Cytokinin catabolism and cytokinin oxidase

Cytokinin oxidase has been highly purified from mature Zea mays kernels. Adenine has been unambiguously identified, by HPLC co-chromatography, UV a     

Cytokinin oxidase is key enzyme of cytokinin degradation

Cytokinin oxidase (EC 1.5.99.12) is an enzyme that catalyzes the irreversible degradation of cytokinin phytohormones that are extremely necessary for growth, development, and differentiation of plants. Cytokinin oxidase plays an important role in the regulation of quantitative level of cytokinins and their distribution in plant tissues. This review generalizes the available information on the structure, properties, and functional role of this enzyme in plant ontogeny under conditions of normal growth and under the influence of unfavorable environmental factors.     

Cytokinin metabolism and the control of cytokinin activity

The roles of the different cytokinin structures are discussed in relation to our knowledge of their biological activities, endogenous occurrence and metabolism.     

Cytokinin oxidase/dehydrogenase activity as a tool in gibberellic acid/cytokinin cross talk

Changes in endogenous cytokinin (CK) content and cytokinin oxidase/dehydrogenase activity (CKX) in response to gibberellic acid (GA₃) in two pea cultivars with different life span were assessed. The control leaves of cv. Scinado, which developed faster, had higher initial cytokinin content and lower CKX activity, while opposite trend was observed in cv. Manuela with longer life span. Increased CKX and decreased CK content were detected in leaves of cv. Scinado after treatments with 0.5, 1 and 5 μM GA₃. Changes in CK content and CKX activity in GA₃-treated cv. Manuela leaves were reciprocal to those in cv. Scinado. CK content and CKX activity in roots were not significantly influenced by the application of GA₃. The slight repression of CKX activity in some of the root samples was accompanied by increased isopentenyladenine and isopentenyladenine riboside content. Obtained results suggest that CKX was responsible for the changes in endogenous cytokinin pool in GA₃-treated plants and most probably this enzyme represents an important link in GA/cytokinin cross talk.     

Cytokinin oxidase/cytokinin dehydrogenase assay: optimized procedures and applications

1H NMR spectroscopy has been used to assess long-term toxicological effects of a rare earth. Male Wistar rats were administrated orally with La(NO3)3 at doses of 0.1, 0.2, 2.0, 10, and 20 mg/kg body wt, resp., for 3-6 months. Urine was collected at 1, 2, and 3 months and serum samples were taken after 6 months. Numerous low-M(r) metabolites in rats serum and rats urine, including creatinine, citrate, glucose, ketone bodies, trimethylamine N-oxide (TMAO), and various amino acids, were identified on 400- and 500-MHz 1H NMR spectra. La3+-induced renal and liver damage is characterized by an increase in the amounts of the excreted ketone bodies, amino acids, lactate, ethanol, succinate, TMAO, dimethylamine, and taurine and a decrease in citrate, glucose, urea, and allantoin. Information on the molecular basis of the long-term toxicity of La(NO>3)3 was derived from the abnormal patterns of metabolite excretions. An assay of some biochemical indexes and analysis of some enzymes in plasma supported NMR results.     

Cytokinin metabolism and the modulation of cytokinin activity in radish

The metabolism of zeatin, N⁶-(Δ²-isopentenyl) adenine, dihydrozeatin, zeatin-O-glucoside and dihydrozeatin-O-glucoside has been studied using derooted radish seedlings. The metabolites were identified by UV and GC/MS. The patterns of metabolism are compared and provide evidence that the O-glucosyl conjugates may be storage forms of the cytokinins.     

Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals.

An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains. New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein. The purified wheat enzyme is a monomer 60 kDa, its N-terminal amino-acid sequence shows similarity to hypothetical cytokinin oxidase genes from Arabidopsis thaliana, but not to the enzyme from maize. N6-isopentenyl-2-(2-hydroxyethylamino)-9-methyladenine is the best substrate from all the cytokinins tested. Interestingly, oxygen was not required and hydrogen peroxide not produced during the catalytic reaction, so the enzyme behaves as a dehydrogenase rather than an oxidase. This was confirmed by the ability of the enzyme to transfer electrons to artificial electron acceptors, such as phenazine methosulfate and 2,6-dichlorophenol-indophenol. 2,3-Dimethoxy-5-methyl-1,4-benzoquinone, a precursor of the naturally occurring electron acceptor ubiquinone, readily interacts with the enzyme in micromolar concentrations. Typical flavoenzyme inhibitors such as acriflavine and diphenyleneiodonium inhibited this enzyme activity. Presence of the flavin cofactor in the enzyme was confirmed by differential pulse polarography and by measuring the fluorescence emission spectrum. Possible existence of a second redox centre is discussed.     

