbROP1, 3, 4 and 5 showing the highest expression among the six Hevea tissues examined, viz. latex, bud, mature leaf, bark, male flower and seed. When tapping was initiated on previously untapped trees, HbROP3 transcription was substantially down-regulated whereas HbROP5 expression was markedly up-regulated. Transcripts of HbROP3 rose gradually with the development of TPD. Except for the cytokinin 6-benzyl aminopurine that induced a rise in HbROP5 transcripts by more than 2-fold, the other growth regulators tested had little effect on HbROPs expression. The roles of HbROPs in rubber tree are discussed in relation to the diverse functions of ROP homologs reported in other plant species., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Sucrose-metabolizing enzymes in plant leaves have hitherto been investigated mainly in temperate plants, and rarely conducted in tandem with gene expression and sugar analysis. Here, we investigated the sugar content, gene expression, and the activity of sucrose-metabolizing enzymes in the leaves of Hevea brasiliensis, a tropical tree widely cultivated for natural rubber. Sucrose, fructose and glucose were the major sugars detected in Hevea leaves at four developmental stages (I to IV), with starch and quebrachitol as minor saccharides. Fructose and glucose contents increased until stage III, but decreased strongly at stage IV (mature leaves). On the other hand, sucrose increased continuously throughout leaf development. Activities of all sucrose-cleaving enzymes decreased markedly at maturation, consistent with transcript decline for most of their encoding genes. Activity of sucrose phosphate synthase (SPS) was low in spite of its high transcript levels at maturation. Hence, the high sucrose content in mature leaves was not due to increased sucrose-synthesizing activity, but more to the decline in sucrose cleavage. Gene expression and activities of sucrose-metabolizing enzymes in Hevea leaves showed striking differences compared with other plants. Unlike in most other species where vacuolar invertase predominates in sucrose cleavage in developing leaves, cytoplasmic invertase and sucrose synthase (cleavage direction) also featured prominently in Hevea. Whereas SPS is normally responsible for sucrose synthesis in plant leaves, sucrose synthase (synthesis direction) was comparable or higher than that of SPS in Hevea leaves. Mature Hevea leaves had an unusually high sucrose:starch ratio of about 11, the highest reported to date in plants. [ABSTRACT FROM AUTHOR]

In plants, trehalose-6-phosphate (Tre6P) synthesized by the Tre6P synthase (TPS) acts as a signal metabolite in regulating sucrose metabolism in relation to plant growth and development. The Hevea genome predicts a total of 14 TPS genes, and two class I TPS members, HbTPS1 and 2, were explored here for their expressions under various hormone and abiotic stress treatments. HbTPS1 and 2 expressions in latex, cytoplasm of rubber-producing laticifers, were markedly affected by the treatments of ethephon (2-chloroethylphosphonic acid, an ethylene releaser), 2,4-dichlorophenoxyacetic acid, jasmonic acid and salicylic acid although in a distinct manner. HbTPS1 and 2 expressions were significantly up-regulated in a similar manner by cold (5 °C) and heat (40 °C) as examined in Hevea seedling leaves and roots, and by wounding in latex. In comparison, the drought treatment (20% PEG) posed a different effect on HbTPS1 and 2 expressions in Hevea seedlings. For example, in roots, HbTPS1 transcripts accumulated with the treatment, whereas those of HbTPS2 decreased for the first 24 h. Various hormone- and stress-responsive elements could be in silico predicted in HbTPS1 and 2 promoters, and a universal expression was observed for the HbTPS1::GUS activity in all growth stages of transgenic Arabidopsis. These results, coupled with the fact of rubber biosynthesis as a defense response using sucrose as the precursor molecule, propose a potential role of TPS genes in regulating rubber production. [ABSTRACT FROM AUTHOR]

