Kefan Cao

Drought stress severely limits plant growth and productivity, yet the molecular basis of drought tolerance and post-drought recovery remains incompletely understood in many forage legumes. Medicago ruthenica is a perennial legume native to arid and cold regions and exhibits strong drought resilience. Results: We integrated key physiological traits related to stomatal regulation, photosynthesis, osmotic adjustment and antioxidant defense with RNA-seq across four stages (well-watered control, CK; drought for 9 days, D9; drought for 12 days, D12; and rewatering for 4 days, RW). Drought triggered stage-dependent physiological shifts, and transcriptome profiling identified >3000 drought- and rewatering-responsive genes enriched in primary metabolism, redox homeostasis and hormone signaling. WGCNA highlighted two drought-associated modules (MEcyan and MEcoral1) and prioritized three hub transcription factors for functional validation: 861 (AP2/ERF), 22 (WRKY) and 89 (bZIP). Overexpression of each gene in tobacco improved drought tolerance, as indicated by enhanced growth/root traits, increased osmolyte accumulation and antioxidant enzyme activities, and reduced membrane damage. Conclusions: Together, these results provide an integrated view of drought stress response and recovery in M. ruthenica and identify 861, 22 and 89 as candidate regulatory genes for engineering drought resilience in legumes.

Drought severely constrains grassland stability and forage productivity. Allium mongolicum Regel, a dominant species in desert steppes, exhibits high drought tolerance, yet its multi-level adaptive responses remain incompletely understood. Here, drought stress of different intensities (15%, 30%, 45%, and 60% PEG-6000) was simulated, and root phenotypes, physiological traits, and time-series transcriptomic profiles were integrated to characterize its drought-responsive patterns. The results revealed a biphasic phenotypic response: mild to moderate drought (15–30% PEG) promoted root elongation and surface expansion, consistent with a growth-prioritized response pattern, whereas severe drought (60% PEG) suppressed elongation and induced compensatory thickening, suggesting a shift toward a more structural defense-oriented response. Physiological analyses showed progressive MDA accumulation accompanied by increased SOD and POD activities and proline content, indicating enhanced antioxidant and osmotic regulation under stress. Time-series transcriptomics suggested a three-phase transcriptional adjustment pattern, including early alarm, intermediate acclimation, and late compensation. Early-stage DEGs were mainly enriched in photosystem regulation and ROS-related pathways, while intermediate stages were associated with translational control and protein folding. At the late stage, genes involved in mitochondrial metabolism and amino acid catabolism were prominently represented, implying increased respiratory activity under prolonged drought. WGCNA further identified two key modules associated with these shifts: the steelblue module, negatively correlated with root growth traits, and the darkturquoise module, positively correlated with growth-related traits and antioxidant activities. Collectively, these results outline a multi-level adaptive framework for drought responses in A. mongolicum and provide insights into how desert plants may balance growth and defense under water-limited conditions.

Background: Euchresta tubulosa is a medicinal plant in the Fabaceae family, traditionally used in Chinese herbal medicine for treating ailments such as sore throat and esophageal cancer. Despite its pharmacological importance and endangered status, no complete chloroplast genome data had been reported for this species or its genus prior to this study. Chloroplast genomes, being highly conserved and maternally inherited, serve as powerful tools for species identification, phylogenetic inference and genetic diversity assessment in plants. Methods: Fresh leaves of E. tubulosa were collected and genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method. High-throughput sequencing was carried out on the Illumina NovaSeq6000 platform. Clean reads were assembled into a complete chloroplast genome using NovoPlasty, with a related species as the seed reference. Genome annotation was performed using DOGMA and validated manually. Codon usage patterns were analyzed using RSCU values and simple sequence repeats (SSRs) were identified with MISA software. A maximum likelihood phylogenetic tree was constructed using PhyML, based on complete chloroplast genome sequences from 14 related species. Result: The assembled chloroplast genome of E. tubulosa measured 153,960 bp and exhibited a typical quadripartite structure, comprising a large single-copy region (LSC), a small single-copy region (SSC) and two inverted repeats (IRs). The overall GC content was 36.3%. A total of 127 genes were identified, including 44 related to photosynthesis, 72 involved in self-replication, 5 with other biological roles and 6 hypothetical or unknown genes. Codon usage analysis revealed 31 codons with RSCU greater than 1, showing a strong bias toward A/U-ending codons. A total of 109 SSR loci were detected, the majority of which were located in intergenic spacer regions. Phylogenetic analysis clearly positioned E. tubulosa as an independent lineage within the Fabaceae, supported by high bootstrap values. The genomic information provided by this study lays a foundation for future research in molecular taxonomy, phylogenetics and conservation of this endangered medicinal plant.

