Background: Drought is a major limiting factor for plant survival and crop productivity. Stylosanthes angustifolia, a pioneer plant, exhibits remarkable drought tolerance, yet the molecular mechanisms driving its drought resistance remain largely unexplored. Results: We present a chromosome-scale reference genome of S. angustifolia, which provides insights into its genome evolution and drought tolerance mechanisms. The assembled genome is 645.88 Mb in size, containing 319.98 Mb of repetitive sequences and 36,857 protein-coding genes. The high quality of this genome assembly is demonstrated by the presence of 99.26% BUSCO and a 19.49 long terminal repeat assembly index. Evolutionary analyses revealed that S. angustifolia shares a whole-genome duplication (WGD) event with other legumes but lacks recent WGD. Additionally, S. angustifolia has undergone gene expansion through tandem duplication approximately 12.31 million years ago. Through integrative multiomics analyses, we identified 4 gene families—namely, xanthoxin dehydrogenase, 2-hydroxyisoflavanone dehydratase, patatin-related phospholipase A, and stachyose synthetase—that underwent tandem duplication and were significantly upregulated under drought stress. These gene families contribute to the biosynthesis of abscisic acid, genistein, daidzein, jasmonic acid, and stachyose, thereby enhancing drought tolerance. Conclusions: The genome assembly of S. angustifolia represents a significant advancement in understanding the genetic mechanisms underlying drought tolerance in this pioneer plant species. This genomic resource provides critical insights into the evolution of drought resistance and offers valuable genetic information for breeding programs aimed at improving drought resistance in crops.

As a medicinal plant, Sophora tonkinensis is mainly distributed in Guangxi, Yunnan, Guizhou and northern Vietnam. In recent years, due to the strong demand for S. tonkinensis, wild resources have become decreased, which has placed great pressure on their protection. Therefore, the introduction of S. tonkinensis was carried out in Guangxi. Although the flowering period of S. tonkinensis is long, its seed setting rate is low. To promote flower production, it is necessary to do basic research on flowering in S. tonkinensis. In this study, the contents of various mineral elements in the roots, stems and leaves of S. tonkinensis were measured during the period of flowering, and cluster analysis was carried out on the content changes. At the same time, the correlations among the elements were analyzed. It was found that As and Hg inhibited the absorption of some important elements related to flower development. It is necessary to pay attention to the effects of these toxic elements on flowering during cultivation. RNA-seq of flower buds and flower organs was carried out, and the weighted gene co-expression network analysis (WGCNA) method was used to mine the key genes. These genes may play an important role in flowering control and flower development in S. tonkinensis. Supplemental data for this article is available online at https://doi.org/10.1080/13102818.2021.1988707 .

Sophora tonkinensis is a valuable medicinal plant whose cultivation is constrained by drought and cadmium (Cd) contamination. DNA methylation, mediated by cytosine-5 DNA methyltransferases (C5-MTases) and DNA demethylases (dMTases), contributes to plant stress response; however, these gene families have remained uncharacterized in S. tonkinensis. Here, we identified 12 methylation-related genes (four StC5-MTases and eight StdMTases) and analyzed their phylogeny, duplication, promoter cis-elements, and expression patterns under Cd exposure and drought/rehydration. Most duplicated pairs showed Ka/Ks < 1, consistent with purifying selection. StCMT1 and StMET2 were induced by both Cd and drought stress but declined after rehydration, whereas StROS1b and StROSlike3 responded rapidly to both stresses. Heterologous overexpression in Nicotiana benthamiana improved growth under Cd stress for StCMT1 lines and under PEG-induced osmotic stress for StROSlike3 lines, as reflected by plant height and whole-plant fresh weight. Together, these findings establish a genome-wide resource for DNA methylation machinery in S. tonkinensis and provide candidate genes for investigating epigenetic regulation of abiotic stress adaptation.

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

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.