Studies of cytokinin action and metabolism using tobacco plants expressing either the ipt or the GUS gene controlled by a chalcone synthase promoter .2. ipt and GUS gene expression, cytokinin levels and metabolism

The expression of GUS and ipt genes under control of a chalcone synthase (chs) promoter (P-CHS) has been determined in tobacco (Nicotiana tabacum L.) plants and related to the development of plants expressing the chimaeric P-CHS-ipt gene. GUS gene expression, which served as a model for the expression of the ipt gene, was highest in the internal phloem tissue of stems, in mature leaf laminae and in the upper part of corollas when fully open. Expression of the P-CHS-ipt gene was assessed by quantifying the cytokinins produced, by determining incorporation of [H-3]adenine into cytokinins and by quantifying ipt mRNA. Results from these studies were in general agreement with those based on expression of the P-CHS-GUS gene. The chs promoter controlled expression of the ipt gene with some degree of tissue and temporal specificity. Expression of the ipt gene markedly elevated the cytokinin level in mature leaf laminae and the upper stems of flowering plants. The former was associated with retardation of leaf senescence and increased rates of transpiration due to changes in number, size and aperture of stomata, while the latter was associated with development of lateral shoots. In shoot tip cultures, 2-fold elevations in endogenous cytokinin level caused clear changes in development and this is discussed in relation to current concepts concerning the hormonal control of plant development. Using the transgenic tobacco tissues, it was shown that cis-zeatin is a substrate for cytokinin oxidase, that cis-zeatin is not converted to trans-zeatin in these tissues and that the endogenous cytokinin level influences the level of cytokinin oxidase activity in tissue and the rate of degradation of exogenous zeatin riboside to adenosine.