Regulation of cytokinin oxidase activity as a factor affecting the content of cytokinins

In the shoots of 7-day-old seedlings of wheat (Triticum durum Desf., cv. Bezenchukskaya 139), ion deficiency in Hoagland-Arnon nutrient solution induced a decrease in the cytokinin content by the end of the first day, whereas the excision of four out of five primary roots brought about an opposite response (accumulation of cytokinins) as early as during the first hour. It was assumed that changes in the content of cytokinins might depend on the activity of cytokinin oxidase (CKO) and the expression of gene encoding this enzyme. It was shown that tenfold dilution of the nutrient solution activated CKO and raised the level of CKO gene expression, whereas excision of some roots brought about a quick decrease in enzyme activity and gene expression. The role of ABA and arrival of cytokinins from the roots to the shoot as factors affecting CKO activity in the shoot is discussed; arguments for the priority of hormonal signal over the influx of nitrates from the roots are offered. It was concluded that the regulation of CKO activity might be one of the important mechanisms determining plant response to treatments via the changes in the cytokinin concentration.     

Changes in free polyamine level and di- and polyamine oxidase activity in cucumber roots under allelochemical stress conditions

Seven-day-old seedlings of the cucumber (Cucumis sativus L.) cv. Wisconsin were treated with 0.5 mM solutions of phenols (p-coumaric, ferulic, p-hydroxybenzoic and vanillic acids) as stress factors. The level of free polyamines as well as activities of their catabolic enzymes, i.e. di- and polyamine oxidases (DAO - EC 1.4.34 and PAO - EC 1.4.36), were estimated for the first three hours of the stress. Cucumber roots were found to have only the presence of putrescine and spermidine. Root treatment with phenols caused a violent decrease of both amine contents during the first hour of the stress. These changes were associated with the increase of amine oxidase activity.     

Endogenous cytokinin accumulation and cytokinin oxidase activity during shoot organogenesis of Petunia hybrida

Changes in endogenous cytokinin content and cytokinin oxidase activity were characterized in leaf explants from two Petunia hybrida Vilm. genetic lines which differed in their shoot organogenic response to exogenous N⁶-benzyladenine (BA). Endogenous cytokinin content in leaf explants of the highly shoot organogenic line, St40, increased 1.7-fold during the shoot induction phase (days 6–10) and had an additional 2.6-fold cytokinin increase correlated with the shift from induction to the shoot development phase. The cytokinins isopentenyl adenine (iP) and isopentenyl adenosine (iPAR) increased, while the cytokinins zeatin, zeatin riboside and dihydrozeatin remained at consistently low levels. In contrast, isoprenoid cytokinins did not accumulate in petunia TLV1 leaf explants which were incapable of shoot induction during 12 days of culture with BA. Cytokinin oxidase activity continuously increased in leaf explants of both petunia genotypes in response to BA, with a larger increase in St40. These results suggest that the differences in organogenic response in the two petunia genotypes may be the result of differences in BA uptake and metabolism which subsequently affects the accumulation of isoprenoid cytokinins and the activity of cytokinin oxidase in the early stages of shoot development.     

Genetic studies on the acidification capacity of sunflower roots induced under iron stress

Under stress of iron deficiency roots of sunflower (Helianthus annuus L.) increase proton efflux which acidifies the root medium, increase the ferric reducing capacity and the exudation of phenolic compounds. Differences have been found previously among sunflower inbred lines in the capacity of their roots to lower pH and it was also found that this character is under genetic control.This work presents the results of an inheritance study made by crossing two genotypes, one (CMS HA 89) without acidification capacity and another (RHA 271) with it. Plants were grown individually in 75 mL vessels with an aerated solution low in iron. After 4 days, solutions were changed to one without iron and the pH of the medium was measured during the following days. Results from F₁, F₂, and backcross generations can be explained with two pairs of alleles controlling the character, being the relation between alleles of complete dominance at both gene pairs, but either gene, when dominant is epistatic to the other.     

Role of roots in regulating the growth rate and cytokinin content in leaves

The role of roots in the enhancement of cytokinin content and leaf growth of Phaseolus vulgaris plants after decapitation and partial defoliation was investigated. Partial excision of the roots of plants which were decapitated above the primary leaf node resulted in a reduction of leaf growth and soluble proteins accumulation in the primary leaves. Roots excision was done at time of decapitation and repeated 8 days later. Endogenous cytokinins, known to be involved in enhancing shoot growth, accumulated in the leaves and stems of decapitated and partially defoliated plants. Lower levels of cytokinins were detected in the leaves of decapitated plants with only a partial root system. The level of cytokinins in the roots of decapitated plants was reduced by partial root excision. The growth and accumulation of cytokinins in leaves were, however, not totally suppressed by removing a large proportion of the roots. At the commencement of the experiment the stem had a higher cytokinin content than both the leaves and roots. This suggests that the stem could be an alternative source of cytokinins to the leaves. The cytokinin complement in the leaves of decapitated plants is not identical to that in the roots. It appears that cytokinins supplied by the roots are metabolized in the leaves, or that alternatively certain cytokinins are synthesized in the leaves themselves.     

