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The morphological architecture of inflorescence influences seed production. The regulatory mechanisms underlying alfalfa (Medicago sativa) inflorescence elongation remain unclear. Therefore, in this study, we conducted a comparative analysis of the transcriptome, proteome, and metabolome of two extreme materials at three developmental stages to explore the mechanisms underlying inflorescence elongation in alfalfa. We observed the developmental processes of long and short inflorescences and found that the elongation capacity of alfalfa with long inflorescence was stronger than that of alfalfa with short inflorescences. Furthermore, integrative analysis of the transcriptome and proteome indicated that the phenylpropanoid biosynthesis pathway was closely correlated with the structural formation of the inflorescence. Additionally, we identified key genes and proteins associated with lignin biosynthesis based on the differential expressed genes and proteins (DEGs and DEPs) involved in phenylpropanoid biosynthesis. Moreover, targeted hormone metabolome analysis revealed that IAA, GA, and CK play an important role in the peduncle elongation of alfalfa inflorescences. Based on omics analysis, we detected key genes and proteins related to plant hormone biosynthesis and signal transduction. From the WGCNA and WPCNA results, we furthermore screened 28 candidate genes and six key proteins that were correlated with lignin biosynthesis, plant hormone biosynthesis, and signaling pathways. In addition, 19 crucial transcription factors were discovered using correlation analysis that might play a role in regulating candidate genes. This study provides insight into the molecular mechanism of inflorescence elongation in alfalfa and establishes a theoretical foundation for improving alfalfa seed production.

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.

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.