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

BACKGROUND AND AIMS: Extrafloral nectaries are defensive 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. METHODS: 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. KEY RESULTS: The CRC gene was found to encode 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. Passiflora 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. Passiflora alata CRC and P. cincinnata CRC expression peaked during the expanding leaf stage and was limited to the inner regions of the nectary. CONCLUSIONS: CRC presented stage-specific expression and conserved structural domains that resemble other species in which CRC acts as a key regulator. This suggests a possible correlation between CRC and nectary development.