Grain filling pattern and cytokinin content in the grains and roots of rice plants

Grain filling patterns and their relationships withzeatin (Z), zeatin riboside (ZR), indole-3-acetic acid(IAA) and gibberellin (GA) contents in the grains androots during grain development were examined in sixrice (Oryza sativa L.) genotypes grown in thefield and in water culture. Three grain fillingpatterns based on the filling rate of superior andinferior spikelets were observed, i.e., fastsynchronous: all spikelets started filling early andfast at the early filling stage; slowsynchronous: all spikelets filled slowly at the earlyfilling stage and reached the maximum filling ratelate; and asynchronous: superior spikeletsstarted filling and reached the maximum filling ratemuch earlier than the inferior ones. The order ofgrain filling percentage in the three types of grainfilling patterns was: fast synchronous >asynchronous > slow synchronous. Changes in Z + ZRcontents in the superior and inferior spikelets wereassociated with the grain filling patterns. Grainfilling percentage was significantly correlated withZ + ZR contents in the grains and roots at the earlyand middle grain filling stages. IAA and GA(GA¹ + GA³ + GA⁴)contents in the grains and roots were notsignificantly correlated with grain fillingpercentage. The results suggest that cytokinins in thegrains and roots during the early phase of graindevelopment play an important role in regulating grainfilling pattern and consequently influence grainfilling percentage.     

Cadmium modulates NADPH oxidase activity and expression in sunflower leaves

The production of reactive oxygen species (ROS) and the ways by which ROS are generated are very important facts related to heavy metal toxicity in plants. In this work, superoxide anion (O₂ ^·−) generation diminished in cadmium treated sunflower (Helianthus annuus L.) leaf discs, and this reduction was time and Cd-concentration dependent. In line with these findings, we observed that NADPH-dependent oxidase activity was significantly inhibited by 0.1 and 0.5 mM Cd²⁺ treatments and the expression of the NADPH oxidase putative gene related to O₂ ^·− synthesis in sunflower leaves was 83 % inhibited by 0.1 mM CdCl₂ and almost completely depleted by 0.5 mM CdCl₂.     

Cytokinin biosynthesis in roots of corn

Removal of the quiescent center (QC) from the root apex of maize (Zea mays L., cv. Kelvedon 33) initiates a set of events which culiminate in the regeneration of an intact apex with a newly formed QC. Concomitant with the formation of a new QC is a marked reduction in extractable cytokinins in the tissue of the proximal meristem. Replacing the excised QC with a Dowex (acidic cation-exchange resin) bead affects both root growth and QC regeneration. Root growth is inhibited by plain Dowex beads and Dowex beads treated with zeatin; this inhibition is reversed if the beads have been treated with CaCl₂ (±zeatin). Dowex beads treated with zeatin delay the formation of a new QC; this effect is the same whether or not the beads also contain CaCl₂. The results of this investigation support the notions that cytokinin biosynthesis in roots is a result of activities of both the QC and the proximal meristem, and that cytokinins, at least if supplied exogenously, can play a role in root morphogenesis by delaying the regeneration of the QC.Abbreviations used throughout the text PM proximal meristem - QC quiescent center - RC root cap     

Response of cytokinin pool and cytokinin oxidase/dehydrogenase activity to abscisic acid exhibits organ specificity in peas

Changes in cytokinin pool and cytokinin oxidase/dehydrogenase activity (CKX EC: 1.5.99.12) in response to increasing abscisic acid (ABA) concentrations (0.5–10 μM) were assessed in the last fully expanded leaves and secondary roots of two pea (Pisum sativum) varieties with different vegetation periods. Certain organ diversity in CKX response to exogenous ABA was observed. Treatment provoked altered cytokinin pool in the aboveground parts of both studied cultivars. Specific CKX activity was influenced significantly basically in roots of the treated plants. Results suggest that ABA-mediated cytokinin pool changes are leaf-specific and involve certain root signals in which CKX activity presents an important link. This enzymatic activity most probably regulates vascular transport of active cytokinins from roots to shoots.     

Cytokinin oxidase activity in the cellular slime mold, Dictyostelium discoideum

The cytokinin N6-(delta 2-isopentenyl)adenine (i6Ade) is produced during the development of the cellular slime mold, Dictyostelium discoideum, and functions in this organism as the immediate precursor of the spore germination inhibitor, discadenine. The metabolism of i6Ade in axenic cultures of D. discoideum Ax-3 amoebae has been investigated in the present study. An enzyme activity that specifically catalyzes the degradation of i6Ade has been detected in Ax-3 amoebae. This enzyme is similar to the cytokinin oxidases present in higher plant systems and cleaves the N6-side chain of i6Ade to form adenine. Discadenine synthase activity was also detected in axenically cultured Ax-3 amoebae. The cytokinin oxidase activity detected in Dictyostelium decreased during aggregation and development of Ax-3 amoebae and in starving Ax-3 amoebae maintained under either fast-shake (230 rpm) or slow-shake (70 rpm) conditions. In the latter case, the fall in enzyme activity was accelerated by treatment with cyclic AMP. In contrast to these results, discadenine synthase activity in Ax-3 amoebae rose sharply during the culmination phase of development, exhibited little change in starving Ax-3 amoebae maintained under fast-shake conditions, and fell under slow-shake conditions unless the amoebae were treated with cyclic AMP. Possible functions of the Dictyostelium cytokinin oxidase and the significance of the i6Ade metabolism observed in vegetative Dictyostelium amoebae are discussed.