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 mechanism of potato (Solanum tuberosum L.) thermotolerance has been the focus of intensive research for many years because plant growth and tuber yield are highly sensitive to heat stress. However, the linkage between the aerial and belowground parts of potato plants in response to high temperatures is not clear. To disentangle this issue, the aerial and belowground parts of the heat-resistant cultivar Dian187 (D187) and the heat-sensitive cultivar Qingshu 9 (Qs9) were independently exposed to high-temperature (30 °C) conditions using a special incubator. The results indicated that when the belowground plant parts were maintained at a normal temperature, the growth of the aerial plant parts was maintained even when independently exposed to heat stress. In contrast, the treatment that independently exposed the belowground plant parts to heat stress promoted premature senescence in the plant’s leaves, even when the aerial plant parts were maintained at a normal temperature. When the aerial part of the plant was independently treated with heat stress, tuberization belowground was not delayed, and tuberization suppression was not as severe as when the belowground plant parts independently underwent heat stress. Heat stress on the belowground plant parts alone had virtually no damaging effects on the leaf photosynthetic system but caused distinct tuber deformation, secondary growth, and the loss of tuber skin colour. Transcriptome analysis revealed that the treatment of the belowground plant parts at 30 °C induced 3361 differentially expressed genes in the Qs9 cultivar’s expanding tubers, while the D187 cultivar had only 10,148 differentially expressed genes. Conversely, when only the aerial plant parts were treated at 30 °C, there were just 807 DEGs (differentially expressed genes) in the D187 cultivar’s expanding tubers compared with 6563 DEGs in the Qs9 cultivar, indicating that the two cultivars with different heat sensitivities have distinct regulatory mechanisms of tuberization when exposed to heat stress. The information provided in this study may be useful for further exploring the genes associated with high-temperature resistance in potato cultivars.

Potato cultivars are inherently sensitive to high temperatures, and dissecting the mechanisms underlying heat response and tolerance has long been a central focus in potato research. However, the molecular mechanisms governing the short-term heat stress response in potato, as well as the regulatory role of DNA methylation in heat adaptation, remain largely unclear. In this study, we identified breeding line D187 as heat-tolerant and cultivar Q9 as heat-sensitive through microtuber induction under heat stress. We further confirmed that heat-sensitive cultivar Q9 exhibited distinct physiological responses in leaves following 6 h of heat treatment. Comparative transcriptome analysis of leaves exposed to 6 h of heat stress revealed distinct molecular response patterns between D187 and Q9. D187 specifically upregulated genes enriched in heat and other stress response pathways to enhance heat adaptation, whereas Q9 relied on pathways related to RNA modification and splicing, presumably adapting to high temperatures via post-transcriptional regulation. Notably, genes involved in the RdDM pathway were differentially upregulated in both genotypes, and heat stress correspondingly enhanced CHH methylation levels in the vicinity of functional genes in the heat-sensitive cultivar Q9. Treatment with 5-Azacytidine, a DNA methylation inhibitor, exacerbated the inhibition of in vitro tuber formation under high temperatures, indicating that maintaining and enhancing DNA methylation is essential for heat adaptation in potato. Furthermore, overexpression of StAGO4a/b in Nicotiana benthamiana modestly improved heat tolerance, suggesting that StAGO4s act as positive regulators of heat tolerance in potato. Collectively, our results suggest that heat-induced CHH methylation near functional genes via the RdDM pathway contributes positively to heat stress response and tolerance in Q9, providing new insights for identifying heat tolerance regulators from a DNA methylation perspective.