In order to test the possibility of improving tolerance to heat and drought (alone and in combination) by elevation of the osmoprotectant proline (Pro) content, stress responses were compared in tobacco plants constitutively over-expressing a gene for the Pro biosynthetic enzyme Δ(2)-pyrroline-5-carboxylate synthetase (P5CSF129A; EC 2.7.2.11/1.2.1.41) and in the corresponding wild-type. Significantly enhanced Pro production in the transformant coincided with a more negative leaf osmotic potential (both at control conditions and following stress) and enhanced production of protective xanthophyll cycle pigments. Heat stress (40 °C) caused a transient increase in the level of bioactive cytokinins, predominantly N(6)-(2-isopentenyl)adenosine, accompanied by down-regulation of the activity of the main cytokinin degrading enzyme cytokinin oxidase/dehydrogenase (EC 1.4.3.18/1.5.99.12). No significant differences were found between the tested genotypes. In parallel, a transient decrease of abscisic acid was observed. Following drought stress, bioactive cytokinin levels decreased in the whole plants, remaining relatively higher in preferentially protected upper leaves and in roots. Cytokinin suppression was less pronounced in Pro transformants. Exposure to heat stress (40 °C for 2h) at the end of 10-d drought period strongly enhanced the severity of the water stress, as indicated by a drop in leaf water potential. The activity of cytokinin oxidase/dehydrogenase was strongly stimulated in upper leaves and roots of stressed plants, coinciding with strong down-regulation of bioactive cytokinins in whole plants. Combined heat and drought stress resulted in a minor decrease in abscisic acid, but only in non-wilty upper leaves. Both stresses as well as their combination were associated with elevation of free auxin, indolylacetic acid, in lower leaves and/or in roots. Auxin increase was dependent on the stress strength. After rehydration, a marked elevation of bioactive cytokinins in leaves was observed. A greater increase in cytokinin content in Pro transformants indicated a mild elevation of their stress tolerance.

Institute of Experimental Botany AS CR Rozvojova 263 165 02 Prague 6 Czech Republic; Department of Biochemistry Charles University Hlavova 2030 8 12843 Prague 2 Czech Republic

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