Isolation of three homologous AP1-like MADS-box genes in crocus ( Crocus sativus L.) and characterization of their expression

Abstract

We have cloned and characterized the expression of three homologous Apetala1-like ( AP1-like) genes from a crocus variety ( Crocus sativus L) cultivated exclusively in Kozani, at northern Greece. The three different homologous genes were designated CsAP1 ( C. sativus APETALA 1) and each one was named CsAP1a, CsAP1b, and CsAP1c. They are the first reported MADS-box genes isolated from this important monocot species cultivated for its flowers. The deduced amino acid sequence of the three genes indicated high homology with members of the MADS-box family of transcription factors, and particularly with other members of the AP1-like family of MADS-box proteins that control floral-meristem and floral-organ identity. All the isolated sequences lack the typical CaaX-motif that is present in dicot AP1 functional proteins but absent in the monocot homologues thus far examined. CsAP1a and CsAP1b are more similar having 88.2% identical amino acids while CsAP1c is more divergent having 70.9% similarity with CsAP1a, and 64.5% similarity with CsAP1b. Phylogenetic analysis of the isolated genes at the amino acid level indicated that they form a clade with other monocot AP1-like genes from maize (ZmM28), barley (BM3), and rice (OsMADS18). Southern experiments indicated the presence of additional AP1-like homologues in crocus. Expression analysis indicated the presence of different amount of steady-state mRNAs for all the three homologous genes in leaves, as well as, in the three mature flower parts, namely: tepals, stamen and carpels. Similar expression pattern display many monocot AP1-like MADS-box genes, which comprise a distinct phylogenetic clade of monocot class A MADS-box genes and may reflect a novel, yet unidentified role of their corresponding proteins in these species.