Cytokinins act synergistically with heat acclimation to enhance rice thermotolerance affecting hormonal dynamics, gene expression and volatile emission.

Heat stress is a frequent environmental constraint. Phytohormones can significantly affect plant thermotolerance. This study compares the effects of exogenous cytokinin meta-topolin-9-(tetrahydropyran-2-yl)purine (mT9THP) on rice (Oryza sativa) under control conditions, after acclimation by moderate temperature (A; 37 °C, 2h), heat stress (HS; 45 °C, 6h) and their combination (AHS). mT9THP is a stable cytokinin derivative that releases active meta-topolin gradually, preventing the rapid deactivation reported after exogenous cytokinin application. Under control conditions, mT9THP negatively affected jasmonic acid in leaves and abscisic and salicylic acids in crowns (meristematic tissue crucial for tillering). Exogenous cytokinin stimulated the emission of volatile organic compounds (VOC), especially 2,3-butanediol. Acclimation upregulated trans-zeatin, expression of stress- and hormone-related genes, and VOC emission. The combination of acclimation and mT9THP promoted the expression of stress markers and antioxidant enzymes and moderately increased VOC emission, including 2-ethylhexyl salicylate or furanones. AHS and HS responses shared some common features, namely, increase of ethylene precursor aminocyclopropane-1-carboxylic acid (ACC), cis-zeatin and cytokinin methylthio derivatives, as well as the expression of heat shock proteins, alternative oxidases, and superoxide dismutases. AHS specifically induced jasmonic acid and auxin indole-3-acetic acid levels, diacylglycerolipids with fewer double bonds, and VOC emissions [e.g., acetamide, lipoxygenase (LOX)-derived volatiles]. Under direct HS, exogenous cytokinin mimicked some positive acclimation effects. The combination of mT9THP and AHS had the strongest thermo-protective effect, including a strong stimulation of VOC emissions (including LOX-derived ones). These results demonstrate for the first time the crucial contribution of volatiles to the beneficial effects of cytokinin and AHS on rice thermotolerance.

Abscisic Acid and Cytokinins Are Not Involved in the Regulation of Stomatal Conductance of Scots Pine Saplings during Post-Drought Recovery.

Delayed or incomplete recovery of gas exchange after water stress relief limits assimilation in the post-drought period and can thus negatively affect the processes of post-drought recovery. Abscisic acid (ABA) accumulation and antagonistic action between ABA and cytokinins (CKs) play an important role in regulation of stomatal conductance under water deficit. Specifically, in pine species, sustained ABA accumulation is thought to be the main cause of delayed post-drought gas exchange recovery, although the role of CKs is not yet known. Therefore, we aimed to study the effects of ABA and CKs on recovery of stomatal conductance in greenhouse-grown 3-year-old Scots pine saplings recovering from water stress. We analysed both changes in endogenous ABA and CK contents and the effects of treatment with exogenous CK on stomatal conductance. Drought stress suppressed stomatal conductance, and post-drought stomatal conductance remained suppressed for 2 weeks after plant rewatering. ABA accumulated during water stress, but ABA levels decreased rapidly after rewatering. Additionally, trans-zeatin/ABA and isopentenyladenine/ABA ratios, which were decreased in water-stressed plants, recovered rapidly in rewatered plants. Spraying plants with 6-benzylaminopurine (0.1-100 µM) did not influence recovery of either stomatal conductance or needle water status. It can be concluded that the delayed recovery of stomatal conductance in Scots pine needles was not due to sustained ABA accumulation or a sustained decrease in the CK/ABA ratio, and CK supplementation was unable to overcome this delayed recovery.

Effect of ascorbate and hydrogen peroxide on hormone and metabolite levels during post-germination growth in wheat.

