Expression of a human tRNA isopentenyltransferase in tobacco reveals a developmental role for tRNA isopentenyladenosine.

In addition to their role as plant hormones, cytokinins are also found as structural components in tRNA. Six different tRNA cytokinins have been found in plants, but most other organisms, including humans, have only one-isopentenyladenosine. In an attempt to probe if the different forms have different functionality, we attempted to alter tRNA cytokinin composition by expressing the human tRNA isopentenyltransferase gene (EC 5.1.2.8) in tobacco [Nicotiana tabacum (L.) cv. Wisconsin 38]. The resulting transgenics had ~40% more isopentenyladenosine in tRNA, and an altered phenotype characterised by reduced internode length, increased stem diameter and rigidity, greener leaves, increased axillary bud outgrowth, abnormal flower morphology, and reduced seed viability. The levels of the two other major isoprene adenines of tRNA, cis-zeatin and 2-methyltiolated cis-zeatin, were also increased, but to a lower degree. Nearly all of the increase in isopentenyladenosine was in a single tRNA species. Two quantitatively minor isopentenyladenosine-containing tRNAs had also increased strongly. IPPT: Dimethylallylpyrophosphate.

Effects of senescence-induced alteration in cytokinin metabolism on source-sink relationships and ontogenic and stress-induced transitions in tobacco.

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.

Identification of a tRNA isopentenyltransferase gene from Arabidopsis thaliana.

The tRNA of most organisms contain modified adenines called cytokinins. Situated next to the anticodon, they have been shown to influence translational fidelity and efficiency. The enzyme that synthesizes cytokinins on pre-tRNA, tRNA isopentenyltransferase (EC 2.5.1.8), has been studied in micro-organisms like Escherichia coli and SaccharomYces cerevisiae, and the corresponding genes have been cloned. We here report the first cloning and functional characterization of a homologous gene from a plant, Arabidopsis thaliana. Expression in S. cerevisiae showed that the gene can complement the anti-suppressor phenotype of a mutant that lacks MOD5, the intrinsic tRNA isopentenyltransferase gene. This was accompanied by the reintroduction of isopentenyladenosine in the tRNA. The Arabidopsis gene is constitutively expressed in seedling tissues.