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.

Anthocyanins and proanthocyanins (PAs) are two end products of the flavonoid biosynthesis pathway. They are believed to be synthesized in the endoplasmic reticulum and then sequestered into the vacuole. In Arabidopsis thaliana, TRANSPARENT TESTA 19 (TT19) is necessary for both anthocyanin and PA accumulation. Here, we found that MtGSTF7, a homolog of AtTT19, is essential for anthocyanin accumulation but not required for PA accumulation in Medicago truncatula. MtGSTF7 was induced by the anthocyanin regulator LEGUME ANTHOCYANIN PRODUCTION 1 (LAP1), and its tissue expression pattern correlated with anthocyanin deposition in M. truncatula. Tnt1-insertional mutants of MtGSTF7 lost anthocyanin accumulation in vegetative organs, and introducing a genomic fragment of MtGSTF7 could complement the mutant phenotypes. Additionally, the accumulation of anthocyanins induced by LAP1 was significantly reduced in mtgstf7 mutants. Yeast-one-hybridization and dual-luciferase reporter assays revealed that LAP1 could bind to the MtGSTF7 promoter to activate its expression. Ectopic expression of MtGSTF7 in tt19 mutants could rescue their anthocyanin deficiency, but not their PA defect. Furthermore, PA accumulation was not affected in the mtgstf7 mutants. Taken together, our results show that the mechanism of anthocyanin and PA accumulation in M. truncatula is different from that in A. thaliana, and provide a new target gene for engineering anthocyanins in plants.

The milestone of compound leaf development is the generation of separate leaflet primordia during the early stages, which involves two linked but distinct morphogenetic events: leaflet initiation and boundary establishment for leaflet separation. Although some progress in understanding the regulatory pathways for each event have been made, it is unclear how they are intrinsically coordinated. Here, we identify the PINNATE-LIKE PENTAFOLIATA2 (PINNA2) gene encoding a newly identified GRAS transcription factor in Medicago truncatula. PINNA2 transcripts are preferentially detected at organ boundaries. Its loss-of-function mutations convert trifoliate leaves into a pinnate pentafoliate pattern. PINNA2 directly binds to the promoter region of the LEAFY orthologue SINGLE LEAFLET1 (SGL1), which encodes a key positive regulator of leaflet initiation, and downregulates its expression. Further analysis revealed that PINNA2 synergizes with two other repressors of SGL1 expression, the BEL1-like homeodomain protein PINNA1 and the C2H2 zinc finger protein PALMATE-LIKE PENTAFOLIATA1 (PALM1), to precisely define the spatiotemporal expression of SGL1 in compound leaf primordia, thereby maintaining a proper pattern of leaflet initiation. Moreover, we showed that the enriched expression of PINNA2 at the leaflet-to-leaflet boundaries is positively regulated by the boundary-specific gene MtNAM, which is essential for leaflet boundary formation. Together, these results unveil a pivotal role of the boundary-expressed transcription factor PINNA2 in regulating leaflet initiation, providing molecular insights into the coordination of intricate developmental processes underlying compound leaf pattern formation.