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.

Integrated physiology, transcriptome and proteome analyses highlight the potential roles of multiple hormone-mediated signaling pathways involved in tapping panel dryness in rubber tree.

Currently, one of the most serious threats to rubber tree is the tapping panel dryness (TPD) that greatly restricts natural rubber production. Over-tapping or excessive ethephon stimulation is regarded as the main cause of TPD occurrence. Although extensive studies have been carried out, the molecular mechanism underlying TPD remains puzzled. An attempt was made to compare the levels of endogenous hormones and the profiles of transcriptome and proteome between healthy and TPD trees. Results showed that most of endogenous hormones such as jasmonic acid (JA), 1-aminocyclopropanecarboxylic acid (ACC), indole-3-acetic acid (IAA), trans-zeatin (tZ) and salicylic acid (SA) in the barks were significantly altered in TPD-affected rubber trees. Accordingly, multiple hormone-mediated signaling pathways were changed. In total, 731 differentially expressed genes (DEGs) and 671 differentially expressed proteins (DEPs) were identified, of which 80 DEGs were identified as putative transcription factors (TFs). Further analysis revealed that 12 DEGs and five DEPs regulated plant hormone synthesis, and that 16 DEGs and six DEPs were involved in plant hormone signal transduction pathway. Nine DEGs and four DEPs participated in rubber biosynthesis and most DEGs and all the four DEPs were repressed in TPD trees. All these results highlight the potential roles of endogenous hormones, signaling pathways mediated by these hormones and rubber biosynthesis pathway in the defense response of rubber trees to TPD. The present study extends our understanding of the nature and mechanism underlying TPD and provides some candidate genes and proteins related to TPD for further research in the future.

Heterozygosity analysis of spontaneous 2n female gametes and centromere mapping of the diploid Hevea brasiliensis based on full-sib triploid populations.

KEY MESSAGE: Unreduced megagametophytes via second-division restitution were confirmed through heterozygosity analysis, and four candidate physical centromeres of rubber were located for the first time. The evaluation of maternal heterozygosity restitution (MHR) is vital in identifying the mechanism of 2n gametogenesis and assessing the utilization value of 2n gametes. In this study, three full-sib triploid populations were employed to evaluate the MHR of 2n female gametes of rubber tree clone GT1 and to confirm their genetic derivation. The 2n female gametes of GT1 were derived from second-division restitution (SDR) and transmitted more than half of the parental heterozygosity. In addition, low recombination frequency markers were developed, and four candidate physical centromeres of rubber tree were located for the first time. The confirmation that 2n female gametes of rubber tree clone GT1 are derived from SDR provides insights into the molecular mechanisms of 2n gametogenesis. In addition, the identified centromere location will aid in the development of centromeric markers for the rapid identification of the 2n gametogenesis mechanism.

Population Genetic Structure of the Rubber Tree Powdery Mildew Pathogen (Erysiphe quercicola) from China.

In order to manage agricultural pathogens, it is crucial to understand the population structure underlying epidemics. Rubber tree powdery mildew, caused by Erysiphe quercicola, is a serious threat to rubber plantations worldwide, especially in subtropical environments including all rubber tree-growing regions in China. However, the population structure of the pathogen is uncertain. In this study, 16 polymorphic microsatellite markers were used to genotype powdery mildew samples from the main rubber tree-growing regions including Yunnan (YN), Hainan (HN), western Guangdong (WG), and eastern Guangdong (EG). YN had higher genotypic diversity (Simpson's indices), genotypic evenness, Nei's gene diversity, allelic richness, and private allelic richness than the other regions. Cluster analysis, discriminant analysis of principal components, pairwise divergence, and shared multilocus genotype analyses all showed that YN differed significantly from the other regions. The genetic differentiation was small among the other three regions (HN, WG, and EG). Analysis of molecular variance indicated that the variability among regions accounted for 22.37% of the total variability. Genetic differentiation was significantly positively correlated (Rxy = 0.772, P = 0.001) with geographic distance. Linkage equilibrium analysis suggested possible occurrence of sexual recombination although asexual reproduction predominates in E. quercicola. The results suggested that although significant genetic differentiation of E. quercicola occurred between YN and the other regions, pathogen populations from the other three regions lacked genetic differentiation.

