Altered cytokinin metabolism affects cytokinin, auxin, and abscisic acid contents in leaves and chloroplasts, and chloroplast ultrastructure in transgenic tobacco

Cytokinins (CKs) are involved in the regulation of plant development including plastid differentiation and function. Partial location of CK biosynthetic pathways in plastids suggests the importance of CKs for chloroplast development. The impact of genetically modified CK metabolism on endogenous CK, indole-3-acetic acid, and abscisic acid contents in leaves and isolated intact chloroplasts of Nicotiana tabacum was determined by liquid chromatography/mass spectrometry and two-dimensional high-performance liquid chromatography, and alterations in chloroplast ultrastructure by electron microscopy. Ectopic expression of Sho, a gene encoding a Petunia hybrida isopentenyltransferase, was employed to raise CK levels. The increase in CK levels was lower in chloroplasts than in leaves. CK levels were reduced in leaves of tobacco harbouring a CK oxidase/dehydrogenase gene, AtCKX3. The total CK content also decreased in chloroplasts, but CK phosphate levels were higher than in the wild type. In a transformant overexpressing a maize beta-glucosidase gene, Zm-p60.1, naturally targeted to plastids, a decrease of CK-O-glucosides in chloroplasts was found. In leaves, the changes were not significant. CK-O-glucosides accumulated to very high levels in leaves, but not in chloroplasts, of plants overexpressing a ZOG1 gene, encoding trans-zeatin-O-glucosyltransferase from Phaseolus lunatus. Manipulation of the CK content affected levels of indole-3-acetic and abscisic acid. Chloroplasts of plants constitutively overexpressing Sho displayed ultrastructural alterations including the occasional occurrence of crystalloids and an increased number of plastoglobuli. The other transformants did not exhibit any major differences in chloroplast ultrastructure. The results suggest that plant hormone compartmentation plays an important role in hormone homeostasis and that chloroplasts are rather independent organelles with respect to regulation of CK metabolism.     

Diurnal variation of cytokinin, auxin and abscisic acid levels in tobacco leaves

As many processes are regulated by both light and plant hormones, evaluation of diurnal variations of their levels may contribute to the elucidation of the complex network of light and hormone signal transduction pathways. Diurnal variation of cytokinin, auxin, and abscisic acid levels was tested in tobacco leaves (Nicotiana tabacum L. cv. Wisconsin 38) grown under a 16/8 h photoperiod. The main peak of physiologically active cytokinins (cytokinin bases and ribosides) was found after 9 h of light, i.e. 1 h after the middle of the light period. This peak coincided with the major auxin peak and was closely followed by a minor peak of abscisic acid. Free abscisic acid started to increase at the light/dark transition and reached its maximum 3 h after dark initiation. The content of total cytokinins (mainly N-glucosides, followed by cis-zeatin derivatives and nucleotides) exhibited the main peak after 9 h of light and the minor peak after the transition to darkness. The main, midday peak of active cytokinins was preceded by a period of minimal metabolic conversion of tritiated trans-zeatin (less than 30%). The major cytokinin-degrading enzyme, cytokinin oxidase/dehydrogenase (EC 1.5.99.12), exhibited maximal activity after the dark/light transition and during the diminishing of the midday cytokinin peak. The former peak might be connected with the elimination of the long-distance cytokinin signal. These cytokinin oxidase/dehydrogenase peaks were accompanied by increased activity of beta-glucosidase (EC 3.2.1.21), which might be involved in the hydrolysis of cytokinin O-glucosides and/or in fine-tuning of active cytokinin levels at their midday peak. The achieved data indicate that cytokinin metabolism is tightly regulated by the circadian clock.     