The modulation of hormone and metabolite levels by ascorbate (ASA) and hydrogen peroxide (H2 O2 ) was compared during post-germination growth in shoots of wheat. Treatment with ASA resulted in a greater reduction of growth than the addition of H2 O2 . ASA also had a larger effect on the redox state of the shoot tissues as shown by the higher ASA and glutathione (GSH) levels, lower glutathione disulfide (GSSG) content and GSSG/GSH ratio compared to the H2 O2 treatment. Apart from common responses (i.e., increase of cis-zeatin and its O-glucosides), the contents of several compounds related to cytokinin (CK) and abscisic acid (ABA) metabolism were greater after ASA application. These differences in the redox state and hormone metabolism following the two treatments may be responsible for their distinct influence on various metabolic pathways. Namely, the glycolysis and citrate cycle were inhibited by ASA and they were not affected by H2 O2 , while the amino acid metabolism was induced by ASA and repressed by H2 O2 based on the changes in the level of the related carbohydrates, organic and amino acids. The first two pathways produce reducing power, while the last one needs it; therefore ASA, as a reductant may suppress and induce them, respectively. H2 O2 as an oxidant had different effect, namely it did not alter glycolysis and citrate cycle, and inhibited the formation of amino acids.

What determines root-sprouting ability: Injury or phytohormones?

PREMISE: Root-sprouting (RS) is an evolutionarily independent alternative to axillary stem branching for a plant to attain its architecture. Root-sprouting plants are better adapted to disturbance than non-RS plants, and their vigor is frequently boosted by biomass removal. Nevertheless, RS plants are rarer than plants that are not root-sprouters, possibly because they must overcome developmental barriers such as intrinsic phytohormonal balance or because RS ability is conditioned by injury to the plant body. The objective of this study was to identify whether phytohormones or injury enable RS. METHODS: In a greenhouse experiment, growth variables, root respiration, and phytohormones were analyzed in two closely related clonal herbs that differ in RS ability (spontaneously RS Inula britannica and rhizomatous non-RS I. salicina) with and without severe biomass removal. RESULTS: As previously reported, I. britannica is a root-sprouter, but injury did not boost its RS ability. Root respiration did not differ between the two species and decreased continuously with time irrespectively of injury, but their phytohormone profiles differed significantly. In RS species, the auxins-to-cytokinins ratio was low, and injury further decreased it. CONCLUSIONS: This first attempt to test drivers behind different plant growth forms suggests that intrinsic phytohormone regulation, especially the auxins-to-cytokinins ratio, might be behind RS ability. Injury, causing a phytohormonal imbalance, seems to be less important in spontaneously RS species than expected for RS species in general.

Light Quality and Intensity Modulate Cold Acclimation in Arabidopsis.

Plant survival in temperate zones requires efficient cold acclimation, which is strongly affected by light and temperature signal crosstalk, which converge in modulation of hormonal responses. Cold under low light conditions affected Arabidopsis responses predominantly in apices, possibly because energy supplies were too limited for requirements of these meristematic tissues, despite a relatively high steady-state quantum yield. Comparing cold responses at optimal light intensity and low light, we found activation of similar defence mechanisms-apart from CBF1-3 and CRF3-4 pathways, also transient stimulation of cytokinin type-A response regulators, accompanied by fast transient increase of trans-zeatin in roots. Upregulated expression of components of strigolactone (and karrikin) signalling pathway indicated involvement of these phytohormones in cold responses. Impaired response of phyA, phyB, cry1 and cry2 mutants reflected participation of these photoreceptors in acquiring freezing tolerance (especially cryptochrome CRY1 at optimal light intensity and phytochrome PHYA at low light). Efficient cold acclimation at optimal light was associated with upregulation of trans-zeatin in leaves and roots, while at low light, cytokinin (except cis-zeatin) content remained diminished. Cold stresses induced elevation of jasmonic acid and salicylic acid (in roots). Low light at optimal conditions resulted in strong suppression of cytokinins, jasmonic and salicylic acid.

Heat Stress Targeting Individual Organs Reveals the Central Role of Roots and Crowns in Rice Stress Responses.

