Senescence and reserve mobilization are integral components of plant development, are basic strategies in stress mitigation, and regulated at least in part by cytokinin. In the present study the effect of altered cytokinin metabolism caused by senescence-specific autoregulated expression of the Agrobacterium tumefaciens IPT gene under control of the P(SAG12) promoter (P(SAG12)-IPT) on seed germination and the response to a water-deficit stress was studied in tobacco (Nicotiana tabacum L.). Cytokinin levels, sugar content and composition of the leaf strata within the canopy of wild-type and P(SAG12)-IPT plants confirmed the reported altered source-sink relations. No measurable difference in sugar and pigment content of discs harvested from apical and basal leaves was evident 72 h after incubation with (+)-ABA or in darkness, indicating that expression of the transgene was not restricted to senescing leaves. No difference in quantum efficiency, photosynthetic activity, accumulation of ABA, and stomatal conductance was apparent in apical, middle and basal leaves of either wild-type or P(SAG12)-IPT plants after imposition of a mild water stress. However, compared to wild-type plants, P(SAG12)-IPT plants were slower to adjust biomass allocation. A stress-induced increase in root:shoot ratio and specific leaf area (SLA) occurred more rapidly in wild-type than in P(SAG12)-IPT plants reflecting delayed remobilization of leaf reserves to sink organs in the transformant. P(SAG12)-IPT seeds germinated more slowly even though abscisic acid (ABA) content was 50% that of the wild-type seeds confirming cytokinin-induced alterations in reserve remobilization. Thus, senescence is integral to plant growth and development and an increased endogenous cytokinin content impacts source-sink relations to delay ontogenic transitions wherein senescence in a necessary process.

A rapid methodology for the simultaneous analysis of a large number of cytokinins is presented. The cross-reactivity of a mixture of polyclonal antibodies against zeatin riboside and isopentenyladenosine was exploited in a protocol that can be used for immunoaffinity purification of 23 additional cytokinins. Ligands include the cytokinin bases zeatin, dihydrozeatin, isopentenyladenine, benzyl-adenine and kinetin, and their corresponding nucleoside, nucleoside-5'-monophosphate, and 9-glucoside derivatives, as well as cis-zeatin, cis-zeatin riboside, the 2-methylthiol derivatives of isopentenyladenosine and zeatin riboside, and benzyl-adenine-3-glucoside. Mixtures of cytokinins could be retained with high recoveries of all the components. Immunoaffinity purification of extracts of Arabidopsis thaliana (L.) Heynh. and Solarium tuberosum L. gave fractions clean enough, as verified by gas chromatographymass spectrometry (GC-MS), to allow analysis of endogenous cytokinins using a single high-performance liquid chromatography (HPLC) step with on-line UV-spectrum detection. The detection limit was 4-6 pmol. The procedure described forms a routine assaying technique that is faster and simpler, yet yields better qualitative and quantitative information than the commonly used procedure of immunoassaying of HPLC fractions.

A compound was isolated from potato (Solanum tuberosum 1. cv Bintje) tuber sprouts by immunoaffinity chromatography with antibodies against the cytokinins zeatin riboside and isopentenyladenosine. Analysis by ultraviolet spectroscopy and gas chromatography-mass spectrometry of derivatives identified the compound as a 9-glucoside of 6-[(Z)-4-hydroxy-3-methyl-2-butenylaminolpurine (cis-zeatin). Kglucosides have often been reported as metabolites of other cytokinins, but to our knowledge, they have never before been found for cis-zeatin. The finding gives proof that cis-zeatin, a modified base in tRNA, also exists as a free substance in plants, since the glucoside, unlike other tRNA-free cis-zeatins described earlier by others, cannot arise by enzymatic degradation of tRNA during plant extraction.

A Pantoea agglomerans isolate from barley seeds, selected for inducing growth promotion in tomato test plants, was found to excrete several hormones of the cytokinin class into its culture medium. In addition to isopentenyladenine, isopentenyladenosine and the 2-methylthiol derivates of these, an unknown compound with affinity to anticytokinin antibodies was also isolated. Mass spectroscopy indicated the structure of this to be a deoxyisopentenyladenosine. The structure of 9-(5 0 -deoxy-b-D-ribofuranosyl)-6-(3-methyl-2-butenylamino)purine was verified after synthesis of standards and analysis with GC-MS. The synthesized 5 0 -deoxyisopentenyl-adenosine showed activity in the Amaranthus bioassay, specific for cytokinins. To our knowledge this is the first report of a naturally occurring cytokinin containing 5 0 -deoxyribose.

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