The petals of Medicago sativa ssp. sativa and M. sativa ssp. falcata are purple and yellow, respectively. Free hybridization between M. sativa ssp. sativa and M. sativa ssp. falcata has created hybrids with various flower colors in nature. Moreover, the flower colors of alfalfa are closely correlated with yield, nutritional quality, stress tolerance and other agronomic characteristics. To elucidate the underlying mechanisms of flower color formation in M. sativa ssp. sativa and M. sativa ssp. falcata, we conducted an integrative analysis of the transcriptome and metabolome of alfalfa with three different petal colors (purple, yellow and cream). The metabolic profiles suggested that anthocyanins and carotenoids are the crucial pigments in purple and yellow flowers, respectively. A quantitative exploration of the anthocyanin and carotenoid components indicated that the accumulations of cyanidin, delphinidin, peonidin, malvidin, pelargonidin and petunidin derivatives are significantly higher in purple flowers than in cream flowers. In addition, the content of carotenes (phytoene, α-carotene and β-carotene) and xanthophylls (α-cryptoxanthin, lutein, β-cryptoxanthin, zeaxanthin, antheraxanthin and violaxanthin derivatives) was markedly higher in yellow flowers than in cream flowers. Furthermore, we found that delphinidin-3,5-O-diglucoside and lutein were the predominant pigments accumulated in purple and yellow flowers, respectively. The transcriptomic results revealed that twenty-five upregulated structural genes (one C4H, three 4CL, twelve CHS, two CHI, one F3H, one F3′H, one F3′5′H and four DFR) are involved in the accumulation of anthocyanins in purple flowers, and nine structural genes (two PSY, one ZDS, two CRTISO, two BCH, one ZEP and one ECH) exert an effect on the carotenoid biosynthesis pathway in yellow flowers. The findings of this study reveal the underlying mechanisms of anthocyanin and carotenoid biosynthesis in alfalfa with three classic flower colors.

Floral bud growth influences seed yield and quality; however, the molecular mechanism underlying the development of floral buds in alfalfa (Medicago sativa) is still unclear. Here, we comprehensively analyzed the transcriptome and targeted metabolome across the early, mid, and late bud developmental stages (D1, D2, and D3) in alfalfa. The metabolomic results revealed that gibberellin (GA), auxin (IAA), cytokinin (CK), and jasmonic acid (JA) might play an essential role in the developmental stages of floral bud in alfalfa. Moreover, we identified some key genes associated with GA, IAA, CK, and JA biosynthesis, including CPS, KS, GA20ox, GA3ox, GA2ox, YUCCA6, amid, ALDH, IPT, CYP735A, LOX, AOC, OPR, MFP2, and JMT. Additionally, many candidate genes were detected in the GA, IAA, CK, and JA signaling pathways, including GID1, DELLA, TF, AUX1, AUX/IAA, ARF, GH3, SAUR, AHP, B-ARR, A-ARR, JAR1, JAZ, and MYC2. Furthermore, some TFs related to flower growth were screened in three groups, such as AP2/ERF-ERF, MYB, MADS-M-type, bHLH, NAC, WRKY, HSF, and LFY. The findings of this study revealed the potential mechanism of floral bud differentiation and development in alfalfa and established a theoretical foundation for improving the seed yield of alfalfa.

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.

Introduction: Caucasian clover (Trifolium ambiguum Bieb.), a perennial legume forage grass, exhibits strong adaptability and resistance to adverse conditions. Owing to its rhizome specificity, it cannot nodulate or fix nitrogen outside of its original location, which limits its promotion and cultivation. The phenomenon of spontaneous nodulation of Caucasian clover was observed for the first time in Inner Mongolia, and investigation of its nodulation and nitrogen fixation mechanisms assists in the subsequent promotion of Caucasian clover cultivation from a theoretical perspective.Methods: In this study, rhizobia extracted from Caucasian clover were inoculated into the field, and the nodulation part of the root system was investigated during the regreening stage of the second year using transcriptomics and proteomics techniques.Results and Discussion: The study identified 70,280 differentially expressed genes (DEGs) and 770 differentially expressed proteins (DEPs) in total, among which carbonic anhydrase, cyanate lyase, phenylalanine, caffeate/5-hydroxyferulate 3-O-methyltransferase, caffeoyl CoA 3-O methyltransferase, chalcone synthase, and chalcone isomerase may have been the main factors affecting the nodulation and nitrogen fixation of Caucasian clover. This study theoretically contributes to the future genetic validation and selection of Caucasian clover varieties with a strong ability to nodulate and fix nitrogen.

Cold stress is one of the major environmental challenges faced by plants, severely affecting their growth, development, and yield. With the intensification of global climate change, the impact of cold stress on plants is becoming increasingly significant. Plants respond to cold stress through complex metabolic regulatory networks, including the accumulation of protective metabolites, modulation of antioxidant defenses, and activation of secondary metabolic pathways. This study aims to explore the dynamic metabolic and transcriptomic responses of T. ambiguum under long-term cold stress, and to reveal its adaptive mechanisms. By integrating metabolomics and transcriptomics, this study provides an indepth analysis of gene expression and metabolic responses of T. ambiguum under different cold stress treatments (2 h, 6 h, 12 h), focusing on key metabolic pathways and signal transduction mechanisms involved in cold stress adaptation. To further investigate the relationship between genes and metabolites, weighted gene co-expression network analysis (WGCNA) was applied to construct the gene-metabolite coexpression network under cold stress. Several functional modules that play significant roles in cold response were identified. Notably, the pink module was found to be associated with lipid metabolism, sugar metabolism, and signal transduction pathways, while the black module was closely linked to plant hormone signaling and antioxidant responses. Additionally, KEGG enrichment analysis revealed that key pathways such as glycerophospholipid metabolism, proline metabolism, and plant hormone signal transduction work synergistically in cold stress adaptation, regulating cellular homeostasis and maintaining energy supply under prolonged cold stress. The results indicate that T. ambiguum enhances its cold tolerance through dynamic coordination between its transcriptomic and metabolic responses. This study provides new molecular biological evidence for the cold adaptation mechanisms of T. ambiguum and offers theoretical support for improving cold tolerance in related crops.

In order to create new forage variety, caucasian clover (Trifolium ambiguum Bieb.) with strong tolerance to drought and cold introduced from New Zealand as the female parent was hybridized with a white clover (T. repens L.) collected from northeast China, which has strong nitrogen fixation capacity. Hybrid embryos of caucasian clover×white clover were successfully raised using embryo rescue. 13.8% of these embryos grew to maturity. The successfully grown plants were transferred to sterilized vermiculite after 5-6 weeks and then to field after 3-4 months. The hybrid plant showed intermediate morphological features and a chromosomal number of 2n=5X=40. In order to obtain more plants for backcross, a tissue culture system was established for the hybrids, using MS+0.1mg l-1 2, 4-D+2mg l-1 6-BA, MS +0.5mg l-1 NAA+1mg l-1 6-BA+1mg l-1 KT and 1/2MS as the medium for bud-induction, differentiation and root-induction respectively. 80% of the cultured plantlets survived these growing conditions. Hybridity of the plant was also confirmed by ISSR markers using 16 primers.