Hormonal control of nitrogen acquisition: roles of auxin, abscisic acid, and cytokinin

Nitrogen is the mineral nutrient that often limits plant growth and development. In response to changes in nitrogen supply, plants display elaborate responses at both physiological and morphological levels to adjust their growth and development. Because higher plants consist of multiple organs with different functions and nutritional requirements, they rely on local and long-distance signalling pathways to coordinate the responses at the whole-plant level. Phytohormones have been considered as signalling substances of such pathways. Amongst phytohormones, abscisic acid, auxin, and cytokinins have been closely linked to nitrogen signalling. Recent evidence has provided some insights into how nitrogen and the phytohormone signals are integrated to bring about changes in physiology and morphology. In this review, the evidence is summarized, mostly focusing on examples related to nitrogen acquisition.     

Interaction of coumarin, gibberellic acid and abscisic acid in the translocation of auxin in bean seedlings

The effects of coumarin on the translocation of 2,4,5-trichlorophenoxyaceticacid (2,4,5-T) in bean (Phaseolus vulgaris L. cv. StringlessGreenpod) seedlings was determined. ¹⁴C-labeled 2,4,5-T wasinjected in the stem tissue at the cotyledonary node along withthe coumarin or the coumarin was added to the nutrient solutionprior to, or at the time of, 2,4,5-T treatment. The amount oftranslocation of radioactive 2,4,5-T to plant parts was thendetermined at various times after treatment. An immediate effectof coumarin was to enhance acropetal 2,4,5-T translocation tothe young shoots. This effect occurred at low 2,4,5-T treatmentlevels and appeared to be specific for 2,4,5-T since sucrosetranslocation was not affected. Prolonged treatment with coumarininhibited acropetal and basipetal 2,4,5-T translocation in amanner similar to prolonged treatment with abscisic acid (ABA).Gibberellin A₃ (GA) reversed the inhibitory effects of coumarinand ABA on 2,4,5-T acropetal translocation. ¹ Journal article 3244 of the Agricultural Experiment Station,Oklahoma State University. (Received December 6, 1976; )     

Jasmonic acid and abscisic acid in shoots,coleoptiles, and roots of wheat seedlings

The endogenous levels of abscisic acid (ABA) and jasmonic acid (JA) were analyzed in wheat seedlings grown in water, a system which in the past has been used to test the effects of these plant growth inhibitors. The levels in different plant parts and in the medium were measured by gas chromatography-mass spectrometry-selected ion monitoring, using [²H₃]ABA and [²H₆]JA as internal standards. In every plant part, JA levels were about 2 orders of magnitude greater than those of ABA. The exudation of JA from roots per seedling was about 14,000-fold greater than that of ABA, although the roots contained only about 170 times more JA than ABA. It is suggested that JA is a possible allelopathic compound.     

Spatio-temporal changes in endogenous abscisic acid contents during etiolated growth and photomorphogenesis in tomato seedlings

The role of abscisic acid (ABA) during early development was investigated in tomato seedlings. The endogenous content of ABA in particular organs was analyzed in seedlings grown in the dark and under blue light. Our results showed that in dark-grown seedlings, the ABA accumulation was maximal in the cotyledons and elongation zone of hypocotyl, whereas under blue-light, the ABA content was distinctly reduced. Our data are consistent with the conclusion that ABA promotes the growth of etiolated seedlings and the results suggest that ABA plays an inhibitory role in de-etiolation and photomorphogenesis in tomato.     

Changes in endogenous abscisic acid and cold hardiness in Actinidia treated with triazole growth retardants

Soil drench of either paclobutrazol or uniconazole (0.4 mg/pot) was applied to plants of Actinidia arguta, Ananasnaja, to determine the effect on endogenous abscisic acid measured in November, January, and March, and the concomittant cold hardiness of the treated plants. Both paclobutrazol and uniconazole treatments significantly increased cold hardiness. Paclobutrazol was more effective in increasing ABA levels compared to uniconazole. Abscisic acid was found to be highest in January, corresponding to deep dormancy, and least in March when plants were undergoing vegetative bud break. Paclobutrazol delayed vegetative bud break by 6.3 days, while uniconazole delayed bud break by 2.9 days.     

