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.

The establishment of dorsoventral polarity is a critical step in leaf morphogenesis, enabling the transition from radial primordia to flattened laminae. The MYB domain transcription factor ASYMMETRIC LEAVES1 (AS1) plays a central role in this process by regulating leaf polarity and developmental transitions, primarily through the repression of Class I KNOX genes. Here, four AS1 paralogs were identified in soybean (Glycine max), two of which showed collinearity with Arabidopsis thaliana and Medicago truncatula. The AS1 proteins of soybean and Arabidopsis exhibit high conservation, whereas the four GmAS1 genes in soybean display different tissue-specific expression patterns. Strikingly, each GmAS1 gene was able to fully rescue the defective phenotype of the Arabidopsis as1 mutant, indicating that GmAS1 genes are functionally conserved in leaf polarity regulation. Promoter analysis further indicated that GmAS1 genes are enriched in cis-acting elements related to light response, hormone regulation, development, and stress response, suggesting potential subfunctionalization among these paralogs. In conclusion, these findings demonstrate that GmAS1 genes are evolutionarily conserved in function but potentially diversified in regulation, providing new insights into their role in leaf polarity and stress adaptation.