Tobacco leaves and roots differ in the expression of proline metabolism-related genes in the course of drought stress and subsequent recovery.

In plants, members of gene families differ in function and mode of regulation. Fine-tuning of the expression of individual genes helps plants to cope with a variable environment. Genes encoding proline dehydrogenase (PDH), the key enzyme in proline degradation, and the proline biosynthetic enzyme, Δ(1)-pyrroline-5-carboxylate synthetase (P5CS), play an important role in responses to osmotic and drought stresses. We compared the expression patterns of three PDH and two putative P5CS genes during drought stress progression and subsequent recovery. Whereas the NtPDH1 gene was affected little by dehydration or rehydration, the NtPDH2 gene responded rapidly to both conditions, and was down-regulated under drought. The CIG1 gene, encoding cytokinin-inducible PDH, exhibited an intermediate transcription pattern. Whereas P5CS B was not affected by the stress conditions, the P5CS A gene was highly up-regulated during drought stress. CIG1 and NtPDH1 transcription was not activated, and P5CS A was only partially reduced in leaves within 24-h after rehydration, a re-watering period sufficient for large physiological changes to occur. The lack of activation of tobacco PDH genes and incomplete reduction of the P5CS A gene in leaves within 24-h of rehydration may reflect the need for the protection of plants to potential subsequent stresses. The data indicate that recovery is a specific physiological process following different patterns in leaves and roots.

Comparison of hormonal responses to heat, drought and combined stress in tobacco plants with elevated proline content.

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.

The role of cytokinins in responses to water deficit in tobacco plants over-expressing trans-zeatin O-glucosyltransferase gene under 35S or SAG12 promoters.

The impact of water deficit progression on cytokinin (CK), auxin and abscisic acid (ABA) levels was followed in upper, middle and lower leaves and roots of Nicotiana tabacum L. cv. Wisconsin 38 plants [wild type (WT)]. ABA content was strongly increased during drought stress, especially in upper leaves. In plants with a uniformly elevated total CK content, expressing constitutively the trans-zeatin O-glucosyltransferase gene (35S::ZOG1), a delay in the increase of ABA was observed; later on, ABA levels were comparable with those of WT. As drought progressed, the bioactive CK content in leaves gradually decreased, being maintained longer in the upper leaves of all tested genotypes. Under severe stress (11 d dehydration), a large stimulation of cytokinin oxidase/dehydrogenase (CKX) activity was monitored in lower leaves, which correlated well with the decrease in bioactive CK levels. This suggests that a gradient of bioactive CKs in favour of upper leaves is established during drought stress, which might be beneficial for the preferential protection of these leaves. During drought, significant accumulation of CKs occurred in roots, partially because of decreased CKX activity. Simultaneously, auxin increased in roots and lower leaves. This indicates that both CKs and auxin play a role in root response to severe drought, which involves the stimulation of primary root growth and branching inhibition.