Proper activity of the age-dependent miR156 is required for leaf heteroblasty and extrafloral nectary development in...

Passion flower extrafloral nectaries (EFNs) protrude from leaves and facilitate mutualistic interactions with insects; however, how age cues control EFN growth remains poorly understood. Here, we examined leaf and EFN morphology and development of two Passiflora species with distinct leaf shapes, and compared the phenotype of these to transgenics with manipulated activity of the age-dependent miR156, which targets several SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factors. Low levels of miR156 correlated with leaf maturation and EFN formation in Passiflora edulis and P. cincinnata. Accordingly, manipulating miR156 activity affected leaf heteroblasty and EFN development. miR156-overexpressing leaves exhibited less abundant and tiny EFNs in both Passiflora species. EFN abundance remained mostly unchanged when miR156 activity was reduced, but it led to larger EFNs in P. cincinnata. Transcriptome analysis of young leaf primordia revealed that miR156-targeted SPLs may be required to properly express leaf and EFN-associated genes. Importantly, altered miR156 activity impacted sugar profiles of the nectar and modified ecological relationships between EFNs and ants. Our work provides evidence that the miR156/SPL module indirectly regulates EFN development in an age-dependent manner and that the EFN development program is closely associated with the heteroblastic developmental program of the EFN-bearing leaves.