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.

Plant height is a vital agronomic trait that greatly determines crop yields because of the close relationship between plant height and lodging resistance. Legumes play a unique role in the worldwide agriculture; however, little attention has been given to the molecular basis of their height. Here, we characterized the first dwarf mutant mini plant 1 (mnp1) of the model legume plant Medicago truncatula. Our study found that both cell length and the cell number of internodes were reduced in a mnp1 mutant. Using the forward genetic screening and subsequent whole-genome resequencing approach, we cloned the MNP1 gene and found that it encodes a putative copalyl diphosphate synthase (CPS) implicated in the first step of gibberellin (GA) biosynthesis. MNP1 was highly homologous to Pisum sativum LS. The subcellular localization showed that MNP1 was located in the chloroplast. Further analysis indicated that GA₃ could significantly restore the plant height of mnp1-1, and expression of MNP1 in a cps1 mutant of Arabidopsis partially rescued its mini-plant phenotype, indicating the conservation function of MNP1 in GA biosynthesis. Our results provide valuable information for understanding the genetic regulation of plant height in M. truncatula.

Nyctinastic leaf movement of Fabaceae is driven by the tiny motor organ pulvinus located at the base of the leaf or leaflet. Despite the increased understanding of the essential role of ELONGATED PETIOLULE1 (ELP1)/PETIOLE LIKE PULVINUS (PLP) orthologs in determining pulvinus identity in legumes, key regulatory components and molecular mechanisms underlying this movement remain largely unclear. Here, we used WT pulvinus and the equivalent tissue in the elp1 mutant to carry out transcriptome and proteome experiments. The omics data indicated that there are multiple cell biological processes altered at the gene expression and protein abundance level during the pulvinus development. In addition, comparative analysis of different leaf tissues provided clues to illuminate the possible common primordium between pulvinus and petiole, as well as the function of ELP1. Furthermore, the auxin pathway, cell wall composition and chloroplast distribution were altered in elp1 mutants, verifying their important roles in pulvinus development. This study provides a comprehensive insight into the motor organ of the model legume Medicago truncatula and further supplies a rich dataset to facilitate the identification of novel players involved in nyctinastic movement.

The morphology of soybean (Glycine max) leaves is a key agricultural trait that determines the planting density and photosynthetic efficiency. In soybean-producing regions of Northern China, narrow-leaf varieties carrying loss-of-function mutations in the Ln gene are widely cultivated and have been incorporated into breeding programs, as the narrow-leaf trait enhances light-use efficiency under high-density planting conditions. However, the molecular mechanism underlying mediolateral axis development of soybean leaf is largely unknown. The WUSCHEL-RELATED HOMEOBOX transcription factor WOX1 plays a key role in mediolateral leaf development. Here, we identified four WOX1 homologs in soybean and determined that GmWOX1a and GmWOX1b are specifically and highly expressed in the middle domain of the leaf primordium. Heterologous expression of GmWOX1a fully complemented the leaf defects of the Nicotiana sylvestris lam1 mutant. Gmwox1acd and Gmwox1bcd triple mutant produced by gene editing exhibited narrow leaves and developmental abnormalities. However, Gmwox1a, Gmwox1b, and Gmwox1cd displayed no visibly altered phenotypes, suggesting functional redundancy among the WOX1 homologs. These findings demonstrate that the four WOX1 homologs redundantly regulate mediolateral axis development in soybean leaves, providing a basis for future studies on leaf morphology and soybean breeding.

Cytokinins comprise a class of phytohormones that promote cell division and participate in diverse developmental processes. Cytokinin activity and steady-state levels in tissues are maintained through biosynthesis, signaling, degradation, side-chain modification, transport, and storage. Mutating HEADLESS (HDL), a Medicago truncatula homolog of Arabidopsis WUSCHEL (WUS), terminates the shoot apical meristem (SAM); thus, hdl mutants continuously generate leaves. In this study, we isolated a direct target of HDL [(HDL Target 1, HDT1)] based on RNA-seq transcriptome analyses using inducible HDL overexpression lines and the hdl mutant. HDT1 encodes a putative cytokinin hydroxylase homologous to Arabidopsis Cytochrome P450 mono-oxygenase CYP735A1 and CYP735A2. HDT1 trans-hydroxylates the prenyl side chain of cytokinins, converting the isopentenyl-adenine (iP)-type into trans-Zeatin (tZ)-type cytokinins. We show that HDL directly binds to the HDT1 promoter and represses its transcription, promoting the preferential accumulation of iP-type cytokinins. Overexpressing HDT1 slowed plant growth, leading to shorter plants caused by inhibited internode elongation, which is reminiscent of the hdl mutant phenotype albeit less severe. Both the hdl mutant and HDT1 overexpression lines accumulated lower levels of the iP-type but higher levels of the tZ-type cytokinins in shoots, indicating that HDT1 promotes tZ-type cytokinin biosynthesis and that HDL represses this process. Overall, this study uncovers the role of HDL in cytokinin biosynthesis and suggests that iP is preferred over tZ for SAM function in M. truncatula, opening avenues of research to better understand HDL function and the molecular mechanisms that regulate cytokinin biosynthesis.