Alfalfa (Medicago sativa L.) is the most important legume forage crop worldwide with high nutritional value and yield. For a long time, the breeding of alfalfa was hampered by lacking reliable information on the autotetraploid genome and molecular markers linked to important agronomic traits. We herein reported the de novo assembly of the allele-aware chromosome-level genome of Zhongmu-4, a cultivar widely cultivated in China, and a comprehensive database of genomic variations based on resequencing of 220 germplasms. Approximate 2.74 Gb contigs (N50 of 2.06 Mb), accounting for 88.39% of the estimated genome, were assembled, and 2.56 Gb contigs were anchored to 32 pseudo-chromosomes. A total of 34,922 allelic genes were identified from the allele-aware genome. We observed the expansion of gene families, especially those related to the nitrogen metabolism, and the increase of repetitive elements including transposable elements, which probably resulted in the increase of Zhongmu-4 genome compared with Medicago truncatula. Population structure analysis revealed that the accessions from Asia and South America had relatively lower genetic diversity than those from Europe, suggesting that geography may influence alfalfa genetic divergence during local adaption. Genome-wide association studies identified 101 single nucleotide polymorphisms (SNPs) associated with 27 agronomic traits. Two candidate genes were predicted to be correlated with fall dormancy and salt response. We believe that the allele-aware chromosome-level genome sequence of Zhongmu-4 combined with the resequencing data of the diverse alfalfa germplasms will facilitate genetic research and genomics-assisted breeding in variety improvement of alfalfa.

Bioactive gibberellic acids (GAs) are diterpenoid plant hormones that are biosynthesized through complex pathways and control various aspects of growth and development. Although GA biosynthesis has been intensively studied, the downstream metabolic pathways regulated by GAs have remained largely unexplored. We investigated Tnt1 retrotransposon insertion mutant lines of Medicago truncatula with a dwarf phenotype by forward and reverse genetics screening and phylogenetic, molecular, biochemical, proteomic and metabolomic analyses. Three Tnt1 retrotransposon insertion mutant lines of the gibberellin 3-beta-dioxygenase 1 gene (GA3ox1) with a dwarf phenotype were identified, in which the synthesis of GAs (GA₃ and GA₄) was inhibited. Phenotypic analysis revealed that plant height, root and petiole length of ga3ox1 mutants were shorter than those of the wild type (Medicago truncatula ecotype R108). Leaf size was also much smaller in ga3ox1 mutants than that in wild-type R108, which is probably due to cell-size diminution instead of a decrease in cell number. Proteomic and metabolomic analyses of ga3ox1/R108 leaves revealed that in the ga3ox1 mutant, flavonoid isoflavonoid biosynthesis was significantly up-regulated, while nitrogen metabolism was down-regulated. Additionally, we further demonstrated that flavonoid and isoflavonoid biosynthesis was induced by prohexadione calcium, an inhibitor of GA3ox enzyme, and inhibited by exogenous GA₃. In contrast, nitrogen metabolism was promoted by exogenous GA₃ but inhibited by prohexadione calcium. The results of this study further demonstrated that GAs play critical roles in positively regulating nitrogen metabolism and transport and negatively regulating flavonoid biosynthesis through GA-mediated signaling pathways in leaves.

Background: Leaf size affects crop canopy morphology and photosynthetic efficiency, which can influence forage yield and quality. It is of great significance to mine the key genes controlling leaf development for breeding new alfalfa varieties. In this study, we mapped leaf length (LL), leaf width (LW), and leaf area (LA) in an F1 mapping population derived from a cultivar named ZhongmuNo.1 with larger leaf area and a landrace named Cangzhou with smaller leaf area. Results: This study showed that the larger LW was more conducive to increasing LA. A total of 24 significant quantitative trait loci (QTL) associated with leaf size were identified on both the paternal and maternal linkage maps. Among them, nine QTL explained about 11.50–22.45% phenotypic variation. RNA-seq analysis identified 2,443 leaf-specific genes and 3,770 differentially expressed genes. Combining QTL mapping, RNA-seq alalysis, and qRT-PCR, we identified seven candidate genes associated with leaf development in five major QTL regions. Conclusion: Our study will provide a theoretical basis for marker-assisted breeding and lay a foundation for further revealing molecular mechanism of leaf development in alfalfa.