Effect of paclobutrazol on abscisic acid levels in wheat seedlings

Contradictory results have been reported for the effects of triazoles on abscisic acid (ABA) levels in plants. Paclobutrazol reduced the height, fresh weight, and ABA levels of wheat seedlings. The magnitude of the inhibitory effect of paclobutrazol on ABA levels was dependent on the length of time after application. ABA levels in plants as determined by gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS) were reduced by 50–60% 2 days after soil application. A further 15% reduction occurred by the seventh day after treatment. ABA level analyses began at the time of treatment with 4-day-old seedlings and continued until 21 days after treatment when only a 20% reduction was detected. These determinations using GC-SIM-MS should increase the understanding of triazole effects on ABA levels.     

Effect of paclobutrazol on abscisic acid levels in wheat seedlings

Contradictory results have been reported for the effects of triazoles on abscisic acid (ABA) levels in plants. Paclobutrazol reduced the height, fresh weight, and ABA levels of wheat seedlings. The magnitude of the inhibitory effect of paclobutrazol on ABA levels was dependent on the length of time after application. ABA levels in plants as determined by gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS) were reduced by 50–60% 2 days after soil application. A further 15% reduction occurred by the seventh day after treatment. ABA level analyses began at the time of treatment with 4-day-old seedlings and continued until 21 days after treatment when only a 20% reduction was detected. These determinations using GC-SIM-MS should increase the understanding of triazole effects on ABA levels.     

Contents of abscisic acid and cytokinins in shoots during dehydration of wheat seedlings

Accumulation of ABA in shoots during 30-min dehydration of wheat seedlings was accompanied by the decline in the content of zeatin nucleotide and the accumulation of zeatin 9-N-glucoside. The total content of zeatin derivatives as well as the content of free base of zeatin remained almost constant. This revised version was published online in July 2006 with corrections to the Cover Date.     

Some growth responses of apple seedlings to abscisic acid and growth stimulators

Antonovka seedlings were obtained from the embryos soaked in abscisic acid (ABA) alone, gibberellin A₄₊₇, benzyladenine or the mixtures of these regulators. The inhibitory effect of ABA on the growth of the seedlings was of temporary duration. The height of the 12 week-old seedlings was dependent only on the degree of seed after-ripening and not on the ABA treatment. The growth stimulators studied were unable to overcome the inhibitory effect of ABA observed in the early stages of seedling growth. The growth stimulators did not substitute, for cold treatment of apple seeds which is the only treatment so far known to overcome the dwarf conditions of the seedlings obtained from non-after-ripened embryos. ABA greatly suppressed the early stages of apple seedling growth (up to 6 weeks) during which they resembled physiological dwarfs. The highest concentrations of ABA (2·0 μg ml^?1) greatly modified the root system of the seedlings, and produced a larger percentage of seedlings with unbranched roots.     

Regulation of o-diphenolase activity by auxin and abscisic acid in germinating wheat embryos

During germination o-diphenolase activity increased several fold ( x 13) in wheat embryos. The enzyme activity and the number of multiple forms wer     

Changes in growth regulator content of radish seedlings, Raphanus sativus, infested with the aphid Myzus persicae

Following 10 days of infestation by the aphid Myzus persicae there was an increase in the amount of growth inhibiting substances and a decrease in cytokinins, gibberellins and auxins in infested as compared with similar uninfested radish seedlings. Even after previously infested seedlings have been freed of aphids for 10 days, differences in the hormone balance remained. The possible relationships between the changes in hormonal balance and the effect of the aphid infestation on growth, translocation and wilting are discussed.     

Stomatal behaviour and water movement through roots of wheat plants treated with abscisic acid

Abstract Experiments with isolated roots of wheat plants suggested that when water uptake rates are low, low concentrations of abscisic acid (ABA) may increase the flux of water into roots. This increase was recorded despite an ABA-stimulated reduction in the hydraulic conductance of the whole root system. Hydraulic conductances were measured under steady-state conditions. A system is described where the stomatal behaviour and water movement through roots of a single intact plant may be concurrently monitored. Experiments with intact plants confirmed that application of ABA could increase the rate of water movement into roots when uptake rates were low. No such increase was observed at high flux rates. Application of ABA to roots caused partial stomatal closure and caused conductance to oscillate around a reduced mean value. An ABA-stimulated increase in the turgor sensitivity of stomata is postulated and the significance of this effect is discussed.     

