The rubber tree (Hevea brasiliensis) is a widely cultivated crop in tropical acidic soil that is tolerant to high concentration of aluminum and the aluminum-activated malate transporter (ALMT) plays an important role in plant aluminum detoxification. However, the effects of ALMT on rubber tree aluminum tolerance, growth performance, and latex production are unclear. In this study, 17 HbALMT genes were identified from the genome of rubber trees. The physiological and biochemical characteristics, phylogenetic relationships, gene structures, conserved motifs, cis-elements of promoter, and expression patterns of the identified HbALMT genes were studied. Phylogenetic relationships indicated that these genes were divided into four clusters and genes in the same cluster have similar gene structures and conserved motifs. The promoters of HbALMT genes contain many cis-elements associated with biotic stress and abiotic stress. Quantitative real-time PCR analysis revealed HbALMTs showed various expression patterns in different tissues, indicating the functional diversity of HbALMT genes in different tissues of rubber trees. Transcriptome analysis and qRT-PCR assay showed that most of the HbALMT genes responded to aluminum stress, and among the 17 HbALMTs, HbALMT1, HbALMT2, HbALMT13, and HbALMT15 displayed higher expression levels in roots after two or five days of Al treatments, indicating their potential involvement in aluminum detoxification. Taken together, this study laid a foundation for further understanding the molecular evolution of the ALMT genes and their involvement in rubber tree aluminum adaption.

Farnesyl diphosphate synthase (FPS) is a key enzyme of isoprenoids biosynthesis. However, knowledge of the FPSs of euphorbiaceous species is limited. In this study, ten FPSs were identified in four euphorbiaceous plants. These FPSs exhibited similar exon/intron structure. The deduced FPS proteins showed close identities and exhibited the typical structure of plant FPS. The members of the FPS family exhibit tissue expression patterns that vary among several euphorbiaceous plant species under normal growth conditions. The expression profiles reveal spatial and temporal variations in the expression of FPSs of different tissues from Euphorbiaceous plants. Our results revealed wide conservation of FPSs and diverse expression in euphorbiaceous plants during growth and development.

Valine-glutamine motif-containing proteins (VQ proteins) play important roles in plant growth, development and response to stress. However, information on the VQ gene family in rubber tree (Hevea brasiliensis Muell. Arg.) is limited. In this study, a total of 21 VQ protein genes (named HbVQ1 ~ HbVQ21) were identified in rubber trees and divided into six subfamilies. Gene structure analysis revealed that most of HbVQs had no introns except for HbVQ5 and HbVQ20. Gene expression analysis revealed that HbVQ4, 5, and 21 were expressed at relatively high levels in latex. In addition, HbVQ4, 5, and 21 interact with the WRKY transcription factor HbWRKY14. Transient co-expression of HbVQ4 or HbVQ5 and HbWRKY14 resulted in relieved HbWRKY14-mediated transcription inhibition of the gene encoding small rubber particle protein (HbSRPP), whereas transient co-expression of HbVQ21 and HbWRKY14 resulted in increased HbWRKY14-mediated HbSRPP transcription inhibition. In summary, HbVQ4, 5, and 21 interact with HbWRKY14 to regulate the expression of HbSRPP. This study provides insight into the functions of VQ proteins in regulating natural rubber biosynthesis.

Plant 14-3-3 proteins mediate a wide range of functionally diverse proteins through protein–protein interactions that are typically phosphorylation-dependent. However, the interactions between 14-3-3 proteins and the major regulators of nature rubber (NR) biosynthesis in H. brasiliensis have not been fully elucidated. In this study, we obtained 81 essential client proteins that interacted with H. brasiliensis 14-3-3 proteins (HbGF14s) through yeast two-hybrid (Y2H) screening. These interaction partners were involved in plant signal transduction, metabolism, development, and NR biosynthesis including small rubber particle protein (SRPP), rubber elongation factor (REF), and MYC2, etc. Furthermore, the interaction of HbGF14c and HbSRPP of H. brasiliensis was confirmed in plants through bimolecular fluorescence complementation (BiFC) assays and in vitro with Pull-down assays. Specifically, the RVSSYLP motif was found to mediate the interaction between HbSRPP and HbGF14c. The findings of this study provide a theoretical basis for the elucidation of the molecular regulation mechanism of the 14-3-3 proteins involved in NR biosynthesis, which could be used to enhance the production of rubber trees through genetic improvement.

The rubber elongation factor (REF) is the most abundant protein in the latex of Hevea brasiliensis, which is closely related to natural rubber biosynthesis. In order to gain a deeper understanding of the transcriptional regulation mechanism of HbREF1, a 1758 bp genomic DNA fragment of the HbREF1 promoter was isolated. Promoter sequence analysis revealed several transcription factor binding sites in the HbREF1 promoter, such as bZIP, bHLH, EIL, AP2/ERF, MYB, and Trihelix. To assess the promoter activity, a series of HbREF1 promoter deletion derivatives were created and fused with firefly luciferase (LUC). The LUC image demonstrated that all of the HbREF1 promoters exhibited transcriptional activity. Furthermore, the assay revealed the presence of multiple regulatory elements within the promoter region that negatively regulate the transcriptional activity. Subsequent analysis of the transcriptional activity following treatment with phytohormones identified an ABA-responsive element located between −583 bp and −200 bp, an ET-responsive element between −718 bp and −583 bp, a JA-responsive element between −1758 bp and −1300 bp, and a SA-responsive element between −1300 bp and −718 bp. These results were largely consistent with the predictions of cis-acting elements. This study has established significant groundwork for future investigations into the regulatory mechanism of HbREF1.