Testing intra-species variation in allocation to growth and defense in rubber tree (Hevea brasiliensis).

Background: Plants allocate resources to growth, defense, and stress resistance, and resource availability can affect the balance between these allocations. Allocation patterns are well-known to differ among species, but what controls possible intra-specific trade-offs and if variation in growth vs. defense potentially evolves in adaptation to resource availability. Methods: We measured growth and defense in a provenance trial of rubber trees (Hevea brasiliensis) with clones originating from the Amazon basin. To test hypotheses on the allocation to growth vs. defense, we relate biomass growth and latex production to wood and leaf traits, to climate and soil variables from the location of origin, and to the genetic relatedness of the Hevea clones. Results: Contrary to expectations, there was no trade-off between growth and defense, but latex yield and biomass growth were positively correlated, and both increased with tree size. The absence of a trade-off may be attributed to the high resource availability in a plantation, allowing trees to allocate resources to both growth and defense. Growth was weakly correlated with leaf traits, such as leaf mass per area, intrinsic water use efficiency, and leaf nitrogen content, but the relative investment in growth vs. defense was not associated with specific traits or environmental variables. Wood and leaf traits showed clinal correlations to the rainfall and soil variables of the places of origin. These traits exhibited strong phylogenetic signals, highlighting the role of genetic factors in trait variation and adaptation. The study provides insights into the interplay between resource allocation, environmental adaptations, and genetic factors in trees. However, the underlying drivers for the high variation of latex production in one of the commercially most important tree species remains unexplained.

Genetic diversity analysis and core germplasm bank construction in cold resistant germplasm of rubber trees (Hevea brasiliensis).

The rubber tree, Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell. Arg., is the sole plant worldwide utilized for the commercial production of natural rubber. Following years of breeding, there exists a wide array of germplasm differentiation in rubber trees. The exploration of diversity and population structure within rubber tree germplasm resources, alongside the establishment of core germplasm resources, is instrumental in elucidating the genetic background and facilitating the effective utilization and management of these resources. By employing SNP molecular marker technology, 195 rubber tree resources were amplified, their genetic diversity analyzed, and a fingerprint map was subsequently constructed. Through this process, the cold-resistant core germplasm of rubber trees was identified. The results revealed that the PIC, He, and pi values ranged from 0.0905 to 0.3750, 0.095 to 0.5000, and 0.0953 to 0.5013, respectively. Both group structure analysis and cluster analysis delineated the accessions into two groups, signifying a simple group structure. A core germplasm bank was established with a sampling ratio of 10%, comprising 21 accessions divided into two populations. Population G1 consists of 20 accessions, while population G2 comprises 1 accession. The research findings have led to the creation of a molecular database that is anticipated to contribute to the management and subsequent breeding applications of rubber tree accessions.

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

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

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.

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.

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.

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.

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.

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.

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.

The Rubber Tree (Heveae brasiliensis) MLO Protein HbMLO12 Promotes Plant Susceptibility to Sustain Infection by a Powdery Mildew Fungus.

Powdery mildew severely affects several important crops and cash plants. Disruption of mildew resistance locus O (MLO) genes elevates resistance against powdery mildew in several plants. However, whether rubber tree (Heveae brasiliensis) MLO proteins are linked to susceptibility remains unknown, owing to technical limitations in the genetic manipulation of this woody plant. A previous study showed that the H. brasiliensis MLO-like protein HbMLO12 demonstrates high amino acid sequence similarity with the known Arabidopsis MLO protein AtMLO12. In this study, we investigated whether HbMLO12 regulates susceptibility to powdery mildew. H. brasiliensis leaves take up exogenously synthesized double-stranded RNAs (dsRNAs), and foliar application of dsRNA homologous to HbMLO12 gene specifically induces HbMLO12 silencing in H. brasiliensis leaf tissues. Notably, HbMLO12 silencing inhibited fungal infection and elevated the immune response during interaction with the rubber tree powdery mildew fungus. Furthermore, the heterologous expression of HbMLO12 suppressed bacterial flg22- and fungal chitin-induced immune responses and enhanced bacterial infection in Arabidopsis. Our study provides evidence that HbMLO12 contributes to susceptibility to powdery mildew. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

Comprehensive Analysis of the Catalase (CAT) Gene Family and Expression Patterns in Rubber Tree (Hevea brasiliensis) under Various Abiotic Stresses and Multiple Hormone Treatments.

