Isolation and characterization of a cytokinin up-regulated gene from tobacco mesophyll protoplasts

We have isolated a cytokinin up-regulated cDNA clone, H13, from an early stage of cultured tobacco mesophyll protoplasts by a differential display method. The expression of this gene was specifically induced by natural and synthetic cytokinins including N-(2-chloro-4-pyridyl)N'-phenylurea (4PU30), a diphenylurea-type cytokinin, although the simultaneous presence of auxin was also required. It seems that the preceding treatment of the tobacco mesophyll protoplasts by auxin is necessary for the gene to respond to cytokinin, The addition of a cytokinin antagonist, compound 182, which suppressed the induction of cell division in tobacco mesophyll protoplasts, completely abolished the expression of this gene, Though the predicted gene product of H13 did not suggest us any sequences of defined functions, two domains of the predicted sequence had significant homology to several reported sequences in the data base. The gene product of H13 is proposed to have a role in regenerating cell wall in cultured protoplasts, since a cDNA clone E6, from cotton fiber cells, which has the most closely related structure to H13, has been isolated from cells which showed active cellulose synthesis. This supposition is supported by the evidence that in the absence of cytokinin, cell wall regeneration was significantly suppressed, resulting in failure of the induction of cell division. Thus, the gene product of H13 is supposed to have a role in regenerating cell walls and facilitating the progression of the cell cycle, resulting in the sustained cell division of tobacco mesophyll protoplasts.