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Medicago, a genus in the Leguminosae or Fabaceae family, includes the most globally significant forage crops, notably alfalfa (Medicago sativa). Its close diploid relative Medicago truncatula serves as an exemplary model plant for investigating legume growth and development, as well as symbiosis with rhizobia. Over the past decade, advances in Medicago genomics have significantly deepened our understanding of the molecular regulatory mechanisms that underlie various traits. In this review, we comprehensively summarize research progress on Medicago genomics, growth and development (including compound leaf development, shoot branching, flowering time regulation, inflorescence development, floral organ development, and seed dormancy), resistance to abiotic and biotic stresses, and symbiotic nitrogen fixation with rhizobia, as well as molecular breeding. We propose avenues for molecular biology research on Medicago in the coming decade, highlighting those areas that have yet to be investigated or that remain ambiguous. 

Leaves are the most significant parts of forage crops such as alfalfa. Senescence is the terminal stage of leaf development and is controlled by an integrated myriad of endogenous signals and environmental stimuli. WRKY transcription factors (TFs) play essential roles in regulating leaf senescence; however, only a few studies on the analysis and identification of the WRKY TF family in Medicago Sativa have been reported. In this study, we identified 198 WRKY family members from the alfalfa (M. sativa L.) cultivar ’XinjiangDaye’ using phylogenetic analysis and categorized them into three subfamilies, Groups I, II, and III, based on their structural characteristics. Group II members were further divided into five subclasses. In addition, several hormone- and stress-related cis-acting elements were identified in the promoter regions of MsWRKYs. Furthermore, 14 aging-related MsWRKYs genes from a previous transcriptome in our laboratory were selected for RT-qPCR validation of their expression patterns, and subsequently cloned for overexpression examination. Finally, MsWRKY5, MsWRKY66, MsWRKY92, and MsWRKY141 were confirmed to cause leaf yellowing in Nicotiana benthaminana using a transient expression system. Our findings lay a groundwork for further studies on the mechanism of M. sativa leaf aging and for the creation of new germplasm resources. 

Seed physical dormancy (hard-seededness) is an interesting ecological phenomenon and important agronomic trait. The loss of seed coat impermeability/hard-seededness is a key target trait during the domestication of leguminous crops which allows seeds to germinate rapidly and uniformly. In this study, we examined the mutation of quantitative trait locus (QTL) genes, GmHs1-1 and GmqHS1, in 18 wild soybean (G. soja) and 23 cultivated soybean (G. max) accessions. The sequencing results indicate that a G-to-T substitution in GmqHS1 and a C-to-T substitution in GmHs1-1 occurred in all 23 cultivated soybean accessions but not in any of the 18 wild soybean accessions. The mutations in the two genes led to increased seed coat permeability in cultivated soybean. Therefore, we provide evidence that two genes, GmHs1-1 and GmqHS1, simultaneously contribute to the domestication of hard-seededness in soybeans. This finding is of great significance for genetic analysis and improved utilization of the soybean hard-seededness trait. 

Physical dormancy of seeds is a form of dormancy due to the presence of an impermeable seed coat layer, and it represents a feature for plants to adapt to environmental changes over an extended period of phylogenetic evolution. However, in agricultural practice, physical dormancy is problematic. because it prevents timely and uniform seed germination. Therefore, physical dormancy is an important agronomical trait to target in breeding and domestication, especially for many leguminous crops. Compared to the well-characterized physiological dormancy, research progress on physical dormancy at the molecular level has been limited until recent years, due to the lack of suitable research materials. This review focuses on the structure of seed coat, factors affecting physical dormancy, genes controlling physical dormancy, and plants suitable for studying physical dormancy at the molecular level. Our goal is to provide a plethora of information for further molecular research on physical dormancy. 

Leaves are a key forage part for livestock, and the aging of leaves affects forage biomass and quality. Preventing or delaying premature leaf senescence leads to an increase in pasture biomass accumulation and an improvement in alfalfa quality. NAC transcription factors have been reported to affect plant growth and abiotic stress responses. In this study, 48 NAC genes potentially associated with leaf senescence were identified in alfalfa under dark or salt stress conditions. A phylogenetic analysis divided MsNACs into six subgroups based on similar gene structure and conserved motif. These MsNACs were unevenly distributed in 26 alfalfa chromosomes. The results of the collinearity analysis show that all of the MsNACs were involved in gene duplication. Some cis-acting elements related to hormones and stress were screened in the 2-kb promoter regions of MsNACs. Nine of the MsNAC genes were subjected to qRT-PCR to quantify their expression and Agrobacterium-mediated transient expression to verify their functions. The results indicate that Ms.gene031485, Ms.gene032313, Ms.gene08494, and Ms.gene77666 might be key NAC genes involved in alfalfa leaf senescence. Our findings extend the understanding of the regulatory function of MsNACs in leaf senescence. 

Abstract Compound leaf development requires the coordination of genetic factors, hormones, and other signals. In this study, we explored the functions of Class ⅡKNOTTED‐like homeobox (KNOXII) genes in the model leguminous plantMedicago truncatula. Phenotypic and genetic analyses suggest thatMtKNOX4,5are able to repress leaflet formation, whileMtKNOX3,9,10are not involved in this developmental process. Further investigations have shown that MtKNOX4 represses the CK signal transduction, which is downstream of MtKNOXⅠ‐mediated CK biosynthesis. Additionally, two boundary genes,FUSED COMPOUND LEAF1(orthologue ofArabidopsisClass MKNOX) andNO APICAL MERISTEM(orthologue ofArabidopsis CUP‐SHAPED COTYLEDON), are necessary for MtKNOX4‐mediated compound leaf formation. These findings suggest, that among the members of MtKNOXⅡ, MtKNOX4 plays a crucial role in integrating the CK pathway and boundary regulators, providing new insights into the roles of MtKNOXⅡ in regulating the elaboration of compound leaves inM. truncatula. 

Abstract Plant cuticles are composed of hydrophobic cuticular waxes and cutin. Very long-chain fatty acids (VLCFAs) are components of epidermal waxes and the plasma membrane and are involved in organ morphogenesis. By screening a barrelclover (Medicago truncatula) mutant population tagged by the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified two types of mutants with unopened flower phenotypes, named unopened flower1 (uof1) and uof2. Both UOF1 and UOF2 encode enzymes that are involved in the biosynthesis of VLCFAs and cuticular wax. Comparative analysis of the mutants indicated that the mutation in UOF1, but not UOF2, leads to the increased number of leaflets in M. truncatula. UOF1 was specifically expressed in the outermost cell layer (L1) of the shoot apical meristem (SAM) and leaf primordia. The uof1 mutants displayed defects in VLCFA-mediated plasma membrane integrity, resulting in the disordered localization of the PIN-FORMED1 (PIN1) ortholog SMOOTH LEAF MARGIN1 (SLM1) in M. truncatula. Our work demonstrates that the UOF1-mediated biosynthesis of VLCFAs in L1 is critical for compound leaf patterning, which is associated with the polarization of the auxin efflux carrier in M. truncatula.