Catalase (CAT) is one of the key enzymes involved in antioxidant defense systems and mainly scavenges H2O2 and plays a vital role in plant growth, development, and various adverse stresses. To date, a systematic study of the CAT gene family in rubber tree has not been reported. In this study, five HbCAT gene family members were identified from the rubber tree genome, and these were mainly clustered into two subfamilies. Gene structure and motif analysis showed that exon-intron and motif patterns were conserved across different plant species. Sequence analysis revealed that HbCAT proteins contain one active catalytic site, one heme-ligand signature sequence, three conserved amino acid residues (His, Tyr, and Asn), and one peroxisome-targeting signal 1 (PTS1) sequence. Fragment duplication is a selection pressure for the evolution of the HbCAT family based on Ka/Ks values. Analysis of cis-acting elements in the promoters indicated that HbCAT gene expression might be regulated by abscisic acid (ABA), salicylic acid (SA), and MYB transcription factors; furthermore, these genes might be involved in plant growth, development, and abiotic stress responses. A tissue-specific expression analysis showed that HbCATs gradually increased with leaf development and were highly expressed in mature leaves. Gene expression profiling exhibited the differential expression of the HbCATs under cold, heat, drought, and NaCl stresses. Our results provide comprehensive information about the HbCAT gene family, laying the foundation for further research on its function in rubber tree.

Cloning, Expression Analysis, and Functional Characterization of Candidate Oxalate Transporter Genes of HbOT1 and HbOT2 from Rubber Tree (Hevea brasiliensis).

Secretion of oxalic acid from roots is an important aluminum detoxification mechanism for many plants such as Hevea brasiliensis (rubber tree). However, the underlying molecular mechanism and oxalate transporter genes in plants have not yet been reported. In this study, the oxalate transporter candidate genes HbOT1 and HbOT2 from the rubber tree were cloned and preliminarily identified. It was found that HbOT1 had a full length of 1163 bp with CDS size of 792 bp, encoding 263 amino acids, and HbOT2 had a full length of 1647 bp with a CDS region length of 840 bp, encoding 279 amino acid residues. HbOT1 and HbOT2 were both stable hydrophobic proteins with transmembrane structure and SNARE_assoc domains, possibly belonging to the SNARE_assoc subfamily proteins of the SNARE superfamily. qRT-PCR assays revealed that HbOT1 and HbOT2 were constitutively expressed in different tissues, with HbOT1 highly expressed in roots, stems, barks, and latex, while HbOT2 was highly expressed in latex. In addition, the expressions of HbOT1 and HbOT2 were up-regulated in response to aluminum stress, and they were inducible by metals, such as copper and manganese. Heterologous expression of HbOT1 and HbOT2 in the yeast mutant AD12345678 enhanced the tolerance to oxalic acid and high concentration aluminum stress, which was closely correlated with the secretion of oxalic acid. This study is the first report on oxalate transporter genes in plants, which provides a theoretical reference for the study on the molecular mechanism of oxalic acid secretion to relieve aluminum toxicity and on aluminum-tolerance genetic engineering breeding.

A divide-and-conquer approach for genomic prediction in rubber tree using machine learning.

Rubber tree (Hevea brasiliensis) is the main feedstock for commercial rubber; however, its long vegetative cycle has hindered the development of more productive varieties via breeding programs. With the availability of H. brasiliensis genomic data, several linkage maps with associated quantitative trait loci have been constructed and suggested as a tool for marker-assisted selection. Nonetheless, novel genomic strategies are still needed, and genomic selection (GS) may facilitate rubber tree breeding programs aimed at reducing the required cycles for performance assessment. Even though such a methodology has already been shown to be a promising tool for rubber tree breeding, increased model predictive capabilities and practical application are still needed. Here, we developed a novel machine learning-based approach for predicting rubber tree stem circumference based on molecular markers. Through a divide-and-conquer strategy, we propose a neural network prediction system with two stages: (1) subpopulation prediction and (2) phenotype estimation. This approach yielded higher accuracies than traditional statistical models in a single-environment scenario. By delivering large accuracy improvements, our methodology represents a powerful tool for use in Hevea GS strategies. Therefore, the incorporation of machine learning techniques into rubber tree GS represents an opportunity to build more robust models and optimize Hevea breeding programs.