HbBIN2 Functions in Plant Cold Stress Resistance through Modulation of HbICE1 Transcriptional Activity and ROS Homeostasis in Hevea brasiliensis.

Cold stress poses significant limitations on the growth, latex yield, and ecological distribution of rubber trees (Hevea brasiliensis). The GSK3-like kinase plays a significant role in helping plants adapt to different biotic and abiotic stresses. However, the functions of GSK3-like kinase BR-INSENSITIVE 2 (BIN2) in Hevea brasiliensis remain elusive. Here, we identified HbBIN2s of Hevea brasiliensis and deciphered their roles in cold stress resistance. The transcript levels of HbBIN2s are upregulated by cold stress. In addition, HbBIN2s are present in both the nucleus and cytoplasm and have the ability to interact with the INDUCER OF CBF EXPRESSION1(HbICE1) transcription factor, a central component in cold signaling. HbBIN2 overexpression in Arabidopsis displays decreased tolerance to chilling stress with a lower survival rate and proline content but a higher level of electrolyte leakage (EL) and malondialdehyde (MDA) than wild type under cold stress. Meanwhile, HbBIN2 transgenic Arabidopsis treated with cold stress exhibits a significant increase in the accumulation of reactive oxygen species (ROS) and a decrease in the activity of antioxidant enzymes. Further investigation reveals that HbBIN2 inhibits the transcriptional activity of HbICE1, thereby attenuating the expression of C-REPEAT BINDING FACTOR (HbCBF1). Consistent with this, overexpression of HbBIN2 represses the expression of CBF pathway cold-regulated genes under cold stress. In conclusion, our findings indicate that HbBIN2 functions as a suppressor of cold stress resistance by modulating HbICE1 transcriptional activity and ROS homeostasis.

Construction and analysis of the tapping panel dryness-related lncRNA/circRNA-miRNA-mRNA ceRNA network in latex of Hevea brasiliensis.

Tapping panel dryness (TPD) results in a severe reduction in latex yield in Hevea brasiliensis. However, the molecular regulatory mechanisms of TPD occurrence are still largely unclear. In this study, whole-transcriptome sequencing was carried out on latex from TPD and healthy trees. In total, 7078 long noncoding RNAs (lncRNAs), 3077 circular RNAs (circRNAs), 4956 miRNAs, and 25041 mRNAs were identified in latex, among which 435 lncRNAs, 68 circRNAs, 320 miRNAs, and 1574 mRNAs were differentially expressed in the latex of TPD trees. GO and KEGG analyses indicated that plant hormone signal transduction, MAPK signaling pathway, and ubiquitin-mediated proteolysis were the key pathways associated with TPD onset. Phytohormone profiling revealed significant changes in the contents of 28 hormonal compounds, among which ACC, ABA, IAA, GA, and JA contents were increased, while SA content was reduced in TPD latex, suggesting that hormone homeostasis is disrupted in TPD trees. Furthermore, we constructed a TPD-related competitive endogenous RNA (ceRNA) regulatory network of lncRNA/circRNA-miRNA-mRNA with 561 edges and 434 nodes (188 lncRNAs, 5 circRNAs, 191 miRNAs, and 50 mRNAs) and identified two hub lncRNAs (MSTRG.11908.1 and MSTRG.8791.1) and four hub miRNAs (hbr-miR156, miR156-x, miRf10477-y, and novel-m0452-3p). Notably, the lncRNA-miR156/157-SPL module containing three hubs probably plays a crucial role in TPD onset. The expression of network hubs and the lncRNA-miR156/157-SPL module were further validated by qRT-PCR. Our results reveal the TPD-associated ceRNA regulatory network of lncRNA/circRNA-miRNA-mRNA in latex and lay a foundation for further investigation of molecular regulatory mechanisms for TPD onset in H. brasiliensis.

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.

Comparative transcriptome analysis of high- and low-embryogenic Hevea brasiliensis genotypes reveals involvement of phytohormones in somatic embryogenesis.