In mammals, nucleostemin (NS), a nucleolar GTPase, is involved in stem cell proliferation, embryogenesis and ribosome biogenesis. Arabidopsis NUCLEOSTEMIN-LIKE 1 (NSN1) has previously been shown to be essential for plant growth and development. However, the role of NSN1 in cell proliferation is largely unknown. Using nsn1, a loss-of-function mutant of Arabidopsis NSN1, we investigated the function of NSN1 in plant cell proliferation and cell cycle regulation. Morphologically, nsn1 exhibited developmental defects in both leaves and roots, producing severely reduced vegetative organs with a much smaller number of cells than those in the wild type. Dynamic analysis of leaf and root growth revealed a lower cell proliferation rate and slower cell division in nsn1. Consistently, the transcriptional levels of key cell cycle genes, including those regulating the transition of G1-S and G2-M, were reduced drastically in nsn1. The introduction of CYCLIN B1::GUS into nsn1 resulted in confined expression of GUS in both the leaf primordia and root meristem, indicating that cell proliferation was hampered by the mutation of NSN1. Upon subjection to treatment with bleomycin and methyl methanesulfonate (MMS), nsn1 plants exhibited hypersensitivity to the genotoxic agents. In the nucleus, NSN1 interacted with nucleosome assembly protein1 (AtNAP1;1), a highly conserved histone chaperone functioning in cell proliferation. Notably, the N-terminal conserved domains of Arabidopsis NSN1 were critical for the physical interaction. As a conserved homolog of mammalian nucleostemin, Arabidopsis NSN1 plays pivotal roles in embryogenesis and ribosome biogenesis. In this study, NSN1 was found to function as a positive regulator in cell cycle progression. The interaction between NSN1 and histone chaperone AtNAP1;1, and the high resemblance in sensitivity to genotoxics between nsn1 and atnap1;1 imply the indispensability of the two nuclear proteins for cell cycle regulation. This work provides an insight into the delicate control of cell proliferation through the cooperation of a GTP-binding protein with a nucleosome assembly/disassembly protein in Arabidopsis.

Introduction: This study aimed to investigate the structure characterization and antiinflammatory activity of a novel polysaccharide, PKP2-1, from the rhizomes of Polygonatum kingianum Coll. and Hemsl.Methods: We isolated a novel polysaccharide, PKP2-1, from the rhizomes of Polygonatum kingianum Coll. and Hemsl. for the first time, which was then successively purified through hot-water extraction, 80% alcohol precipitation, anion exchange and gel permeation chromatography. The in vitro anti-inflammatory activity of PKP2-1 in MH7A cells was assessed using a CCK-8 kit assay.Results: Monosaccharide composition assay revealed that PKP2-1 was mainly composed of glucose, galactose, mannose, and glucuronic acid at an approximate molar ratio of 6:2:2:1. It had a molecular weight of approximately 17.34 kDa. Structural investigation revealed that the backbone of PKP2-1 consisted of (→2, 3)-α-D-Galp(4→, →2)-α-D-Manp(3→, →2)-β-D-Glcp(4→) and α-D-Glcp(3→) residues with side chains (→2)-β-D-Glcp(4→, →1)-α-D-Galp(4→) and α-D-Glcp(3→) branches located at O-3 position of (→2, 3)-α-D-Galp(4→). The in vitro anti-inflammatory activity of PKP2-1 in MH7A cells revealed that PKP2-1 could reduce the expression of IL-11β and IL-6, increase the expression of IL-10 and induce apoptosis of synovial fibroblasts.Conclusion: The PKP2-1 could inhibit MH7A cell growth and potentially be exploited as an anti-inflammatory agent.