Guangxi Medicinal Botanical Garden

Summary The N-terminal signal sequences of plant cytochrome P450 enzymes are recognized as critical determinants for subcellular localization and functional diversification, yet their evolutionary drivers and mechanisms remain largely unresolved. In this study, the evolutionary trajectories of these signals were systematically decoded through the integration of the protein language model ESM-2 with phylogenetic and selection analyses. A conserved functional fingerprint was identified. This region may serve as the essential endoplasmic reticulum targeting signal and be evolutionarily decoupled from adjacent surfaces under positive selection during lineage-specific expansions. A ‘functional-adaptive decoupling’ model is proposed to explain this pattern, wherein a conserved functional core is maintained while surrounding interfaces diversify. This evolutionary architecture is interpreted as the outcome of a two-step cycle: an initial phase of positive selection driving functional innovation, followed by pervasive neutral evolution that facilitates structural exploration and potentiates future adaptations. This work demonstrates how interpretable machine learning can be integrated with evolutionary theory to reconcile neutralist and selectionist perspectives on protein evolution. A novel framework is thus provided for understanding the layered evolution of protein modules, where structural constraint, adaptive innovation, and neutral drift operate on distinct tiers to generate functional diversity.

Sophora tonkinensis is a key medicinal plant endemic to the calcium-rich karst regions along the China-Vietnam border. This study investigated how calcium regulates the growth and biosynthesis of bioactive compounds in S. tonkinensis tissue culture seedlings by exposing them to a gradient of calcium concentrations. Our findings demonstrate that a moderate calcium level (T2, 2.99 mmol·L-1) promoted root development, increasing root dry weight, and significantly elevated the content of matrine and oxymatrine. In contrast, a high calcium level (T4, 5.98 mmol·L-1) inhibited root growth, reducing root length, but triggered a distinct metabolic shift, markedly increasing the accumulation of trifolirhizin and maackiain. Integrated transcriptomic, proteomic, and metabolomic analyses revealed that calcium exerts systemic regulation through multiple functional pathways. We identified four key signaling pathways-phytohormone, plant immunity, MAPK, and phospholipid signaling-as central hubs coordinating genetic information processing, vesicular transport, and metabolic reprogramming. These results provide valuable insights into the calcium-mediated regulatory networks and offer valuable targets for optimizing cultivation practices to enhance the yield of bioactive compounds in S. tonkinensis.

Gynostemma pentaphyllum, a perennial medicinal herb, is widely distributed and exhibits remarkable adaptability to various environments. WRKY transcription factors play critical roles in regulating plant growth and development, as well as abiotic stress responses. However, the WRKY genes in G. pentaphyllum and their expression patterns under different tissues and Cd stress have not been thoroughly investigated, hindering our understanding of their role. In this study, 64 WRKY transcription factors identified in the G. pentaphyllum genome (GpWRKY) were phylogenetically grouped into three major groups and five subgroups and assigned a systematic nomenclature reflecting their positions across the 11 chromosomes. Comprehensive analyses of conserved domains, gene structures and motif features revealed a high degree of conservation with the gene family. Furthermore, seven pairs of segmental and tandem duplication events were detected, suggesting a contribution to the expansion of family. Cis-acting element analysis revealed the presence of hormone-related, stress-related, light-related, and development-related elements in the promoters of the majority of GpWRKY genes. Expression profiling analysis demonstrates that 64 GpWRKY genes were distinct in tissue-specific expression patterns and were substantially induced under Cd stress. Heterologous expression assay confirmed that GpWRKY48, one of the genes highly induced by Cd, positively regulates Cd tolerance. In this study, GpWRKY genes in G. pentaphyllum for the first time systematically identified and further investigated their physicochemical properties, evolution, and expression patterns, providing a theoretical basis for future studies on the functional characterization of G. pentaphyllum WRKY genes during plant growth and development, as well as Cd stress responses.

Cucurbitaceae is an economically important family with a wide geographical distribution range. The phylogenetic backbone of Cucurbitaceae has not been well resolved, and conflicting results have been reported in previous phylogenetic studies. Trichosanthes is the largest genus in Cucurbitaceae; however, its infrageneric classification remains controversial. Here, we used both plastome and nuclear datasets to reconstruct the phylogeny of Cucurbitaceae with emphasis on Trichosanthes. The plastome dataset includes 137 taxa, representing 15 tribes and 35 genera. The 1,244 single-copy nuclear genes (SCNs) dataset includes 143 samples from all 15 tribes and 68 genera of Cucurbitaceae. Phylograms of plastome dataset and 1,244 SCNs essentially agreed with each other with the exception of first branching tribes and Tr. Bryonieae. The phylogram of the plastome dataset was used to illustrate phylogeny and molecular dating. Cucurbitaceae was subdivided into nine clades along the phylogram backbone. The first branching clade of Cucurbitaceae included four tribes: Actionstemmateae, Fevilleeae, Gomphogyneae, and Zanonieae. Actionstemmateae was sister to the remaining three tribes, with Gomphogyneae and Fevilleeae being successive sister to Zanonieae. Six tribes, including Indofevilleeae, Thladiantheae, Momordiceae, Siraitieae, Joliffieae, and Bryonieae, formed successive and well-supported clades along the backbone of Cucurbitaceae. Cucurbitaceae originated in the Late Cretaceous (ca. 94.186 million years ago [MYA]), and diversified during the Late Cretaceous to the Eocene. Diversification of all tribes occurred during the later Cretaceous to Eocene. Trichosanthes originated ca. 30.089 MYA, and the section diversification occurred in middle Miocene approximately 6 million years (20.839 - 14.804 MYA). Climate optimum in the Mid-Eocene and Mid-Miocene was probably among the important drivers of the diversification of Trichosanthes complex. Our results provide new insights into the phylogeny of Cucurbitaceae, including members of the first branching clade and the relative phylogenetic positions of Indofevilleeae, Thladiantheae, and Momordiceae, and show that Trichosanthes is paraphyletic with three genera nested within it. Our results suggest that the previously proposed infrageneric classification systems for Trichosanthes need to be revised. Section Involucraria was subdivided into two distantly related groups. Trichosanthes originated in the Oligocene and experienced rapid diversification in the Middle Miocene and throughout the Pliocene. Long-fringed petals might have arisen once in a clade formed by Trichosanthes and Hodgsonia.