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.

Genome-wide identification of rubber tree pathogenesis-related 10 (PR-10) proteins with biological relevance to plant defense.

Pathogenesis-related 10 (PR-10) is a group of small intracellular proteins that is one of 17 subclasses of pathogenesis-related proteins in plants. The PR-10 proteins have been studied extensively and are well-recognized for their contribution to host defense against phytopathogens in several plant species. Interestingly, the accumulation of PR-10 proteins in the rubber tree, one of the most economically important crops worldwide, after being infected by pathogenic organisms has only recently been reported. In this study, the homologous proteins of the PR-10 family were systemically identified from the recently available rubber tree genomes in the NCBI database. The sequence compositions, structural characteristics, protein physical properties, and phylogenetic relationships of identified PR-10 proteins in rubber trees support their classification into subgroups, which mainly consist of Pru ar 1-like major allergens and major latex-like (MLP) proteins. The rubber tree PR10-encoding genes were majorly clustered on chromosome 15. The potential roles of rubber tree PR-10 proteins are discussed based on previous reports. The homologous proteins in the PR-10 family were identified in the recent genomes of rubber trees and were shown to be crucial in host responses to biotic challenges. The genome-wide identification conducted here will accelerate the future study of rubber tree PR-10 proteins. A better understanding of these defense-related proteins may contribute to alternative ways of developing rubber tree clones with desirable traits in the future.

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.

Interactions of REF1 and SRPP1 rubber particle proteins from Hevea brasiliensis with synthetic phospholipids: Effect of charge and size of lipid headgroup.

According to the fatty acid and headgroup compositions of the phospholipids (PL) from Hevea brasiliensis latex, three synthetic PL were selected (i.e. POPA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and POPG: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) to investigate the effect of PL headgroup on the interactions with two major proteins of Hevea latex, i.e. Rubber Elongation Factor (REF1) and Small Rubber Particle Protein (SRPP1). Protein/lipid interactions were screened using two models (lipid vesicles in solution or lipid monolayers at air/liquid interface). Calcein leakage, surface pressure, ellipsometry, microscopy and spectroscopy revealed that both REF1 and SRPP1 displayed stronger interactions with anionic POPA and POPG, as compared to zwitterionic POPC. A particular behavior of REF1 was observed when interacting with POPA monolayers (i.e. aggregation + modification of secondary structure from α-helices to ß-sheets, characteristic of its amyloid aggregated form), which might be involved in the irreversible coagulation mechanism of Hevea rubber particles.

The impact of non-deproteinization on physicochemical and biological properties of natural rubber latex for biomedical applications.

Latex is a colloidal suspension derived from the Hevea brasiliensis tree, derived from natural rubber, poly(isoprene), and assorted constituents including proteins and phospholipids. These constituents are inherent to both natural rubber and latex serum. This investigation was undertaken to examine the impact of the deproteinization process on chemical and biological dynamics of natural rubber latex. Natural Rubber (NR) extracted from the pure latex (LNCP) was obtained through centrifugation, followed by six rounds of solvent purification (LP6). The structure was characterized using Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), swelling test, surface zeta potential (ζ), scanning electron microscopy (SEM) and in vitro assay. The results revealed that the LP6 group presented decreased swelling kinetics, reduced cell adhesion and proliferation, and a smoother surface with decreased negative surface charge. Conversely, the LNCP group shown accelerated swelling, heightened adhesion and cellular growth, and a more negatively charged and rougher surface. As such, the attributes of latex serum and proteins have potential usage across numerous biomedical applications.

Peptidomic Analysis and Antimicrobial Activity of Serum Peptide from Hevea brasiliensis Clone BPM24.

BACKGROUND: Hevea brasiliensis is severely affected by the fungal disease caused by Phytophthora spp. Significant loss of rubber yield is widespread and extensive use of chemical fungicides has resulted in health and environmental problems. OBJECTIVE: This work aims to extract and identify the latex serum peptides from a disease tolerant clone of H. brasiliensis, and study the inhibitory efficacy against pathogenic bacteria and fungi. METHODS: Serum peptides were extracted from H. brasiliensis BPM24 using mixed lysis solution. Low molecular weight peptides were screened and fractionated by solid-phase extraction and then identified by tandem mass spectrometry. Total and fractionated serum peptides were assayed for bacterial and fungal inhibition using broth microdilution and poisoned food methods. An inhibitory control study in the greenhouse was also performed using susceptible clones for pre and postinfection with Phytophthora spp. RESULTS: Forty-three serum peptide sequences were successfully identified. Thirty-four peptides matched with the proteins associated with plant defense response signaling, host resistance, and adverse environmental factors. The inhibitory study of total serum peptides demonstrated antibacterial and anti-fungal properties. The greenhouse study exhibited disease inhibitory efficacy of 60% for the treatment of Phytophthora spp. in post-infected plants and 80% for pre-treated samples. CONCLUSION: Latex serum peptides from disease tolerant H. brasiliensis revealed several proteins and peptides associated with plant defense and disease resistance. The peptides play a vital role for defense against bacteria and fungi pathogens, including Phytophthora spp. Enhanced disease protection can be obtained when the extracted peptides were applied to the susceptible plants before exposure to the fungi. These findings provided an insight and may pave the way for the development of biocontrol peptides from natural resources.

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.

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.

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.

The rubber tree RALF peptide hormone and its receptor protein kinase FER implicates in rubber production.

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.

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.

Evolution and function of ubiquitin-specific proteases (UBPs): Insight into seed development roles in plants.

The tung oil produced by the tung tree (Vernicia fordii) provides resources for the manufacture of biodiesel. Ubiquitin-specific proteases (UBPs) are the largest group of deubiquitinases and play key roles in regulating development and stress responses. Here, 21 UBPs were identified in V. fordii, roughly one-half the number found in Manihot esculenta and Hevea brasiliensis. Most UBP duplications are produced from whole-genome duplication (WGD), and significant differences in gene retention existed among Euphorbiaceae. The great majority of UBP-containing blocks in V. fordii, V. montana, Ricinus communis, and Jatropha curcas exhibited extensive conservation with the duplicated regions of M. esculenta and H. brasiliensis. These blocks formed 14 orthologous groups, indicating they shared WGD with UBPs in M. esculenta and H. brasiliensis, but most of these UBPs copies were lost. The UBP orthologs contained significant functional divergence which explained the susceptibility of V. fordii to Fusarium wilt and the resistance of V. montana to Fusarium wilt. The expression patterns and experiments suggested that Vf03G1417 could affect the seed-related traits and positively regulate the seed oil accumulation. This study provided important insights into the evolution of UBPs in Euphorbiaceae and identified important candidate VfUBPs for marker-assisted breeding in V. fordii.