Don’t be so crabby: characterization and RNA expression of the <i>CRABS CLAW</i> gene in petiolar nectaries of <i>Passiflora alata</i> and <i>Passiflora cincinnata</i>

Extrafloral nectaries are defense structures that protect plants against herbivores. In Passiflora, extrafloral nectaries are prominent features, and the YABBY family transcription factor CRABS CLAW (CRC) has been proposed to regulate their development. Here, we investigated the morphoanatomy of petiolar nectaries and characterized the CRC gene at distinct stages of nectary development in Passiflora alata and Passiflora cincinnata. We carried out bioinformatics and phylogenetic analyses, followed by scanning electron microscopy, anatomy, in situ hybridization, and RT-qPCR assessment of nectaries sampled at three leaf developmental stages. The CRC gene was found to contain a highly conserved C2C2 zinc finger motif (composed of three β-sheets and a potential fourth β-sheet or α-helix) and a YABBY domain (two α-helices); along with variable regions. Passiflora-CRC protein sequences clustered closely to Rosids and Asterids. P. cincinnata presented short-patelliform nectaries, whereas those of P. alata were broader and cupuliform. Both species exhibited a similar multiseriate secretory epidermis in the crater region, an underlying nectariferous parenchyma with highly vacuolated, compact cells, and a subnectariferous parenchyma with vascular bundles that terminated before reaching the nectariferous zone. Meristematic activity was pronounced in the early stages and in fully developed nectaries, indicating progressive tissue differentiation. Pa-CRC and Pc-CRC expression peaked during the expanding leaf stage and was limited to the inner regions of the nectary. CRC presented stage-specific expression and conserved structural domains that resembles other species where CRC acts as a key regulator. This suggests a possible correlation between CRC and nectary development.