Inter-organ communication and the heat stress (HS; 45°C, 6 h) responses of organs exposed and not directly exposed to HS were evaluated in rice (Oryza sativa) by comparing the impact of HS applied either to whole plants, or only to shoots or roots. Whole-plant HS reduced photosynthetic activity (F v /F m and QY_Lss ), but this effect was alleviated by prior acclimation (37°C, 2 h). Dynamics of HSFA2d, HSP90.2, HSP90.3, and SIG5 expression revealed high protection of crowns and roots. Additionally, HSP26.2 was strongly expressed in leaves. Whole-plant HS increased levels of jasmonic acid (JA) and cytokinin cis-zeatin in leaves, while up-regulating auxin indole-3-acetic acid and down-regulating trans-zeatin in leaves and crowns. Ascorbate peroxidase activity and expression of alternative oxidases (AOX) increased in leaves and crowns. HS targeted to leaves elevated levels of JA in roots, cis-zeatin in crowns, and ascorbate peroxidase activity in crowns and roots. HS targeted to roots increased levels of abscisic acid and auxin in leaves and crowns, cis-zeatin in leaves, and JA in crowns, while reducing trans-zeatin levels. The weaker protection of leaves reflects the growth strategy of rice. HS treatment of individual organs induced changes in phytohormone levels and antioxidant enzyme activity in non-exposed organs, in order to enhance plant stress tolerance.

Heat Acclimation and Inhibition of Cytokinin Degradation Positively Affect Heat Stress Tolerance of Arabidopsis.

In order to pinpoint phytohormone changes associated with enhanced heat stress tolerance, the complex phytohormone profiles [cytokinins, auxin, abscisic acid (ABA), jasmonic acid (JA), salicylic acid and ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC)] were compared in Arabidopsis thaliana after direct heat shock (45°C, 3 h) and in heat-stressed pre-acclimated plants (1 h at 37°C followed by 2 h at optimal temperature 20°C). Organ-specific responses were followed in shoot apices, leaves, and roots immediately after heat shock and after 24-h recovery at 20°C. The stress strength was evaluated via membrane ion leakage and the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) and antioxidant enzymes [superoxide dismutases, guaiacol peroxidases (POD), catalases, ascorbate peroxidases (APX)]. Heat acclimation diminished negative effects of heat stress, especially in apices and roots, no significant differences being observed in leaves. Low NOX1-3 activities indicated diminished production of reactive oxygen species. Higher activity of APX, POD1, and the occurrence of POD3-4 reflected acclimation-stimulated readiness of the antioxidant system. Acclimation diminished heat shock-induced changes of ABA, JA, cytokinin, and auxin levels in apices. Excess of ABA catabolites suggested an early stress response. The strong up-regulation of ABA and ACC in roots indicated defense boost in roots of acclimated plants compared to the non-acclimated ones. To evaluate the possibility to enhance stress tolerance by cytokinin pool modulation, INCYDE-F, an inhibitor of cytokinin oxidase/dehydrogenase, was applied. As cytokinin effects on stress tolerance may depend on timing of their regulation, INCYDE was applied at several time-points. In combination with acclimation, INCYDE treatment had a slight positive effect on heat stress tolerance, mainly when applied after 2-h period of the optimal temperature. INCYDE increased contents of cytokinins trans-zeatin and cis-zeatin in roots and auxin in all tissues after heat shock. INCYDE also helped to suppress the content of ABA in leaves, and ethylene in apices and roots. INCYDE application to non-acclimated plants (applied before or after heat shock) strengthened negative stress effects, probably by delaying of the repair processes. In conclusion, pre-treatment with moderately elevated temperature enhanced heat stress tolerance and accelerated recovery after stress. Inhibition of cytokinin degradation by INCYDE slightly improved recovery of acclimated plants.

Light Regulates the Cytokinin-Dependent Cold Stress Responses in Arabidopsis.

