Age-dependent miR156-targeted SPLs are required for extrafloral nectary development in Passiflora spp.

- Passion flower extrafloral nectaries (EFNs) protrude from adult leaves and facilitate mutualistic interactions with insects, but how age cues control EFN establishment remains poorly understood. - Here, we combined genetic and molecular studies to investigate how leaf development and EFN patterning are regulated through the age-dependent miR156-SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) module in two EFN-containing Passiflora species with distinct leaf shapes. - Low levels of miR156 correlate with leaf maturation and EFN formation in Passiflora edulis and P. cincinnata. Consistently, overexpression of miR156 (miR156-OE), which leads to low levels of SPLs, affected leaf ontogeny and EFN development in both species. Laminar EFNs were underdeveloped and less abundant in both P. edulis and P. cincinnata miR156-OE leaves. Importantly, the ecological relationships established by EFNs and their sugar profiles were negatively regulated by high levels of miR156. Moreover, transcriptome analysis of young leaf primordia revealed that miR156-targeted SPLs may be required for proper expression of leaf and EFN development- associated genes in P. edulis and P. cincinnata. - Our work provides the first evidence that the highly conserved miR156/SPL module regulates EFN development in an age-dependent manner and that the program responsible for EFN development is closely associated with the heteroblastic developmental program of the EFN-bearing leaves.