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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).)

Background: Root-associated microbes of the mangrove trees play important roles in protecting and maintaining mangrove ecosystems. At present, most of our understanding of mangrove root-related microbial diversity is obtained from specific mangrove species in selected geographic regions. Relatively little is known about the composition of the bacterial microbiota existing in disparate mangrove species microenvironments, particularly the relationship among different mangrove species in tropical environments., Methods: We collected the root, rhizosphere soil, and non-rhizosphere soil of four mangrove trees ( Acanthus ilicifolius , Bruguiera gymnorrhiza , Clerodendrum inerme, and Lumnitzera racemosa ) and detected the 16S rRNA gene by a conventional PCR. We performed high throughput sequencing using Illumina Novaseq 6000 platform (2 × 250 paired ends) to investigate the bacterial communities related with the different mangrove species., Results: We analyzed the bacterial diversity and composition related to the diverse ecological niches of mangrove species. Our data confirmed distinct distribution patterns of bacterial communities in the three rhizocompartments of the four mangrove species. Microbiome composition varied with compartments and host mangrove species. The bacterial communities between the endosphere and the other two compartments were distinctly diverse independent of mangrove species. The large degree of overlap in critical community members of the same rhizocompartment across distinct mangrove species was found at the phylum level. Furthermore, this is the first report of Acidothermus found in mangrove environments. In conclusion, understanding the complicated host-microbe associations in different mangrove species could lay the foundation for the exploitation of the microbial resource and the production of secondary metabolites., Competing Interests: The authors declare there are no competing interests., (©2023 Sui et al.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. 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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xiao, Mo, Sui, Lin, Long, Qin, Fang and Tang.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fan, Qi, Xiao, Li, Lan, Huang, Yang, Zhang, Zhang, Tao and Tang.)

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.

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 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., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

A novel method based on the monolithic column solid-phase extraction (SPE) coupled with high performance liquid chromatography in online mode was developed for the determination of trace forchlorfenuron in fruits. The enrichment factor of SPE for forchlorfenuron was 214. The method was fully validated. It showed a linear range of 0.01-50.00 microg/L and a limit of detection of 25 ng/L, with the relative standard deviation (RSD) of 3.9%. The proposed method was successfully applied for the determination of forchlorfenuron in grape, kiwifruit and watermelon samples, with the recoveries of 87.0%-120.7% and the RSDs of 0.6%-9.2%. This online method was proved to be selective, sensitive and convenient for the determination of trace forchlorfenuron in complex samples.

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.)

Increasing demand for natural rubber prompts studies into the mechanisms governing the productivity of rubber tree (Heveabrasiliensis). It is very interesting to notice that a rubber tree of clone PR107 in Yunnan, China is reported to yield more than 20 times higher than the average rubber tree. This super-high-yielding (SHY) rubber tree (designated as SY107), produced 4.12 kg of latex (cytoplasm of rubber producing laticifers, containing about 30% of rubber) per tapping, more than 7-fold higher than that of the control. This rubber tree is therefore a good material to study how the rubber production is regulated at a molecular aspect. A comprehensive cDNA-AFLP transcript profiling was performed on the latex of SY107 and its average counterparts by using the 384 selective primer pairs for two restriction enzyme combinations (ApoI/MseI and TaqI/MseI). A total of 746 differentially expressed (DE) transcript-derived fragments (TDFs) were identified, of which the expression patterns of 453 TDFs were further confirmed by RT-PCR. These RT-PCR confirmed TDFs represented 352 non-redundant genes, of which 215 had known or partially known functions and were grouped into 10 functional categories. The top three largest categories were transcription and protein synthesis (representing 24.7% of the total genes), defense and stress (15.3%), and primary and secondary metabolism (14.0%). Detailed analysis of the DE-genes suggests notable characteristics of SHY phenotype in improved sucrose loading capability, rubber biosynthesis-preferred sugar utilization, enhanced general metabolism and timely stress alleviation. However, the SHY phenotype has little correlation with rubber-biosynthesis pathway genes.

Gas chromatography (GC) is a widely used analytical technique in many fields. Sample preparation is very important in GC analysis due to its time consumed and deviations produced. In the present paper, the progress of some typical sample preparation techniques in gas chromatography, including purge and trap, solid phase extraction, solid phase microextraction, liquid phase microextraction, microwave assisted extraction, ultrasonic-assisted extraction, etc., are reviewed.

Real-time RT-PCR (RT-qPCR) is a sensitive and precise method of quantifying gene expression, however, suitable reference genes are required. Here, a systematic reference gene screening was performed by RT-qPCR on 22 candidate genes in Hevea brasiliensis. Two ubiquitin-protein ligases (UBC2a and UBC4) were the most stable when all samples were analyzed together. A mitosis protein (YLS8) and a eukaryotic translation initiation factor (eIF1Aa) were the most stable in response to tapping. UBC2b and UBC1 were the most stable among different genotypes. UBC2b and a DEAD box RNA helicase (RH2b) were the most stable across individual trees. YLS8 and RH8 were most stably expressed in hormone-treated samples. Expression of the candidate reference genes varied significantly across different tissues, and at least four genes (RH2b, RH8, UBC2a and eIF2) were needed for expression normalization. In addition, examination of relative expression of a sucrose transporter HbSUT3 in different RNA samples demonstrated the importance of additional reference genes to ensure accurate quantitative expression analysis. Overall, our work serves as a guide for selection of reference genes in RT-qPCR gene expression studies in H. brasiliensis., (Published by Elsevier Ireland Ltd.)

cDNA amplified fragment length polymorphism (cDNA-AFLP) is a powerful transcript-profiling tool widely used in diverse plant species. When applied to a new biological system, however, existing protocols usually require substantial modifications. Furthermore, the usage of radioactive isotope in typical protocols excludes their application in many labs. Latex, as the cytoplasm of rubber-producing cells sees a critical role in elucidating rubber biosynthesis and its regulation in rubber tree (Hevea brasiliensis). This paper describes a detailed step-by-step silver-staining cDNA-AFLP procedure, which is suitable to latex transcript profiling analysis. Theoretical analysis revealed that with the combination of two restriction enzyme pairs (ApoI/MseI and TaqI/MseI), approximately 94% of latex whole transcriptome could be visualized. After varying multiple parameters, including the amounts of primary and secondary template usage, pre-amplification cycle number and gel development, we obtained a high-quality silver-staining fingerprint. In the ApoI/MseI system, an average of 88.6 discernable bands (100-1,000 bp) was produced for each selective primer pair, and 97.2 bands for another system (TaqI/MseI). TaqI/MseI was the first pair of 4-bp cutters used in cDNA-AFLP analysis and proved to be efficient and reliable. The sensitivity and reliability of our method were further verified by an application example in detecting differential gene expression in the latex of Hevea tree.