This study investigated whether an increased production of the plant hormone cytokinin in roots, the main site of its synthesis and putative signaling organ, can influence developmental events, such as growth of axillary shoot meristems or leaf senescence, in the plant shoot. To this end, transgenic tobacco plants (Nicotiana tabacum L.) were generated that conditionally overproduce cytokinins. These plants harbour the ipt gene under the transcriptional control of a modified 35S promoter that is repressed in plants with high titers of tetracycline repressor protein. De-repression of transcription led to a rapid more than 50-fold increase of hormone concentration. The time course of changes in the steady-state levels of 16 different cytokinin metabolites, as a consequence of IPT enzyme activity, was monitored in different plant tissues. Zeatin riboside was the first and most dramatically increased product; zeatin, dihydrozeatin and glucosides accumulated later. The consequences of enhanced cytokinin synthesis remained mainly restricted to the site of hormone production. For example, de-repression of ipt gene transcription in lateral buds caused the growth of single buds only at the site of tetracycline application. In reciprocal grafts of transgenic plants with wild-type plants, no biological cytokinin effects, i.e. growth of lateral shoot meristems or sequential leaf senescence, were observed in the non-transgenic plant part. Also, the increase in steady-state levels of cytokinins remained restricted mainly to the transgenic part, despite a specific increase of the zeatin riboside concentration in the transpiration stream. These results question the role of cytokinins as a long-range root-to-shoot signal in correlative control of apical dominance and sequential leaf senescence of tobacco, and support the assumption that this hormone is relevant to paracrine signaling.

Metabolic control of cytokinin oxidase by its substrate was investigated in planta using wild-type (WT) and conditionally ipt gene-expressing transgenic (IPT) tobacco (Nicotiana fabacum 1.) callus cultures and plants. The derepression of the tetracycline (Tc)-dependent ipt gene transcription was followed by a progressive, more than 100-fold increase in total cytokinin content in IPT calli. The activity of cytokinin oxidase extracted from these calli began to increase 16 t o 20 h after gene derepression, and after 13 d it was 10-fold higher than from Tc-treated WT calli. An increase in cytokinin oxidase activity, as a consequence of elevated cytokinin levels, was found in detached leaves (8-fold after 4 d) and i n roots of intact plants (4-fold after 3 d). The partially purified cytokinin oxidase from WT, repressed IPT, and Tc-derepressed IPT tobacco calli exhibited similar characteristics. It had the same broad pH optimum (pH 6.5-8.5), its activity in vitro was enhanced 4-fold in the presence of copper-imidazole, and the apparent K,,,(N6-[A2iso-pentenylladenine) values were in the range of 3.1 to 4.9 PM. The increase in cytokinin oxidase activity in cytokinin-overproducing tissue was associated with the accumulation of a glycosylated form of the enzyme. The present data indicate the substrate induction of cytokinin oxidase activity in different tobacco tissues, which may contribote t o hormone homeostasis.

SummaryThe rolC gene of Agrobacterium rhizogenes T-DNA plays an essential role in the establishment of hairy root disease and its overexpression in transgenic plants causes pleiotropic developmental alterations. This study investigated whether the biological activity of the rolC 13-glucosidase is due to an alteration of the cytokinin balance in planta.HPLC radiocounting assays of [3H]-Iabeled cytokinin glucosides fed exogenously to tobacco leaf disks, to rolC expressing Escherichia coil cells or cell-free extracts showed that cytokinin N3-and O-glucosides are the preferred substrate of the rolC protein. Hydrolysis of N7-and N9-glucosides was not detected at substrate concentrations close to physiological levels. Furthermore, these conjugates were also not active as cytokinins in biotests when fed to rolC-expressing tissues. For analysis of the rolC activity on endogenous cytokinin conjugates the gene was expressed under the transcriptional control of a modified tetracycline-inducible 35S promoter. This was done to avoid possible interference with secondary effects or plant homeostatic mechanisms which could mask primary in planta events when transgenes are expressed constitutively. No changes in the endogenous pool of different cytokinin glucosides, as determined by a newly developed electrospray tandem mass spectroscopy directly coupled to high performance liquid chromatography, were found following chemical induction of the rolC gene. Also the levels of free cytokinins remained unchanged after gene induction. Hybrid tobacco plants expressing the cytokinin-

Blueberries are an increasingly important h~ticultural crop in New Zealand and other coun~es. Fruit ripen over an extended period, with considerable differences in storage and other qualities. Tissue concentrations of abscisic acid (ABA), 1-aminocyciopmpane-l-carboxylic acid (ACC), malonyl-ACC and also ACC oxidase activity and ethylene evolution were determined at 5 stages of colour development on 2 harvest dates. These were related to &y matter, sucrose and reducing sugar content. ACC concentrations, and the proportion conjugated varied between harvest dates, while ethylene evolution and ABA concentrations were similar. ABA concentrations were high relative to other fruit, exceeding 10 nmole/g fw prior to full ripeness. ABA was present in higher concentration in mesocarp than in seeds or pericarp/cuticle. It is proposed that ABA is implicated in dry matter acquisition, but is not a primary determinant of colour development or ethylene regulation.