To elucidate the effect of light intensity on the cold response (5°C; 7 days) in Arabidopsis thaliana, we compared the following parameters under standard light (150 µmol m-2 s-1), low light (20 µmol m-2 s-1), and dark conditions: membrane damage, photosynthetic parameters, cytokinin oxidase/dehydrogenase (CKX) activity, phytohormone levels, and transcription of selected stress- and hormone-related genes and proteome. The impact of cytokinins (CKs), hormones directly interacting with the light signaling pathway, on cold responses was evaluated using transformants overexpressing CK biosynthetic gene isopentenyl transferase (DEX:IPT) or CK degradation gene HvCKX2 (DEX:CKX) under a dexamethasone-inducible promoter. In wild-type plants, cold treatment under light conditions caused down-regulation of CKs (in shoots) and auxin, while abscisic acid (ABA), jasmonates, and salicylic acid (SA) were up-regulated, especially under low light. Cold treatment in the dark strongly suppressed all phytohormones, except ABA. DEX:IPT plants showed enhanced stress tolerance associated with elevated CK and SA levels in shoots and auxin in apices. Contrarily, DEX:CKX plants had weaker stress tolerance accompanied by lowered levels of CKs and auxins. Nevertheless, cold substantially diminished the impact from the inserted genes. Cold stress in dark minimized differences among the genotypes. Cold treatments in light strongly up-regulated stress marker genes RD29A, especially in roots, and CBF1-3 in shoots. Under control conditions, their levels were higher in DEX:CKX plants, but after 7-day stress, DEX:IPT plants exhibited the highest transcription. Transcription of genes related to CK metabolism and signaling showed a tendency to re-establish, at least partially, CK homeostasis in both transformants. Up-regulation of strigolactone-related genes in apices and leaves indicated their role in suppressing shoot growth. The analysis of leaf proteome revealed over 20,000 peptides, representing 3,800 proteins and 2,212 protein families (data available via ProteomeXchange, identifier PXD020480). Cold stress induced proteins involved in ABA and jasmonate metabolism, antioxidant enzymes, and enzymes of flavonoid and glucosinolate biosynthesis. DEX:IPT plants up-regulated phospholipase D and MAP-kinase 4. Cold stress response at the proteome level was similar in all genotypes under optimal light intensity, differing significantly under low light. The data characterized the decisive effect of light-CK cross-talk in the regulation of cold stress responses.

Hormonal Responses to Plasmodiophora brassicae Infection in Brassica napus Cultivars Differing in Their Pathogen Resistance.

Hormonal dynamics after Plasmodiophora brassicae infection were compared in two Brassica napus cultivars-more resistant SY Alister and more sensitive Hornet, in order to elucidate responses associated with efficient defense. Both cultivars responded to infection by the early transient elevation of active cytokinins (predominantly cis-zeatin) and auxin indole-3-acetic acid (IAA) in leaves and roots, which was longer in Hornet. Moderate IAA levels in Hornet roots coincided with a high expression of biosynthetic gene nitrilase NIT1 (contrary to TAA1, YUC8, YUC9). Alister had a higher basal level of salicylic acid (SA), and it stimulated its production (via the expression of isochorismate synthase (ICS1)) in roots earlier than Hornet. Gall formation stimulated cytokinin, auxin, and SA levels-with a maximum 22 days after inoculation (dai). SA marker gene PR1 expression was the most profound at the time point where gall formation began, in leaves, roots, and especially in galls. Jasmonic acid (JA) was higher in Hornet than in Alister during the whole experiment. To investigate SA and JA function, SA was applied before infection, and twice (before infection and 15 dai), and JA at 15 dai. Double SA application diminished gall formation in Alister, and JA promoted gall formation in both cultivars. Activation of SA/JA pathways reflects the main differences in clubroot resistance.

Cytokinins: Their Impact on Molecular and Growth Responses to Drought Stress and Recovery in Arabidopsis.