Background: Low-temperature stress is a major abiotic factor that significantly impacts plant growth and productivity, especially in cold climatic conditions. Trifolium ambiguum M. Bieb. (Caucasian clover), a perennial legume known for its cold tolerance and it is an ideal model for studying cold adaptation mechanisms. However, the metabolic pathways that underpin its cold tolerance are not well understood. This study used widely targeted metabolomics to examine the metabolic reprogramming of T. ambiguum under low-temperature stress. Methods: Trifolium ambiguum seedlings were grown under controlled conditions and 14-day-old plants were exposed to two treatments: low-temperature stress (4°C) and control conditions (25°C). Samples were collected two hours after treatment. Widely targeted metabolomics profiling was performed to analyze metabolic changes induced by low-temperature stress. Key pathways and metabolites involved in the low-temperature stress response were identified through kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment and metabolic network analysis. Result: Low-temperature stress induced significant metabolic reprogramming in Trifolium ambiguum, with notable alterations observed in fatty acid metabolism. This enhancement improved cell membrane fluidity and stability, thereby strengthening the plant’s towards cold tolerance. Furthermore, the significant accumulation of proline and glutamate underscores their critical roles in osmotic regulation and antioxidative defense mechanisms. Additionally, the upregulation of secondary metabolites like (+)-Forbesione and flavonoids plays a key role in scavenging reactive oxygen species (ROS) and regulating stress-related signaling. KEGG pathway enrichment analysis revealed that C5-Branched dibasic acid metabolism and alpha-Linolenic acid metabolism play central roles in cold adaptation. C5-Branched dibasic acid metabolism provided essential carbon skeletons and energy substrates, while alpha-linolenic acid metabolism contributed to maintaining membrane stability and supporting antioxidative responses. The enrichment of the TCA cycle and glycerophospholipid metabolism further ensured energy supply and membrane integrity under stress conditions.

Objectives: Light intensity is a critical environmental factor regulating plant growth, development, and stress adaptation. However, the physiological and molecular mechanisms underlying light responses in Aconitum kusnezoffii, a valuable alpine medicinal plant, remain poorly understood. This study aimed to elucidate the adaptive strategies of A. kusnezoffii under different light intensities through integrated physiological and transcriptomic analyses. Methods: Two-year-old A. kusnezoffii plants were exposed to three controlled light regimes (790, 620, and 450 lx). Leaf anatomical traits were assessed via histological sectioning and microscopic imaging. Antioxidant enzyme activities (CAT, POD, and SOD), membrane lipid peroxidation (MDA content), osmoregulatory substances, and carbon metabolites were quantified using standard biochemical assays. Transcriptomic profiling was conducted using Illumina RNA-seq, with differentially expressed genes (DEGs) identified through DESeq2 and functionally annotated via GO and KEGG enrichment analyses. Results: Moderate light (620 lx) promoted optimal leaf structure by enhancing palisade tissue development and epidermal thickening, while reducing membrane lipid peroxidation. Antioxidant defense capacity was elevated through higher CAT, POD, and SOD activities, alongside increased accumulation of soluble proteins, sugars, and starch. Transcriptomic analysis revealed DEGs enriched in photosynthesis, monoterpenoid biosynthesis, hormone signaling, and glutathione metabolism pathways. Key positive regulators (PHY and HY5) were upregulated, whereas negative regulators (COP1 and PIFs) were suppressed, collectively facilitating chloroplast development and photomorphogenesis. Trend analysis indicated a “down–up” gene expression pattern, with early suppression of stress-responsive genes followed by activation of photosynthetic and metabolic processes. Conclusions: A. kusnezoffii employs a coordinated, multi-level adaptation strategy under moderate light (620 lx), integrating leaf structural optimization, enhanced antioxidant defense, and dynamic transcriptomic reprogramming to maintain energy balance, redox homeostasis, and photomorphogenic flexibility. These findings provide a theoretical foundation for optimizing artificial cultivation and light management of alpine medicinal plants.

Gibberellins (GAs) are essential phytohormones that regulate seed dormancy release and germination. Lomatogonium rotatum (L.) Fries ex Nym is a traditional medicinal plant whose seed germination is often hindered by physiological dormancy. In this study, we systematically investigated the effects of exogenous GA₃ on the seed germination of L. rotatum and elucidated the underlying molecular regulatory mechanisms via transcriptomic analysis. GA₃ treatment (500 mg/L for 24 h) significantly improved the germination rate, vigor index, and other germination traits. RNA-seq analysis identified time-dependent transcriptional changes in GA₃-treated seeds across three developmental stages (24 h, 72 h, and 96 h). KEGG enrichment and K-means clustering revealed dynamic actiSvation of hormonal signaling, secondary metabolism, and DNA replication pathways. WGCNA uncovered two hormone-responsive co-expression modules (Red and Lightcyan) corresponding to early and late stages of germination, respectively. Key genes related to ABA and GA biosynthesis and signal transduction showed phase-specific expression, highlighting the coordinated hormonal regulation during seed germination. Our findings provide new insights into the molecular basis of GA₃-regulated seed germination and offer theoretical support for the cultivation and utilization of L. rotatum.

