Isolation and Characterization of a Cytokinin Up-Regulated Gene from Tobacco Mesophyll Protoplasts

We have isolated a cytokinin up-regulated cDNA clone, H13, froman early stage of cultured tobacco mesophyll protoplasts bya differential display method. The expression of this gene wasspecifically induced by natural and synthetic cytokinins includingN-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30), a diphenylurea-typecytokinin, although the simultaneous presence of auxin was alsorequired. It seems that the preceding treatment of the tobaccomesophyll protoplasts by auxin is necessary for the gene torespond to cytokinin. The addition of a cytokinin antagonist,compound 182, which suppressed the induction of cell divisionin tobacco mesophyll protoplasts, completely abolished the expressionof this gene. Though the predicted gene product of H13 did notsuggest us any sequences of defined functions, two domains ofthe predicted sequence had significant homology to several reportedsequences in the data base. The gene product of H13 is proposedto have a role in regenerating cell wall in cultured protoplasts,since a cDNA clone E6, from cotton fiber cells, which has themost closely related structure to H13, has been isolated fromcells which showed active cellulose synthesis. This suppositionis supported by the evidence that in the absence of cytokinin,cell wall regeneration was significantly suppressed, resultingin failure of the induction of cell division. Thus, the geneproduct of H13 is supposed to have a role in regenerating cellwalls and facilitating the progression of the cell cycle, resultingin the sustained cell division of tobacco mesophyll protoplasts. ¹These authors are equally contributed to this work.