BACKGROUND: Rubber plant (Hevea brasiliensis) is one of the major sources of latex. Somatic embryogenesis (SE) is a promising alterative to its propagation by grafting and seed. Phytohormones have been shown to influence SE in different plant species. However, limited knowledge is available on the role of phytohormones in SE in Hevea. The anther cultures of two Hevea genotypes (Yunyan 73477-YT and Reken 628-RT) with contrasting SE rate were established and four stages i.e., anthers (h), anther induced callus (y), callus differentiation state (f), and somatic embryos (p) were studied. UPLC-ESI-MS/MS and transcriptome analyses were used to study phytohormone accumulation and related expression changes in biosynthesis and signaling genes. RESULTS: YT showed higher callus induction rate than RT. Of the two genotypes, only YT exhibited successful SE. Auxins, cytokinins (CKs), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), gibberellins (GAs), and ethylene (ETH) were detected in the two genotypes. Indole-3-acetic acid (IAA), CKs, ABA, and ETH had notable differences in the studied stages of the two genotypes. The differentially expressed genes identified in treatment comparisons were majorly enriched in MAPK and phytohormone signaling, biosynthesis of secondary metabolites, and metabolic pathways. The expression changes in IAA, CK, ABA, and ETH biosynthesis and signaling genes confirmed the differential accumulation of respective phytohormones in the two genotypes. CONCLUSION: These results suggest potential roles of phytohormones in SE in Hevea.

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.

Genomic insight into domestication of rubber tree.

Understanding the genetic basis of rubber tree (Hevea brasiliensis) domestication is crucial for further improving natural rubber production to meet its increasing demand worldwide. Here we provide a high-quality H. brasiliensis genome assembly (1.58 Gb, contig N50 of 11.21 megabases), present a map of genome variations by resequencing 335 accessions and reveal domestication-related molecular signals and a major domestication trait, the higher number of laticifer rings. We further show that HbPSK5, encoding the small-peptide hormone phytosulfokine (PSK), is a key domestication gene and closely correlated with the major domestication trait. The transcriptional activation of HbPSK5 by myelocytomatosis (MYC) members links PSK signaling to jasmonates in regulating the laticifer differentiation in rubber tree. Heterologous overexpression of HbPSK5 in Russian dandelion (Taraxacum kok-saghyz) can increase rubber content by promoting laticifer formation. Our results provide an insight into target genes for improving rubber tree and accelerating the domestication of other rubber-producing plants.

Latex C-serum from Hevea brasiliensis induces apoptotic cell death in a leukemic cell line.

BACKGROUND: Hevea brasiliensis latex is generally cultivated for the use of rubber particles. Previous studies have shown that the antiproliferative activity of C-serum in hepatocellular carcinoma is not induced through the classical apoptotic signaling pathway. However, in a leukemic cell line, the anti-proliferation effect of latex C serum remained unclear. METHODS: Leukemic cell lines (K562 and U937) and human peripheral blood mononuclear cells (PBMCs) were examined for cell viability using the MTT assay. Flow cytometry was used for apoptotic cell detection by annexin V/PI staining. The expression levels of proapoptotic and antiapoptotic marker genes were measured by qRT‒PCR. Moreover, the caspase activities of the extrinsic and intrinsic apoptotic pathways were detected by enzymatic activities. RESULTS: Latex C-serum inhibited cell proliferation in the K562 and U937 leukemic cell lines but did not affect human PBMCs. Latex C-serum significantly induced the percentage of early and late apoptotic cells in the leukemic cell line. The expression levels of the pro-apoptotic marker genes BAD, BAX, and CASPASE3 significantly increased in the leukemic cell line after post-latex C-serum leukemic cell treatment. The extrinsic, intrinsic and common apoptotic pathways were also studied through caspase-8, -9, and -3 activities. Latex C-serum treatment significantly induced caspase-8, -9, and -3 activation in the K562 cell line and U937 cell line compared to the untreated cells. CONCLUSIONS: These results indicate that latex C-serum enhanced anti-proliferation in leukemic cell lines by inducing apoptosis and caspase activation.

Whole genome resequencing reveal patterns of genetic variation within Colletotrichum acutatum species complex from rubber trees in China.