Isoflavones are important secondary metabolites in leguminous plants with significant physiological functions and economic value. However, the genetic variation, transcriptional regulation, and metabolic pathways governing isoflavone biosynthesis in Trifolium pratense remain largely unexplored. In this study, we systematically analyzed 500 accessions of T. pratense for isoflavone content and performed RNA-seq-based transcriptomic profiling to investigate the molecular mechanisms underlying isoflavone biosynthesis. Cluster analysis revealed significant genetic variation, with distinct transcriptional profiles between high- (H1, H2, H3) and low-isoflavone (L1, L2, L3) groups. GO and KEGG pathway enrichment analyses identified key metabolic pathways, including phenylpropanoid metabolism, flavonoid biosynthesis, carbohydrate metabolism, and hormone signaling, which play crucial roles in isoflavone regulation. Weighted gene co-expression network analysis (WGCNA) identified three key gene modules—MEblue, MEturquoise, and MEyellow—strongly correlated with isoflavone content. The MEturquoise and MEyellow modules were upregulated in high-isoflavone groups and were enriched in phenylpropanoid biosynthesis, lipid metabolism, and transcriptional regulation, suggesting that these pathways actively promote isoflavone accumulation. Conversely, the MEblue module, highly expressed in low-isoflavone groups, was enriched in sugar metabolism and MAPK signaling, indicating a potential metabolic flux shift away from secondary metabolism. Moreover, key rate-limiting enzymes (PAL, C4H, 4CL, CHS, and IFS) exhibited higher expression in high-isoflavone groups, highlighting their importance in precursor supply and enzymatic catalysis. Additionally, transcription factors such as MYB, WRKY, and NAC were identified as potential regulators of isoflavone biosynthesis, indicating a complex interplay between hormonal, circadian, and environmental signals. This study provides a comprehensive molecular framework for understanding isoflavone biosynthesis in T. pratense and identifies key regulatory genes and metabolic pathways that could be targeted for genetic improvement, metabolic engineering, and molecular breeding. The findings offer valuable insights into enhancing isoflavone production in legumes for agricultural, nutritional, and pharmaceutical applications.

The hybrid progeny (1-1) resulting from the cross between Caucasian clover and white clover initially demonstrated an inability to fix nitrogen naturally via spontaneous nodulation. However, following inoculation with specific rhizobia strains derived from the Trifolium genus, successful nodulation and nitrogen fixation were observed in the 1-1 progeny, resulting in enhanced biomass production and adaptability. To explore in greater depth the mechanisms driving nitrogen fixation in these hybrid progeny, the inoculation was carried out using the dominant rhizobia strain (No. 5), isolated from Mengnong Clover No. 1. Root samples were collected at 3, 6, and 9 days post inoculation for RNA sequencing. A total of 1755 differentially expressed unigenes were identified between the control and treatment groups. KEGG pathway analysis highlighted key pathways associated with nodule nitrogen fixation. In combination with Weighted Gene Co-expression Network Analysis (WGCNA) and Gene Set Enrichment Analysis (GSEA), several differentially expressed genes were identified, suggesting their potential contribution to nitrogen fixation. Noteworthy among these, the gene TRINITY_DN7551_c0_g1 in the Phenylpropanoid biosynthesis pathway (MAP00940) emerged as a key candidate. This study offers valuable RNA-seq data, contributing significantly to the understanding of the molecular regulatory mechanisms underpinning nodule nitrogen fixation in legumes, thereby laying a solid foundation for future investigations into the hybrid progeny of Caucasian and white clover crosses.

Caucasian clover (Trifolium ambiguum M.Bieb.) is a perennial legume known for its exceptional cold tolerance, commonly used in agriculture and ecosystems in cold climates. Given the impact of climate change, enhancing the cold adaptation of Caucasian clover is crucial for sustaining agricultural productivity. This study employs metabolomics, transcriptomics, and Weighted Gene Co-expression Network Analysis (WGCNA) to investigate the molecular mechanisms of Caucasian clover’s response to low-temperature stress. Metabolomic analysis showed that low-temperature stress triggered the accumulation of fatty acids, amino acids, and antioxidants, which are critical for maintaining membrane stability and antioxidant capacity, thus protecting the plant from oxidative damage. Transcriptomic analysis revealed significant upregulation of genes involved in cold adaptation, particularly those related to antioxidant defense, membrane lipid repair, and signal transduction, including genes in the ABA signaling pathway and antioxidant enzymes, thereby improving cold tolerance. WGCNA identified gene modules closely linked to cold adaptation, especially those involved in antioxidant defense, fatty acid metabolism, signal transduction, and membrane repair. These modules function synergistically, with coordinated gene expression enhancing cold resistance. This study also investigated the isoflavonoid biosynthesis pathway under low-temperature stress, highlighting its role in enhancing antioxidant capacity and cold tolerance. Low-temperature stress induced upregulation of key enzyme genes, such as Isoflavone Synthase (IFS) and Isoflavone-7-O-Glucosyltransferase (IF7GT), promoting antioxidant metabolite accumulation and further enhancing the plant’s cold adaptation. Overall, this study offers novel molecular insights into the cold tolerance mechanisms of Caucasian clover and provides valuable theoretical support for breeding cold-resistant crops in cold climates.