Our phenotyping and hormonal study has characterized the role of cytokinins (CK) in the drought and recovery responses of Arabidopsis thaliana. CK down-regulation was achieved by overexpression of the gene for CK deactivating enzyme cytokinin oxidase/dehydrogenase (CKX): constitutive (35S:CKX) or at the stress onset using a dexamethasone-inducible pOp/LhGR promoter (DEX:CKX). The 35S:CKX plants exhibited slow ontogenesis and higher expression levels of stress-associated genes, e.g., AtP5CS1, already at well-watered conditions. CK down-regulation resulted during drought in higher stress tolerance (indicated by relatively low up-regulation of the expression of drought stress marker gene AtRD29B) accompanied with lower leaf water loss. Nevertheless, these plants exhibited slow and delayed recovery after re-watering. CK levels were increased at the stress onset by stimulation of the expression of CK biosynthetic gene isopentenyl transferase (ipt) (DEX:IPT) or by application of exogenous CK meta-topolin. After water withdrawal, long-term CK elevation resulted in higher water loss in comparison with CKX transformants as well as with plants overexpressing ipt driven by senescence-inducible SAG12 promoter (SAG:IPT), which gradually enhanced CKs during the stress progression. In all cases, CK up-regulation resulted in fast and more vigorous recovery. All drought-stressed plants exhibited growth suppression associated with elevation of abscisic acid and decrease of auxins and active CKs (with the exception of SAG:IPT plants). Apart from the ipt overexpressers, also increase of jasmonic and salicylic acid was found.

Hormonal dynamics during salt stress responses of salt-sensitive Arabidopsis thaliana and salt-tolerant Thellungiella salsuginea.

Salt stress responses in salt-sensitive Arabidopsis thaliana (2-150mM NaCl) and the closely related salt-tolerant Thellungiella salsuginea (Eutrema halophila, 150-350mM NaCl) were compared to identify hormonal and transcriptomic changes associated with enhanced stress tolerance. Phytohormone levels, expression of selected genes, membrane stability, and Na+ and K+ concentrations were measured in shoot apices, leaves, and roots. Thellungiella exhibited higher salt stress tolerance associated with elevated basal levels of abscisic acid and jasmonic acid, and lower levels of active cytokinins (excluding cis-zeatin) in shoot apices. Analysis of the dynamics of the early salt stress response (15min to 24h) revealed that the halophyte response was faster and stronger. Very mild stress, in our hydropony arrangement 2-25mM NaCl, affected the transcription of genes involved in cytokinin metabolism (AtIPTs, AtCKXs). Mild stress induced in Arabidopsis (50mM) stress responses only in shoot apices, while in Thellungiella (150mM) across the whole plant. Arabidopsis exhibited in hydropony evidence of severe stress above 75mM NaCl and died in 150mM, whereas the halophyte only became severely stressed above 225mM. The responses of individual phytohormones (cytokinins, auxin, abscisic acid, jasmonic acid, salicylic acid and their metabolites) to salinity are discussed.

ZnO nanoparticle effects on hormonal pools in Arabidopsis thaliana.

At present, nanoparticles have been more and more used in a wide range of areas. However, very little is known about the mechanisms of their impact on plants, as both positive and negative effects have been reported. As plant interactions with the environment are mediated by plant hormones, complex phytohormone analysis has been performed in order to characterize the effect of ZnO nanoparticles (mean size 30nm, concentration range 0.16-100mgL-1) on Arabidopsis thaliana plants. Taking into account that plant hormones exhibit high tissue-specificity as well as an intensive cross-talk in the regulation of growth and development as well as defense, plant responses were followed by determination of the content of five main phytohormones (cytokinins, auxins, abscisic acid, salicylic acid and jasmonic acid) in apices, leaves and roots. Increasing nanoparticle concentration was associated with gradually suppressed biosynthesis of the growth promoting hormones cytokinins and auxins in shoot apical meristems (apices). In contrast, cis-zeatin, a cytokinin associated with stress responses, was elevated by 280% and 590% upon exposure to nanoparticle concentrations 20 and 100mgL-1, respectively, in roots. Higher ZnO nanoparticle doses resulted in up-regulation of the stress hormone abscisic acid, mainly in apices and leaves. In case of salicylic acid, stimulation was found in leaves and roots. The other stress hormone jasmonic acid (as well as its active metabolite jasmonate isoleucine) was suppressed at the presence of nanoparticles. The earliest response to nanoparticles, associated with down-regulation of growth as well as of cytokinins and auxins, was observed in apices. At higher dose, up-regulation of abscisic acid, was detected. This increase, together with elevation of the other stress hormone - salicylic acid, indicates that plants sense nanoparticles as severe stress. Gradual accumulation of cis-zeatin in roots may contribute to relatively higher stress resistance of this tissue.