• The latex has high gene expression specificity comparing with other tissues. • Natural rubber biosynthesis is a principal metabolic activity in latex. • Ubiquitin-proteasome system is a primary regulation system in latex. • Ethylene and jasmonate signaling pathways perform different functions in latex. Natural rubber, produced in the laticifers of the para rubber tree (Hevea brasiliensis), is a valuable biopolymer of commercial and strategic importance. While the basic pathway of rubber biosynthesis has been established, further understanding of its regulatory mechanisms might be obtained by studying laticifer-specific gene expression. Here, gene expression patterns was investigated in five different tissues of the rubber tree to explore differentially expressed genes (DEGs). The expression of 4122 high expression genes and 6485 low expression genes in the laticifer was enriched in various metabolic pathways related to rubber biosynthesis. The study results suggest that isopentenyl pyrophosphate (IPP), a principal precursor of natural rubber, is supplied mainly via the mevalonate (MVA) pathway in rubber biosynthesis, rather than the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. The ubiquitin-proteasome system (UBS) that is surprisingly active in the laticifer might serve as an important metabolic control mechanism in rubber biosynthesis. The involvement of latex specific transcription factors (TFs) was identified in seven plant hormone signal transductions, suggesting that jasmonate (JA) signaling is active in the positive regulation of rubber biosynthesis. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via the original article. [ABSTRACT FROM AUTHOR]

In Hevea brasiliensis , the laticifers, a green factory of natural rubber, are specialised vessels in which rubber biosynthesis is the main metabolism, and fresh rubber occupies 35–45% volume of latex (the cytoplasm of laticifers). Intriguingly, Latex can be reproduced, drawing on three essential components (i.e., ATP, Acetyl-CoA and NADPH) from glycolysis in laticifers during tapping (a simple collection method). Some related key genes of sucrose loading and cleavage have been identified, such as HbSUT3/HbSUT1a , HbNIN2 , and surely more genes in TCA cycle will be exploited. Besides the aerobic respiration, the anaerobic fermentation is found highly active in laticifers, however, the role of anaerobic metabolism in laticifers remains unclear. In view of the essential role of anaerobic respiration in helping plant survive adverse conditions by supplementing ATP and intermediate metabolites, we hypothesised that it would be involved in rubber biosynthesis in similar way. To assess the role of anaerobic metabolism in laticifers, two main fermentation genes, lactate dehydrogenase (LDH) and pyruvate decarboxylase (PDC) were analysed and characterised on their gene transcription, protein expression, enzyme activities, and the corresponding metabolites flux in response to tapping treatments. Our comprehensive results revealed that the available ATP decreased rapidly with tapping, accompanying by the increasement of dry rubber yield. Moreover, HbLDH1 and HbPDC4 were identified as the key genes in anaerobic metabolism of laticifers, and they were activated by tapping，indicated by a 3.4 - fold decrease in lactate content and a 4.6 - fold increase of acetaldehyde. In addition, the levels of both acetate and acetyl-CoA decreased rapidly. These results suggested that anaerobic metabolism is indispensable to laticifers, and furthermore the synergistic regulation between ethanol and lactate fermentation has been predicted to be involved in rubber biosynthesis by providing ATP or possible intermediate material acetate. A schematic diagram of anaerobic fermentation that contributes to rubber synthesis after tapping was hereby established. • Anaerobic fermentation in laticifers is largely involved in rubber synthesis. • HbLDH1 and HbPDC4 are key genes in anaerobic metabolism of the laticifers. • The transition from lactate to ethanol fermentation occurs with tappings. [ABSTRACT FROM AUTHOR]

Hevea brasiliensis is presently the only commercial source of natural rubber. The use of anthocyanin production as a visual and non-destructive selection marker may be a viable strategy for increasing the transformation efficiency of this species. In this study, high performance liquid chromatography (HPLC) analysis confirmed that the cyanidin-type anthocyanin is a major determinant of red color in Hevea tissues. It was also found that the anthocyanin production in Hevea somatic embryos could be activated by methyl jasmonate (MeJA) treatment. By sequence alignment and phylogenetic analysis, a novel Hevea R2R3-MYB transcription factor (TF) was isolated and designated here as Hevea brasiliensis Anthocyanin 2 (HbAN2). The expressions of HbAN2 correlate well with the anthocyanin levels in Hevea tissues. The regulatory roles of HbAN2 in anthocyanin biosynthesis were functionally characterized in the native and heterologous systems. In tobacco suspension culture cells, the expression of HbAN2 alone or in combination with two HbbHLH genes activated the promoter of NtDFR , a key gene responsible for anthocyanin biosynthesis. In tobacco plants and Hevea embryos, overexpression of HbAN2 upregulated the late stage anthocyanin pathway genes, resulting in increased anthocyanin accumulation in the transformed parts of the explants, which were therefore easily distinguishable from the non transformed parts. Although transgenic Hevea shoots failed to regenerate into whole plants, these results suggest that HbAN2 is a regulator of anthocyanin biosynthesis and has the potential to be used as visual selection marker for Hevea transformation. • Cyanidin-type anthocyanin is the major determinant of red color in Hevea tissues. • Cyanidin production in Hevea embryos could be induced by methyl jasmonate treatment. • Expression of a novel R2R3-MYB, HbAN2 , correlates with anthocyanin levels in Hevea. • HbAN2 alone or in combination with HbbHLHs could activate the promoter of NtDFR. • HbAN2 overexpression promotes anthocyanin biosynthesis in tobacco and Hevea embryos. [ABSTRACT FROM AUTHOR]

