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.

Background: Nitrogen (N) form preference influences responses of invasive plants to soil N form changes caused by global change and the evolution of their invasiveness, and the differences in root uptake and shoot assimilation for different forms of N affect N form utilization in plants. However, the relative roles of shoots and roots in N-form preference are still unclear for invasive plants. Methods: We grew the homo- and heterografted seedlings of the nitrate-preferred invasive plant Xanthium strumarium and the ammonium-preferred native plant X. sibiricum under two levels of nitrate and ammonium. Key Results: Under high N levels, the grafted seedlings with X. strumarium as scion, regardless of the source of the rootstock always preferred nitrate, whereas those with X. sibiricum as scion always preferred ammonium. Higher expression of nitrate reductase genes in leaves of X. strumarium relative to X. sibiricum was associated with its nitrate preference. Regardless of scion origin, the grafted seedlings with X. strumarium as rootstock always accumulated more total biomass and N than those with X. sibiricum as rootstock under both low and high N levels, which was associated with higher root surface area, volume, and NPF6.3 expression in the invasive relative to the native species. Conclusions: Our results indicate that N-form preference was determined primarily by shoot identity, whereas roots contributed to the magnitude of the response, providing a novel perspective for understanding the mechanisms underlying exotic plant invasion from the viewpoint of N form utilization.