Kaiye Liu

Rubber tree is an economically important woody plant, whereas the cellular heterogeneity and regulatory networks underlying its xylem differentiation remain poorly characterised. Here, we built a protocol to isolate differentiating xylem protoplasts and performed scRNA‐seq on two rubber tree cultivars (RK525 and RY73397) with distinct rates of girth growth. The first single‐cell transcriptional atlas of differentiating xylem in the rubber tree was established, providing a cellular basis for growth variations. Ten distinct cell clusters were identified, with cell types—including vessel element, libriform fibre, and ray parenchyma—determined by established marker genes. Pseudotime analysis revealed two clearly developmental trajectories, underscoring fusiform lineages (fibres and vessels) being crucial for growth differences. Specifically, the fast‐growing cultivar RK525 exhibited a cluster of upregulated genes in fibre and vessel cells. Subsequently, differentially expressed genes (DEGs) in fusiform cell clusters between the two cultivars were intersected with subcluster‐upregulated genes and pseudotime‐core DEGs in RK525, highlighting secondary cell wall (SCW) biosynthesis genes that regulate xylem development and promote rapid growth. WGCNA further identified a specific module significantly correlated with fibre cells, which includes six key transcription factors. Moreover, ATAC‐seq revealed variety‐specific chromatin accessibility patterns and transcription factor binding motifs associated with xylem development. Functional validation using Arabidopsis mutants of rubber tree homologues confirmed their crucial roles in vascular development. These findings reveal intricate regulatory programmes in differentiating xylem, and provide valuable resources and targets for genetic improvement of the rubber tree.

Natural rubber (NR), derived from the latex of the Para rubber tree (Hevea brasiliensis), is an irreplaceable raw material for the tire, medical, and engineering industries. Despite its global economic importance, rubber tree breeding has historically been constrained by a narrow genetic base originating from the 1876 Wickham collection and a prolonged juvenile phase. This review synthesizes historical germplasm conservation efforts—most notably the IRRDB 1981 Amazonian expedition—and over a century of clonal selection that has increased NR yields nearly tenfold. We critically assess recent biotechnological advances, including somatic embryogenesis and Agrobacterium-mediated transformation, highlighting their current bottlenecks and the potential of developmental regulators to overcome recalcitrance. A major focus is the rapid acceleration of genomic resources, tracing the progression from early draft assemblies to recent telomere-to-telomere (T2T) haplotype-resolved genomes and pan-genome analyses. We further summarize quantitative trait loci (QTL) mapping and genome-wide association studies (GWAS) that have identified key genes governing growth traits, latex yield, and laticifer differentiation. Finally, we outline future perspectives emphasizing the integration of CRISPR/Cas9-based gene editing with multi-omics-driven genomic selection to drastically shorten breeding cycles. By consolidating these interdisciplinary advancements, this review underscores the transition of rubber tree improvement from traditional selection toward precision molecular breeding, thereby ensuring the sustainable supply of this strategic industrial resource.

Super pangenomes, as complete genome sequencing at the genus level, have provided new insights into the speciation and evolution of functional genes. Genome size (GS) estimation is a critical first step. Although K-mer-based GS evaluators are applied extensively to guide genome assembly process and quality assessment, the results vary substantially with the tools and parameters used, presenting challenges for genus-level genome studies. Here, we investigated K-mer spectra from datasets of species with and without whole genome duplication, revealing that the trade-off in K-mer length amplified the signal of genomic characteristics related to repeat content or heterozygosity. Moreover, GS predictions were influenced by genomic heterozygosity and sequencing accuracy when different K-mer lengths were employed. In contrast, consistent GS predictions were obtained across all HiFi-based evaluations, demonstrating high accuracy of the derived limiting values from the regions of GS evaluation convergence during continuous variation of K. Unlike traditional methods that rely on single predictions, we introduced a closed-loop GS-estimating framework, that incorporates steady-value calculations, leveraging the continuity and accuracy of HiFi reads. Finally, we developed a high-performance pipeline, LVgs ( https://github.com/xingjianfeng100/LVgs ), by integrating FastK and GenomeScope 2.0. The robustness and applicability of LVgs for genus-level species was demonstrated through its application to various diploid and polyploidy species.

Summary: Rubber particles (RPs) are specialized organelles for the biosynthesis and storage of natural rubber in rubber‐producing plants. However, the mechanisms underlying the biogenesis and development of RPs remain unclear. In this study, two latex‐specific cis‐prenyltransferases (CPTs), TkCPT1 and TkCPT2, were identified in Taraxacum kok‐saghyz, with almost identical orthologues retained across other Taraxacum species. For the first time, Tkcpt1 single and Tkcpt1/2 double mutants were successfully generated using the CRISPR/Cas9 system. Rubber biosynthesis was significantly depressed in Tkcpt1 mutants and completely blocked in Tkcpt1/2 mutants. The absence of RPs in the Tkcpt1/2 was confirmed using oil red O and Nile red staining, high‐speed centrifugal stratification, cryo‐SEM and TEM on fresh latex or laticifer cells. Transcriptomic and proteomic analyses revealed that, in the latex of Tkcpt1/2, rubber biosynthesis was blocked at the protein level, while metabolomic profiling indicated an enrichment of lipids and terpenoids. Furthermore, knockout of TkCPTL1, a latex‐specific CPT‐like gene that encodes a rubber transferase activator, resulted in outright disruption of rubber biosynthesis and RP ontogeny, a phenotype similar to that of Tkcpt1/2 mutants. These findings indicate that rubber biosynthesis is a driving force for the biogenesis and development of RPs, providing new insights into rubber production mechanisms.

MicroRNAs (miRNAs) are produced from highly structured primary transcripts (pri-miRNAs) and regulate numerous biological processes in eukaryotes. Due to the extreme heterogeneity of these structures, the initial processing sites of plant pri-miRNAs and the structural rules that determine their processing have been predicted for many miRNAs but remain elusive for others. Here we used semi-active DCL1 mutants and advanced degradome-sequencing strategies to accurately identify the initial processing sites for 147 of 326 previously annotated Arabidopsis miRNAs and to illustrate their associated pri-miRNA cleavage patterns. Elucidating the in vivo RNA secondary structures of 73 pri-miRNAs revealed that about 95% of them differ from in silico predictions, and that the revised structures offer clearer interpretation of the processing sites and patterns. Finally, DCL1 partners Serrate and HYL1 could synergistically and independently impact processing patterns and in vivo RNA secondary structures of pri-miRNAs. Together, our work sheds light on the precise processing mechanisms of plant pri-miRNAs.

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.

Natural rubber (cis-1,4-polyisoprene, NR) is an important raw material utilized widely in the manufacturing of medical, agricultural, and industrial products. Rubber tree (Hevea brasiliensis) and several alternative rubber-producing plants (Taraxacum kok-saghyz, Lactuca sativa, and Parthenium argentatum) have the capability to produce high-quality NR. With the progress of genome sequencing, similar rubber biosynthesis pathways have been discovered among different rubber-producing plant species. NR is synthesized and stored in rubber particles, which are specialized organelles comprising a hydrophobic NR core surrounded by a lipid monolayer and membrane-bound proteins. The rubber transferase complex is considered to be the pivotal enzyme involved in catalyzing NR biosynthesis. However, the exact compositions of the RT complex in rubber-producing plants remain elusive and poorly understood. Here, we review the progress of genome sequencing, natural rubber biosynthesis, and the components of the RT complex in rubber-producing plants. We emphasize that identifying the detailed components of the RT complex holds great significance for exploring the mechanism of NR biosynthesis and accelerating molecular breeding in rubber-producing plants.