The rubber tree, Hevea brasiliensis , is a tropical rubber-yielding crop with economic importance and provides over 90% of natural rubber (NR) for the global rubber industry. NR biosynthesis belongs to a typical secondary metabolism in laticifers. Definitely, NR production is a huge energy-consuming metabolism, especially during latex regeneration. The tricarboxylic acid (TCA) cycle, as the primary energy source, strongly supports NR synthesis in laticifers. In this study, a mitochondrial citrate synthase (CS) gene named as HbCS4 was isolated from rubber tree and related changes of expression patterns, enzyme activity and metabolites assays in response to tapping and ethephon (ethylene-releasing agent) treatments were compared. These molecular analyses were also conducted in two typical varieties Reyan 7–33–97 and PR107 as well as in HbCS4- overexpressing lines. As a result, the expression characteristics of HbCS4 or its enzyme activity tallied with the energy requirement of rubber synthesis in both treatments and the yielding traits of varieties Reyan 7–33–97 and PR107. In addition, in HbCS4-overexpressing lines, the activities of three key enzymes in TCA cycle, namely CS, NAD-IDH and α-KGDH, were all increased simultaneously. To conclude, these results suggested that HbCS4 is the initial enzyme required for energy supply in laticifers, which greatly contributes to rubber production through changing its expression or enzyme activity. • The TCA cycle is the primary energy source for rubber synthesis. • The HbCS4 contributes to latex regeneration by affecting energy supply. • The activity of HbCS4 could be an energy marker for rubber productivity. [ABSTRACT FROM AUTHOR]

• HbHXK2 and 4 are the predominant isoforms in laticifer. • HbHXK2 and 4 have catalytic activities towards glucose, especially higher in HbHXK4. • HbHXK2 and 4 are presumably targeted to the chloroplast and mitochondria respectively. • Two yield-stimulating experiments enhance the HXK activity and HbHXK4 transcripts. • HbHXK4 plays a role in glucose metabolism for latex regeneration, together with HbHXK2. In higher plants, hexokinase (HXK) is a crucial enzyme that phosphorylates glucose, because there is no evidence for the existence of glucokinase (GLK). In the rubber tree laticifers, the phosphorylation of glucose initiates glycolysis and pentose phosphate pathways, which provide energy and other compounds required for latex regeneration. However, the mechanism of how HXKs participate in the latex regeneration process in laticifers is currently unknown. In this study, four HXK cDNAs (HbHXK1-4) were isolated from the rubber tree (Hevea brasiliensis) and sequenced. Nucleotide-derived amino acid sequences revealed the presence of conserved amino acids that are required for catalytic activity and subcellular targeting. Among these genes, HbHXK2 and 4 were found to be the predominant isoforms in latex (cytoplasm of laticifers) when compared with others. Phylogenetic and subcellular localization analysis revealed that HbHXK2 was presumably targeted to the chloroplast and HbHXK4 to the mitochondria. Both HbHXK2 and 4 were confirmed to have catalytic activities towards glucose and fructose with a significantly higher affinity for glucose. In yield-stimulating experiments, HbHXK4 was strongly induced by tapping and ethephon treatments; whereas, HbHXK2 had the opposite expression pattern. The abundance of HbHXK4 transcripts was consistent with the observed catalytic activity and latex yield. Furthermore, in laticifers, HbHXK4 was more efficient in phosphorylating glucose compared with HbHXK2. Based on these results, it is concluded that HbHXK2 and 4 are the key enzymes for glucose metabolism during latex regeneration and may contribute to the productivity of H. brasiliensis (para rubber tree), especially HbHXK4. [ABSTRACT FROM AUTHOR]