The impact of heat stress targeting on the hormonal and transcriptomic response in Arabidopsis.

Targeting of the heat stress (HS, 40°C) to shoots, roots or whole plants substantially affects Arabidopsis physiological responses. Effective stress targeting was proved by determination of the expression of HS markers, HsfA2 and HSA32, which were quickly stimulated in the targeted organ(s), but remained low in non-stressed tissues for at least 2h. When shoots or whole plants were subjected to HS, a transient decrease in abscisic acid, accompanied by a small increase in active cytokinin levels, was observed in leaves, consistent with stimulation of transpiration, the main cooling mechanism in leaves. HS application targeted to part of plant resulted in a rapid stimulation of expression of components of cytokinin signaling pathway (especially of receptor genes) in the non-exposed tissues, which indicated fast inter-organ communication. Under all HS treatments, shoot apices responded by transient elevation of active cytokinin contents and stimulation of transcription of genes involved in photosynthesis and carbohydrate metabolism. Duration of this stimulation was negatively correlated with stress strength. The impact of targeted HS on the expression of 63 selected genes, including those coding regulatory 14-3-3 proteins, was compared. Stimulation of GRF9 (GRF14µ) in stressed organs after 2-6h may be associated with plant stress adaptation.

Enhanced drought and heat stress tolerance of tobacco plants with ectopically enhanced cytokinin oxidase/dehydrogenase gene expression.

Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms.

A somaclonal line SE7 of finger millet (Eleusine coracana) exhibits modified cytokinin homeostasis and increased grain yield.

The SE7 somaclonal line of finger millet (Eleusine coracana) achieved increased grain yield in field trials that apparently resulted from a higher number of inflorescences and seeds per plant, compared with the wild type. Levels of endogenous cytokinins, especially those of highly physiologically active iso-pentenyl adenine, were increased during early inflorescence development in SE7 plants. Transcript levels of cytokinin-degrading enzymes but not of a cytokinin-synthesizing enzyme were also decreased in young leaves, seedlings, and initiating inflorescences of SE7. These data suggest that attenuated degradation of cytokinins in SE7 inflorescences leads to higher cytokinin levels that stimulate meristem activity and result in production of more inflorescences. Gene expression was compared between SE7 and wild-type young inflorescences using the barley 12K cDNA array. The largest fraction of up-regulated genes in SE7 was related to transcription, translation, and cell proliferation, cell wall assembly/biosynthesis, and to growth regulation of young and meristematic tissues including floral formation. Other up-regulated genes were associated with protein and lipid degradation and mitochondrial energy production. Down-regulated genes were related to pathogen defence and stress response, primary metabolism, glycolysis, and the C:N balance. The results indicate a prolonged proliferation phase in SE7 young inflorescences characterized by up-regulated protein synthesis, cytokinesis, floral formation, and energy production. In contrast, wild-type inflorescences are similar to a more differentiated status characterized by regulated protein degradation, cell elongation, and defence/stress responses. It is concluded that attenuated degradation of cytokinins in SE7 inflorescences leads to higher cytokinin levels, which stimulate meristem activity, inflorescence formation, and seed set.

Complex phytohormone responses during the cold acclimation of two wheat cultivars differing in cold tolerance, winter Samanta and spring Sandra.