The Colletotrichum acutatum species complex possesses a diverse number of important traits, such as a wide host range and host preference, different modes of reproduction, and different strategies of host infection. Research using comparative genomics has attempted to find correlations between these traits. Here, we used multi-locus techniques and gene genealogical concordance analysis to investigate the phylogenetic relationships and taxonomic status of the Colletotrichum acutatum species complex using field isolates obtained from rubber trees. The results revealed that the dominant species was C. australisinense, followed by C. bannaense, while strain YNJH17109 was identified as C. laticiphilum. The taxonomic status of strains YNLC510 and YNLC511 was undetermined. Using whole-genome single nucleotide polymorphism data to analyze population structure, 18 strains of C. australisinense were subsequently divided into four populations, one of which was derived from an admixture of two populations. In addition, the strains LD1687, GD1628, and YNLC516, did not belong to any populations, and were considered to be admixtures of two or more populations. A split decomposition network analysis also provided evidence for genetic recombination within Colletotrichum acutatum species complex from rubber trees in China. Overall, a weak phylogeographic sub-structure was observed. Analysis also revealed significant differences in morphological characters and levels of virulence between populations.

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

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

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.

Characterization of Colletotrichum Causing Anthracnose on Rubber Trees in Yunnan: Two New Records and Two New Species from China.

Among the most damaging diseases of rubber trees is anthracnose caused by the genus Colletotrichum, which leads to significant economic losses. Nonetheless, the specific Colletotrichum spp. that infect rubber trees in Yunnan Province, an important natural rubber base in China, have not been extensively investigated. Here, we isolated 118 Colletotrichum strains from rubber tree leaves exhibiting anthracnose symptoms in multiple plantations in Yunnan. Based on comparisons of their phenotypic characteristics and internal transcribed spacer ribosomal DNA sequences, 80 representative strains were chosen for additional phylogenetic analysis based on eight loci (act, ApMat, cal, CHS-1, GAPDH, GS, his3, and tub2), and nine species were identified. Colletotrichum fructicola, C. siamense, and C. wanningense were found to be the dominant pathogens causing rubber tree anthracnose in Yunnan. C. karstii was common, whereas C. bannaense, C. brevisporum, C. jinpingense, C. mengdingense, and C. plurivorum were rare. Among these nine species, C. brevisporum and C. plurivorum are reported for the first time in China, and two species are new to the world: C. mengdingense sp. nov. in the C. acutatum species complex and C. jinpingense sp. nov. in the C. gloeosporioides species complex. Their pathogenicity was confirmed with Koch's postulates by inoculating each species in vivo on rubber tree leaves. This study clarifies the geographic distribution of Colletotrichum spp. associated with anthracnose on rubber trees in representative locations of Yunnan, which is crucial for the implementation of quarantine measures.

Functional characterization of a bark-specific monoterpene synthase potentially involved in wounding- and methyl jasmonate-induced linalool emission in rubber (Hevea brasiliensis).

Rubber (Hevea brasiliensis) is a latex-producing plant that often encounters mechanical wounding, as well as pathogen and pest attacks through wound sites during and after tapping. Terpenoids play an important role in the ecological interactions of many plant species, and their diversity is mainly generated by enzymes known as terpene synthases (TPS). In this study, one cDNA sequence encoding a putative terpene synthase, HbTPS20, was obtained from the bark tissues of H. brasiliensis. The encoded protein contains 610 amino acids with a putative N-terminal plastid transit peptide of approximately 70 residues. It belongs to the TPS-b subfamily. Further phylogenetic analysis showed that HbTPS20 formed a separate branch that diverged from the progenitor of all other potentially functional terpene synthases of the rubber TPS-b subfamily. The truncated HbTPS20 without the signal peptide coding sequence was successfully expressed in E. coli and in vitro enzymatic assays with geranyl diphosphate (GPP) or neryl diphosphate (NPP) as a substrate defined HbTPS20 as an active linalool synthase (HbLIS) with the ability to produce linalool as the principal product. RT-qPCR analysis showed that the highest transcript levels of HbTPS20 were found in barks, and this gene was expressed at 2.26- and 250-fold greater levels in the bark tissues of wounded and MeJA-treated plants, respectively, than in those of the control plants. This indicates that this gene may be involved in the induced stress responses of rubber.

Chromosome-level wild Hevea brasiliensis genome provides new tools for genomic-assisted breeding and valuable loci to elevate rubber yield.