Ectopic Expression of HbRPW8-a from Hevea brasiliensis Improves Arabidopsis thaliana Resistance to Powdery Mildew Fungi (Erysiphe cichoracearum UCSC1).

The RPW8s (Resistance to Powdery Mildew 8) are atypical broad-spectrum resistance genes that provide resistance to the powdery mildew fungi. Powdery mildew of rubber tree is one of the serious fungal diseases that affect tree growth and latex production. However, the RPW8 homologs in rubber tree and their role of resistance to powdery mildew remain unclear. In this study, four RPW8 genes, HbRPW8-a, b, c, d, were identified in rubber tree, and phylogenetic analysis showed that HbRPW8-a was clustered with AtRPW8.1 and AtRPW8.2 of Arabidopsis. The HbRPW8-a protein was localized on the plasma membrane and its expression in rubber tree was significantly induced upon powdery mildew infection. Transient expression of HbRPW8-a in tobacco leaves induced plant immune responses, including the accumulation of reactive oxygen species and the deposition of callose in plant cells, which was similar to that induced by AtRPW8.2. Consistently, overexpression of HbRPW8-a in Arabidopsis thaliana enhanced plant resistance to Erysiphe cichoracearum UCSC1 and Pseudomonas syringae pv. tomato DC30000 (PstDC3000). Moreover, such HbRPW8-a mediated resistance to powdery mildew was in a salicylic acid (SA) dependent manner. Taken together, we demonstrated a new RPW8 member in rubber tree, HbRPW8-a, which could potentially contribute the resistance to powdery mildew.

Germination and in vitro development of mature zygotic embryos and protein profile of seedlings of wild and cultivated Hevea brasiliensis.

The main factors governing Hevea brasiliensis germination and seedling establishment remains unclear. We examined the effect of growth regulators Indole 3-Acetic Acid (IAA) and 6-Benzylaminopurine (BAP), and their interactions on germination and the development of mature zygotic embryos (MZE) and protein profile of Hevea brasiliensis seedlings from wild and cultivated (clone PB 250) genotypes. Embryonic axes excised from seeds (wild and clone PB 250) were inoculated in Murashige and Skoog medium (control) and supplemented with IAA (3 µM) and BAP (6 µM) individually and their combination (3 µM IAA + 6 µM BAP). For both genotypes, the mature embryos displayed a high percentage of germination and establishment, and the seedlings were characterized by protein bands ranging from 7 to 30 kDa. Notably, the wild genotype showed proteins in the 14 kDa range, which may be associated with one of the major rubber elongation factors (REF). The wild and clone genotypes presented different behavior and strategies in relation to the protein profile in the presence of different growth regulators. Although the latex biosynthetic pathway and its mechanisms of regulation still remain largely unknown, our results aid in our understanding of the dynamics of proteins in different rubber tree clones in vitro.

Genome-wide identification and response stress expression analysis of the BES1 family in rubber tree (Hevea brasiliensis Muell. Arg.).

Brassinolide (BR) plays an important role in plant growth, development, and the adaptation adversity process. Moreover, BRI1-EMS-SUPPRESSOR 1 (BES1) genes are crucial transcription factors (TFs) in the BR signaling pathway. To realize the function of HbBES1 family is helpful to improve genetic resources for rubber tree breeding. Based on the rubber tree database, we used bioinformatics to characterize physicochemical properties, gene structure, cis-elements, and expression patterns. These results indicated that there were nine BES1 members in rubber tree, which we named HbBES1-1 to HbBES1-9 and divided into two groups (I and II) based on their genetic relationships. HbBES1 genes in the same group shared similar gene structures and motifs. Cis-acting element analysis showed that the promoter sequences of HbBES1 genes contained many regulator elements that were related to hormone and stress, indicating that HbBES1 genes might be involved in the regulation of hormone and stress signal pathways. Our analysis of tissue specificity revealed that all of the nine HbBES1 members expressed highly in branches. Gene expression profiles under different hormone treatments showed that the HbBES1 gene family was induced to varying degrees under different hormones, HbBES1-3 and HbBES1-9 were extremely induced by ethylene (ETH). These results lay the foundation for further exploration of the molecular mechanism of the BES1 gene family, especially HbBES1-3 and HbBES1-9, regulating plant stress tolerance in rubber tree.