Hormonal changes accompanying the cold stress (4°C) response that are related to the level of frost tolerance (FT; measured as LT50) and the content of the most abundant dehydrin, WCS120, were compared in the leaves and crowns of the winter wheat (Triticum aestivum L.) cv. Samanta and the spring wheat cv. Sandra. The characteristic feature of the alarm phase (1 day) response was a rapid elevation of abscisic acid (ABA) and an increase of protective proteins (dehydrin WCS120). This response was faster and stronger in winter wheat, where it coincided with the downregulation of bioactive cytokinins and auxin as well as enhanced deactivation of gibberellins, indicating rapid suppression of growth. Next, the ethylene precursor aminocyclopropane carboxylic acid was quickly upregulated. After 3-7 days of cold exposure, plant adaptation to the low temperature was correlated with a decrease in ABA and elevation of growth-promoting hormones (cytokinins, auxin and gibberellins). The content of other stress hormones, i.e., salicylic acid and jasmonic acid, also began to increase. After prolonged cold exposure (21 days), a resistance phase occurred. The winter cultivar exhibited substantially enhanced FT, which was associated with a decline in bioactive cytokinins and auxin. The inability of the spring cultivar to further increase its FT was correlated with maintenance of a relatively higher cytokinin and auxin content, which was achieved during the acclimation period.

Distribution, biological activities, metabolism, and the conceivable function of cis-zeatin-type cytokinins in plants.

Cytokinins (CKs) are plant hormones affecting numerous developmental processes. Zeatin and its derivatives are the most important group of isoprenoid CKs. Zeatin occurs as two isomers: while trans-zeatin (transZ) was found to be a bioactive substance, cis-zeatin (cisZ) was reported to have a weak biological impact. Even though cisZ derivatives are abundant in various plant materials their biological role is still unknown. The comprehensive screen of land plants presented here suggests that cisZ-type CKs occur ubiquitously in the plant kingdom but their abundance might correlate with a strategy of life rather than with evolutionary complexity. Changing levels of transZ and cisZ during Arabidopsis ontogenesis show that levels of the two zeatin isomers can differ significantly during the life span of the plant, with cisZ-type CKs prevalent in the developmental stages associated with limited growth. A survey of the bioassays employed illustrates mild activity of cisZ and its derivatives. No cis↔trans isomerization, which would account for the effects of cisZ, was observed in tobacco cells and oat leaves. Differences in uptake between the two isomers resulting in distinct bioactivity have not been detected. In contrast, cisZ and transZ have a different metabolic fate in oat and tobacco. Analysis of a CK-degrading enzyme, cytokinin oxidase/dehydrogenase (CKX), reveals that Arabidopsis possesses two isoforms, AtCKX1 expressed in stages of active growth, and AtCKX7, both of which have the highest affinity for the cisZ isomer. Based on the present results, the conceivable function of cisZ-type CKs as delicate regulators of CK responses in plants under growth-limiting conditions is hypothesized.

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.

Efficiency of different methods of extraction and purification of cytokinins.

The increasing use of advanced methods, such as mass spectrometry, for the determination of cytokinins has raised special requirements for the extraction and purification of this class of plant hormones. Extraction of Arabidopsis thaliana plants with three different solvents, [80% (v/v) MeOH, Bieleski's MCF-7, and modified Bieleski's] provided similar yields of most analyzed cytokinins determined by high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). However, the extraction with a modified Bieleski's solvent (MeOH-HCO2H-H2O [15:1:4, v/v/v]) gave the highest responses of deuterated cytokinins (used as test compounds) in plant extracts as compared to the responses of pure deuterated standards (relative internal standard response, RISR). Purification of cytokinins using Oasis MCX sorbent with reversed-phase and cation-exchange characteristics, in comparison to the DEAE Sephadex RP-C18 method, provided higher levels of zeatin riboside monophosphate and similar levels of cytokinin bases, ribosides and glucosides. Using this method the content of UV-absorbing contaminates was decreased by about 90% and the RISR values of all tested cytokinin standards but riboside monophosphates were increased about two-fold. The former method provided preparations more suitable for HPLC/MS/MS analysis with respect to simplicity and sample purity.