The rubber tree (Hevea brasiliensis) is grown in tropical regions and is the major source of natural rubber. Using traditional breeding approaches, the latex yield has increased by sixfold in the last century. However, the underlying genetic basis of rubber yield improvement is largely unknown. Here, we present a high-quality, chromosome-level genome sequence of the wild rubber tree, the first report on selection signatures and a genome-wide association study (GWAS) of its yield traits. Population genomic analysis revealed a moderate population divergence between the Wickham clones and wild accessions. Interestingly, it is suggestive that H. brasiliensis and six relatives of the Hevea genus might belong to the same species. The selective sweep analysis found 361 obvious signatures in the domesticated clones associated with 245 genes. In a 15-year field trial, GWAS identified 155 marker-trait associations with latex yield, in which 326 candidate genes were found. Notably, six genes related to sugar transport and metabolism, and four genes related to ethylene biosynthesis and signalling are associated with latex yield. The homozygote frequencies of the causal nonsynonymous SNPs have been greatly increased under selection, which may have contributed to the fast latex yield improvement during the short domestication history. Our study provides insights into the genetic basis of the latex yield trait and has implications for genomic-assisted breeding by offering valuable resources in this new domesticated crop.

In Silico Genome-Wide Mining and Analysis of Terpene Synthase Gene Family in Hevea Brasiliensis.

Terpene synthases (TPSs) catalyze terpenoid synthesis and affect the intracellular isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) concentration. In this study, we mined the in silico genome-wide TPS genes of Hevea brasiliensis and identified 47 full-length TPS genes. They had DDXXD, DXDD, NSE/DTE, RR(X)8 W, EA(X)W, and other conserved motifs. The phylogenetic tree analysis revealed that the TPSs of H.brasiliensis (HbTPSs) were divided into five subfamilies, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g. HbTPSs were predicted to have functions in the cellular components, molecular functions, and biological processes. HbTPSs were involved in seven pathways, which were K14173, K14175, K15803, K04120, K04121, K17982, and K12742 in the secondary metabolite pathway prediction. Three-dimensional structures of HbTPSs of 7 pathways were predicted, and DDXXD, NSE/DTE, and EA(X)W conserved motifs near the binding sites were found. Cis-acting elements analysis showed that they had more cis-acting elements related to phytohormone responsiveness, which indicated that terpenoid biosynthesis might be related to phytohormone regulation. RNA-Seq analysis showed that different HbTPSs were expressed differentially in different tissues. This study's results help reveal the role of HbTPSs and their molecular mechanism and help resolve the regulatory mechanism of terpenoid biosynthesis in H.brasiliensis.

Downregulation of HbFPS1 affects rubber biosynthesis of Hevea brasiliensis suffering from tapping panel dryness.

Tapping panel dryness (TPD) is a century-old problem that has plagued the natural rubber production of Hevea brasiliensis. TPD may result from self-protective mechanisms of H. brasiliensis in response to stresses such as excessive hormone stimulation and mechanical wounding (bark tapping). It has been hypothesized that TPD impairs rubber biosynthesis; however, the underlying mechanisms remain poorly understood. In the present study, we firstly verified that TPD-affected rubber trees exhibited lower rubber biosynthesis activity and greater rubber molecular weight compared to healthy rubber trees. We then demonstrated that HbFPS1, a key gene of rubber biosynthesis, and its expression products were downregulated in the latex of TPD-affected rubber trees, as revealed by transcriptome sequencing and iTRAQ-based proteome analysis. We further discovered that the farnesyl diphosphate synthase HbFPS1 could be recruited to small rubber particles by HbSRPP1 through protein-protein interactions to catalyze farnesyl diphosphate (FPP) synthesis and facilitate rubber biosynthesis initiation. FPP content in the latex of TPD-affected rubber trees was significantly decreased with the downregulation of HbFPS1, ultimately resulting in abnormal development of rubber particles, decreased rubber biosynthesis activity, and increased rubber molecular weight. Upstream regulator assays indicated that a novel regulator, MYB2-like, may be an important regulator of downregulation of HbFPS1 in the latex of TPD-affected rubber trees. Our findings not only provide new directions for studying the molecular events involved in rubber biosynthesis and TPD syndrome and contribute to rubber management strategies, but also broaden our knowledge of plant isoprenoid metabolism and its regulatory networks.