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.

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.

The Transcriptomic Response of Arabidopsis thaliana to Zinc Oxide: A Comparison of the Impact of Nanoparticle, Bulk, and Ionic Zinc.

The impact of nanosize was evaluated by comparing of the transcriptomic response of Arabidopsis thaliana roots to ZnO nanoparticles (nZnO), bulk ZnO, and ionic Zn(2+). Microarray analyses revealed 416 up- and 961 down-regulated transcripts (expression difference >2-fold, p [FDR] < 0.01) after a seven-day treatment with nZnO (average particle size 20 nm, concentration 4 mg L(-1)). Exposure to bulk ZnO resulted in 816 up- and 2179 down-regulated transcripts. The most dramatic changes (1711 transcripts up- and 3242 down-regulated) were caused by the presence of ionic Zn(2+) (applied as ZnSO4.7H20 at a concentration of 14.14 mg L(-1), corresponding to the amount of Zn contained in 4 mg L(-1) ZnO). Genes involved in stress response (e.g., to salt, osmotic stress or water deprivation) were the most relatively abundant group of gene transcripts up-regulated by all three Zn treatments while genes involved in cell organization and biogenesis (e.g., tubulins, arabinogalactan proteins) and DNA or RNA metabolism (e.g., histones) were the most relatively abundant groups of down-regulated transcripts. The similarity of the transcription profiles and the increasing number of changed transcripts correlating with the increased concentration of Zn(2+) in cultivation medium indicated that released Zn(2+) may substantially contribute to the toxic effect of nZnO because particle size has not demonstrated a decisive role.

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.

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.

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.