Artificially improving traits of cultivated alfalfa (Medicago sativaL.), one of the most important forage crops, is challenging due to the lack of a reference genome and an efficient genome editing protocol, which mainly result from its autotetraploidy and self-incompatibility. Here, we generate an allele-aware chromosome-level genome assembly for the cultivated alfalfa consisting of 32 allelic chromosomes by integrating high-fidelity single-molecule sequencing and Hi-C data. We further establish an efficient CRISPR/Cas9-based genome editing protocol on the basis of this genome assembly and precisely introduce tetra-allelic mutations into null mutants that display obvious phenotype changes. The mutated alleles and phenotypes of null mutants can be stably inherited in generations in a transgene-free manner by cross pollination, which may help in bypassing the debate about transgenic plants. The presented genome and CRISPR/Cas9-based transgene-free genome editing protocol provide key foundations for accelerating research and molecular breeding of this important forage crop.

Plant-specific WOX family transcription factors play important roles ranging from embryogenesis to lateral organ development. The WOX1 transcription factors, which belong to the modern clade of the WOX family, are known to regulate outgrowth of the leaf blade specifically in the mediolateral axis; however, the role of WOX1 in compound leaf development remains unknown. Phylogenetic analysis of the whole WOX family in tomato (Solanum lycopersicum) indicates that there are 10 members that represent the modern, intermediate, and ancient clades. Using phylogenetic analysis and a reverse genetic approach, in this study we identified SlLAM1 in the modern clade and examined its function and tissue-specific expression pattern. We found that knocking out SlLAM1 via CRISPR/Cas9-mediated genome editing led to narrow leaves and a reduced number of secondary leaflets. Overexpression of tomato SlLAM1 could rescue the defects of the tobacco lam1 mutant. Anatomical and transcriptomic analyses demonstrated that floral organ development, fruit size, secondary leaflet initiation, and leaf complexity were altered due to loss-of-function of SlLAM1. These findings demonstrate that tomato SlLAM1 plays an important role in the regulation of secondary leaflet initiation, in addition to its conserved function in blade expansion.

The metabolism of monoterpene indole alkaloids (MIAs) is an outstanding example of how plants shape chemical diversity from a single precursor. Here we report the discovery of novel enzymes from the Alstonia scholaris tree, a cytochrome P450, an NADPH dependent oxidoreductase and a BAHD acyltransferase that together synthesize the indole alkaloid akuammiline with a unique methanoquinolizidine cage structure. The two paralogous cytochrome P450 enzymes rhazimal synthase (AsRHS) and geissoschizine oxidase (AsGO) catalyse the cyclization of the common precursor geissoschizine and they direct the MIA metabolism towards to the two structurally distinct and medicinally important MIA classes of akuammilan and strychnos alkaloids, respectively. To understand the pathway divergence, we investigated the catalytic mechanism of the two P450 enzymes by homology modelling and reciprocal mutations. Upon conducting mutant enzyme assays, we identified a single amino acid residue that mediates the space in active sites, switches the enzymatic reaction outcome and impacts the cyclization regioselectivity. Our results represent a significant advance in MIA metabolism, paving the way for discovery of downstream genes in akuammilan alkaloid biosynthesis and facilitating future synthetic biology applications. We anticipate that our work presents, for the first time, insights at the molecular level for plant P450 catalytic activity with a significant key role in the diversification of alkaloid metabolism, and provides the basis for designing new drugs.

The size of leaf and seed organs, determined by the interplay of cell proliferation and expansion, is closely related to the final yield and quality of forage and crops. Yet the cellular and molecular mechanisms underlying organ size modulation remain poorly understood, especially in legumes. Here, MINI ORGAN1 (MIO1), which encodes an F-box protein SMALL LEAF AND BUSHY1 (SLB1) recently reported to control lateral branching in Medicago truncatula, was identified as a key regulator of organ size. We show that loss-of-function of MIO1/SLB1 severely reduced organ size. Conversely, plants overexpressing MIO1/SLB1 had enlarged organs. Cellular analysis revealed that MIO1/SLB1 controlled organ size mainly by modulating primary cell proliferation during the early stages of leaf development. Biochemical analysis revealed that MIO1/SLB1 could form part of SKP1/Cullin/F-box (SCF) E3 ubiquitin ligase complex, to target BIG SEEDS1 (BS1), a repressor of primary cell division, for degradation. Interestingly, we found that MIO1/SLB1 also played a key role in pulvinus development and leaf movement by modulating cell proliferation of the pulvinus as leaves developed. Our study not only demonstrates a conserved role of MIO1/SLB1 in the control of organ size in legumes, but also sheds light on the novel function of MIO1/SLB1 in leaf movement.

The BRASSINAZOLE-RESISTANT (BZR) family of transcription factors (TFs) are positive regulators in the biosynthesis of brassinosteroids. The latter is a class of steroid hormones that affect a variety of developmental and physiological processes in plants. BZR TFs play essential roles in the regulation of plant growth and development, including multiple stress-resistance functions. However, the evolutionary history and individual expression patterns of the legume BZR genes has not been determined. In this study, we performed a genome-wide investigation of the BZR gene family in seven legume species. In total, 52 BZR genes were identified and characterized. By analyzing their phylogeny, we divided these BZR genes into five groups by comparison with orthologs/paralogs in Arabidopsis thaliana. The intron/exon structural patterns and conserved protein motifs of each gene were analyzed and showed high group-specificities. Legume BZR genes were unevenly distributed among their corresponding genomes. Genome and gene sequence comparisons revealed that gene expansion of the BZR TF family in legumes mainly resulted from segmental duplications and that this family has undergone purifying selection. Synteny analysis showed that BZR genes tended to localize within syntenic blocks conserved across legume genomes. The expression patterns of BZR genes among various legume vegetative tissues and in response to different abiotic stresses were analyzed using a combination of public transcriptome data and quantitative PCR. The patterns indicated that many BZR genes regulate legume organ development and differentiation, and significantly respond to drought and salt stresses. This study may provide valuable information for understanding the evolution of BZR gene structure and expression, and lays a foundation for future functional analysis of the legume BZR genes by species and by gene.