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.

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.

Flower bud differentiation is the commencement of plant reproductive development. Flower organ specialization follows flower bud differentiation. This biological mechanism controls whether plants reproduce successfully via seed. Caucasian clover (Trifolium ambiguum) is used in mixed temperate pastures. However, its low natural seed setting rate contributes to a low seed yield. This study aimed to elucidate how the reproductive biology of Caucasian clover could affect seed production. Caucasian clover flower bud differentiation and flower organ development were systematically examined. Flower bud differentiation has six consecutive stages: pre‐differentiation, differentiation of the inflorescence, the floret, the perianth, the stamen, and finally the pistil primordium. The anther has two chambers. Pollen grains have six development phases: the tetraspore, early‐uninucleate, late‐uninucleate, vegetative cell and germ‐cell form, binucleate, and maturation phase. The microspore tetrad is tetrahedral, and the mature pollen grains are bicellular. Pollen grain viability was found 35%. The Caucasian clover monocarpelate ovary contains 1–2 ovules that are covered with a 2‐layer integument. The ovule is anatropous. The embryo sac development type is a polygonum. The floral formula is “⚥↑K₍₅₎ C₁₊₁₊₂ A₍₉₎₊₁ G_(1:1:1−2)”. These genetic traits of two pollen sacs and low viability pollen grains may be responsible for the low natural seed setting. Poor seed setting may also be due to embryo sac dysplasia that leads to ovule sterility. Therefore, the breeding aim should be to improve pollen viability, to decrease embryo sac dysplasia and to reduce embryo abortion that could be targeted for genetic improvement to enhance seed yields.