Monoclonal antibodies for the detection and quantitation of the endogenous plant growth regulator, abscisic acid

Monoclonal antibodies (mAB) have been produced which recognize the physiologically active 2-cis-(S-form of the endogenous plant growth regulator, abscisic acid (ABA). Cross-reaction with the ABA-catabolites, phaseic and dihydrophaseic acid, is negligible, and (R)-ABA, 2-trans-ABA, the ABA-conjugate, ABA-β-D-glucopyranosyl ester, as well as the putative ABA precursor, xanthoxin, are totally unreactive. In addition to being very specific, the mAB exhibit high affinites for 2-cis-(S)-ABA: the K values were 7.9 × 10⁹ l/mol and 3.7 × 10⁹ l/mol for antibodies from two different clones. By mAB-radioimmunoassay (RIA), 4 pg 2-cis-(S)-ABA (99.5% confidence level) can be detected. mAB-RIA can be used to quantitate ABA directly in unprocessed plant extracts.     

Changes in the composition of phospholipids in nuclear subfractions of wheat seedlings treated with gibberellin

Macromolecule transport between the cytoplasm and nucleus occurs through the nuclear pore complexes with the help of soluble transport factors. The receptors of GA are not yet identified, and the molecular mechanism of plant response to GA is not known so far. We compared the content of phospholipids in the soluble nuclear fraction and nuclear membrane of wheat seedlings on the third and fourth days of germination after treatment with GA. It was shown that GA induced differently directed changes in the composition of phospholipids in nuclear subfractions tested. Changes in the composition of the nuclear membrane are supposed to be involved in the control of gene expression by GA.     

Effect of PEG-treatment on the leaf growth response and auxin content in shoots of wheat seedlings

Addition of Polyethylene glycol(PEG) to the nutrient medium of wheatseedlings led to a rapid cessation of leaf growth and shrinkage, measured withahighly sensitive growth sensor, followed by a partial restoration of extensionin the subsequent 40 min. This osmotic shock resulted in IAAaccumulation in the shoots. Leaf extensibility, measured by addition of 5g as a counterweight to an extensiometer, did not change for 20min after the start of the treatment, but then increased andbecametwice as high as the initial value. Changes in leaf extensibility coincidedwitha restoration of leaf growth. Leaf shrinkage, which was observed immediatelyafter addition of the osmoticum, suggests that the initial growth response wasdue to a water deficit, which led to a decrease in cell turgor. Howeverrestoration of growth during water stress could be due to changes in cell wallrheological properties, which could be triggered by IAA accumulation.     

Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought, salt and abscisic acid responses, and abscisic acid biosynthesis

Cytokinins (CKs) regulate plant growth and development via a complex network of CK signaling. Here, we perform functional analyses with CK-deficient plants to provide direct evidence that CKs negatively regulate salt and drought stress signaling. All CK-deficient plants with reduced levels of various CKs exhibited a strong stress-tolerant phenotype that was associated with increased cell membrane integrity and abscisic acid (ABA) hypersensitivity rather than stomatal density and ABA-mediated stomatal closure. Expression of the Arabidopsis thaliana ISOPENTENYL-TRANSFERASE genes involved in the biosynthesis of bioactive CKs and the majority of the Arabidopsis CYTOKININ OXIDASES/DEHYDROGENASES genes was repressed by stress and ABA treatments, leading to a decrease in biologically active CK contents. These results demonstrate a novel mechanism for survival under abiotic stress conditions via the homeostatic regulation of steady state CK levels. Additionally, under normal conditions, although CK deficiency increased the sensitivity of plants to exogenous ABA, it caused a downregulation of key ABA biosynthetic genes, leading to a significant reduction in endogenous ABA levels in CK-deficient plants relative to the wild type. Taken together, this study provides direct evidence that mutual regulation mechanisms exist between the CK and ABA metabolism and signals underlying different processes regulating plant adaptation to stressors as well as plant growth and development.