Hevea brasiliensis is an economically important tree species that provides the only commercial source of natural rubber. The replacement of the CaMV35S promoter by endogenous polyubiquitin promoters may be a viable way to improve the genetic transformation of this species. However, no endogenous polyubiquitin promoters in Hevea have been reported yet. Here, we identified three Hevea polyubiquitin genes HbUBI10.1, HbUBI10.2 and HbUBI10.3, which encode ubiquitin monomers having nearly identical amino acid sequences to that of AtUBQ10. The genomic fragments upstream of these HbUBI genes, including the signature leading introns, were amplified as putative HbUBI promoters. In silico analysis showed that a number of cis-acting elements which are conserved within strong constitutive polyubiquitin promoters were presented in these HbUBI promoters. Transcriptomic data revealed that HbUBI10.1 and HbUBI10.2 had a constitutive expression in Hevea plants. Semi-quantitative RT-PCR showed that these three HbUBI genes were expressed higher than the GUS gene driven by CaMV35S in transgenic Hevea leaves. All three HbUBI promoters exhibited the capability to direct GFP expression in both transient and stable transformation assays, although they produced lower protoplast transformation efficiencies than the CaMV35S promoter. These HbUBI promoters will expand the availability of promoters for driving the transgene expression in Hevea genetic engineering.

DNA methylation is an epigenetic modification that plays a crucial role in the regulation of gene expression, genome defense, and numerous biological processes. DNA methyltransferase (MTase) is the pivotal enzyme catalyzing the DNA methylation reaction. To explore the regulation mechanism of DNA methylation in rubber trees (Hevea brasiliensis), we identified 13 genome-wide MTase genes containing conserved structural domains of DNA methyltransferase based on the rubber tree reference genome. Through phylogenetic analysis, these genes were classified into four subfamilies: MET1, CMT, DRM, and DNMT2. A significant expansion of DNA methyltransferase genes was found in rubber trees, especially in the DRM subfamily. Notably, among the four members of the MET1 subfamily, only HbMET1-1 contains the complete UBA and RFD domains, suggesting its critical role in the function of MET1 in plants, while the other members may have developed different functions during evolution. Gene expression pattern analysis revealed that most DNA methyltransferases were specifically expressed at low levels in latex. However, following tapping from unharvested rubber trees, the expression levels of HbMTase genes are altered, and these alterations exhibit variability. Among them, HbMET1-1 and HbDRM2-1 exhibited a response to tapping stress, with their expression levels rapidly increasing after stress application and subsequently decreasing gradually. The expression levels of HbCMT3-1 and HbCMT3-2 continued to increase as the rubber tapping process progressed, which was consistent with the observed changes in HbMTase enzyme activity. These findings suggest that tapping, as a form of mechanical stress, affects the expression of particular genes. Through subcellular localization analysis, we found that HbDNMT2 is the only DNA methyltransferase located in the cytoplasm its expression level gradually decreases during the rubber tapping process. It is hypothesized that this gene activates a multitude of genes involved in the rubber biosynthesis pathway and participates actively in rubber biosynthesis. In conclusion, the comprehensive analysis of the structural characterization, conserved domains analysis, cis-regulatory elements, subcellular localization, expression profiling, and HbMTase enzyme activity detection provides critical insights into the functional characteristics of DNA methyltransferases in rubber trees and initially reveals the DNA methylation response in rubber tree tapping.

Propagation of rubber tree (Hevea brasiliensis) via secondary somatic embryogenesis (SSEis) is a reliable method. However, its efficiency is relatively low. The aim of this study was to understand more about the factors related to SSEis in rubber trees, trying to improve the efficiency of somatic embryo (SE) yield. Our study showed that the orientations of explants, i.e., the fragments of primary SE (PSE), on the medium affected secondary SE (SSE) yield significantly. Among five experimental tests, the highest yield was 2.6 ± 0.9 secondary somatic embryos (SSEs) per explant, which was achieved by orienting the abaxial side of the explant in contact with the medium and then the adaxial side after a period of culture time. Based on histological evidence, SSEis was induced from the epidermal cells and adjacent cells on the adaxial side of the explants. A remarkable difference in embryogenic capacity difference existed among individual PSE. The concentrations of soluble proteins, starch, soluble sugars, and the superoxide dismutase activity (SOD) levels in the explants were measured during a 25-day long SSEis induction treatment and compared between explants of high and low embryogenic capacity. This study proves that the explant orientation toward the culture medium plays a crucial role in SSEis, while the concentration changes of these biochemical compounds correlate to morphological changes in the explants during induction, as do the changes in SOD activity. Furthermore, the trend of the dynamic changes in the explants reflected a process of de-differentiation and re-differentiation, which started from mature SE tissues during SSE induction.