Isolation, Characterization, and Expression Analysis of an NAP-Like cDNA from Crocus (Crocus sativus L.)

Abstract

In Arabidopsis, it has been shown that the B-class MADS-box genes are expressed in the developing whorl 2 petals and whorl 3 stamens throughout the ontogeny of these organs. APETALA3/PISTILLATA (AP3/PI) heterodimers act as an inducer of a regulator-coding gene called NAP, a homologue to NAC-like transcription factor genes, required for meristem establishment, separation of floral organs, and leaf senescence. In monocots like crocus (Crocus sativus) cultivated for saffron production, the expression of B-class MADS-box CsatAP3/CsatPI genes extends to whorl 1 where petaloid tepals are formed. We report here for the first time the cloning and characterization of an NAC-like gene, named CsatNAP, from crocus. The sequence alignment indicated that CsatNAP protein contains the typical domain structure of plant NAC proteins consisted of the conserved five subdomains of the N-terminal NAC domain. Phylogenetic analysis revealed that CsatNAP protein falls in the subgroup NAP of the NAC proteins. Expression analysis of crocus NAP indicates that it is expressed in whorl 1, supporting the hypothesis that also in crocus, AP3/PI-like is associated with the expression of Crocus NAP gene. The expression patterns of CsatNAP cDNA were studied in flowers and different flower organs as well as in leaves and different part of leaves during senescence. The CsatNAP transcripts were detected in whole flowers and flower organs. Promoter analysis of CsatNAP revealed a number of putative common cis-regulatory elements and, among them, two CArG boxes indicative of its control by MADS box transcription factors known to bind on CArG sites. Micro-RNA (miRNA) target analysis showed that the NAP sequence of Crocus contain a possible target site for miRNA164. Furthermore, CsatNAP showed increased expression in senescence leaves compared to the green ones.