Molecular cloning and characterization of cellulose synthase genes expressed during tracheary elements differentiation in cultures of Zinnia elegans

Isolated mesophyll cells of Zinnia elegans induced to differentiate into tracheary elements (TEs) semi-synchronously in culture are a valuable experimental system for research on cellulose synthesis. To explore a possible cellulose synthase gene family that might be specific to secondary wall thickening, a RACE (Rapid Amplification of cDNA Ends) strategy was applied using total RNA from Zinnia cells cultured in differentiation medium for 60 hours. Three cDNA fragments, designated ZeCesAl (AF323039), ZeCesA2 (AF323040), and ZeCesA3 (AF323041), that were mainly expressed during TE differentiation and not during primary wall synthesis were isolated from 3' RACE technique. A cDNA fragment corresponding to the 5' part of ZeCesAl gene was obtained from the 5' RACE technique. A full-length ZeCesAl cDNA sequence was constructed by overlapping the 5' and 3'-RACE fragments. In common with known plant cellulose synthase genes iCesAs), ZeCesAl encodes a predicted membrane protein having conserved motifs and domain structure. Based on amino acid sequence comparisons and phylogenetic analyses, ZeCesAl-3 show a closer relationship to secondary wall-specific CesAs, especially to GhCesAl, AtCesAS, and PtCesA2, than to primary wallspecific CesAs. This indicates that ZeCesAl 3 are orthologs of CesA genes from other species that belong to the secondary wall clade. ZeCesAl-3 represent very similar sequences and define a set of paralogous genes, probably duplicated from the same original gene. Northern blot analyses and tissue printing revealed that ZeCesAl-3 were expressed in a close association with TE differentiation in vitro, and ZeCesAl was expressed in regions with developing vascular bundles in stems and leaves.