Degradation of cytokinins by maize cytokinin dehydrogenase is mediated by free radicals generated by enzymatic oxidation of natural benzoxazinones

Hydroxamic acid 2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐one (DIMBOA) was isolated from maize phloem sap as a compound enhancing the degradation of isopentenyl adenine by maize cytokinin dehydrogenase (CKX), after oxidative conversion by either laccase or peroxidase. Laccase and peroxidase catalyze oxidative cleavage of DIMBOA to 4‐nitrosoresorcinol‐1‐monomethyl ether (coniferron), which serves as a weak electron acceptor of CKX. The oxidation of DIMBOA and coniferron generates transitional free radicals that are used by CKX as effective electron acceptors. The function of free radicals in the CKX‐catalyzed reaction was also verified with a stable free radical of 2,2′‐azino‐bis‐3‐ethylbenzothiazoline‐6‐sulfonic acid. Application of exogenous cytokinin to maize seedlings resulted in an enhanced benzoxazinoid content in maize phloem sap. The results indicate a new function for DIMBOA in the metabolism of the cytokinin group of plant hormones.     

Regulation of cytokinin content in plant cells

Cytokinin levels in plant cells are dependent on cytokinin biosynthesis and/or uptake from extracellular sources, metabolic interconversions, inactivation and degradation. Cytokinin conversion to compounds differing in polarity seems to be decisive for their entrapment within the cell and intracellular compartmentation, which affects their metabolic stability. Increase in cytokinin levels, resulting either from their uptake or intracellular biosynthesis, may promote further autoinductive accumulation of cytokinins which may function in the induction of cytokinin-initiated physiological processes. Accumulated cytokinins are capable of inducing cytokinin oxidase which consequently decreases cytokinin levels. This seems to be the mechanism of re-establishment and maintenance of cytokinin homeostasis required for further development of physiological events induced by transient cytokinin accumulation. Auxin may influence cytokinin levels by down regulation of cytokinin biosynthesis and/or by promotion of cytokinin degradation. A model of the regulation of cytokinin levels in plant cells based on these phenomena is presented and its physiological role(s) is discussed.     

Dynamics of expression and distribution of cytokinin oxidase/dehydrogenase in developing maize kernels

Maturing maize kernels are a rich source of cytokinins and cytokinin oxidase/dehydrogenase activity, but the relationship between kernel development, cytokinin levels, the induction of cytokinin oxidase/dehydrogenase and the control of cell division is not known. Using polyclonal antibodies raised against recombinant maize cytokinin oxidase/dehydrogenase, we investigated the appearance of cytokinin oxidase/dehydrogenase (ZmCKX1) in both hybrid and inbred maize kernels as a function of time after pollination. Cytokinin oxidase/dehydrogenase was detected by five days after pollination (5 DAP) in a hybrid line, but significantly later in inbred lines. The bulk of the cytokinin oxidase/dehydrogenase detected was associated with the embryo and placental/chalazal region of the kernels rather than with the endosperm. We identified additional maize sequences in the database that appear to encode cytokinin oxidase/dehydrogenase gene family members and correspond closely with a subset of the ten cytokinin oxidase/dehydrogenase genes identified in the rice genome. Gene expression of Zmckx1 was examined by RT-PCR in immature kernels and compared with that of three putative maize cytokinin oxidase/dehydrogenase homologs. We conclude that the manipulation of kernel cytokinin levels to increase endosperm cell division will require a more detailed understanding of specific expression patterns and localization of multiple cytokinin oxidase/dehydrogenases within kernels.     

Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.     

Sink Effect of Cytokinin in Detached Leaves Is Not Caused by Structural Rearrangements of Phloem

The sink effect of cytokinin is manifested as a decrease in source capacity and the induction of sink activity in the phytohormone-treated region of a mature excised leaf. In order to find out whether this effect was due to the direct action of cytokinin on the phloem structure, two types of phloem terminals were examined. In pumpkin (Cucurbita pepo L.) leaves, the phloem terminals are open; i.e., they are linked to mesophyll by numerous symplastic connections, which are located in narrow areas called plasmodesmal pit fields. In broad bean (Vicia faba L.) leaves, the phloem terminals belong to the closed type and have no symplastic links with mesophyll. The electron microscopic study of terminal phloem did not reveal any structural changes in the companion cells, which could account for the suppression of assimilate export. The treatment of leaves with cytokinin neither disturbed the structure of plasmodesmal pit fields in pumpkin leaves nor eliminated the wall protuberances (the ingrowths promoting phloem loading) in bean leaves. No evidence was obtained that the cytokinin-induced import of assimilates in mature leaves is caused by the recovery of meristematic activity, i.e., by either formation of new phloem terminals having immature sieve elements capable of unloading or by the development of new sieve elements within the existing veins. Cytokinin did not induce de novo formation of phloem elements. Structural characteristics of the leaf phloem, such as the number of branching orders in the venation pattern, the number of vein endings per areole, the number of areoles per leaf, the area of one areole, and the number of sieve elements per bundle remained unaltered. It is concluded that the sink effect of cytokinin in excised leaves cannot be determined by alteration of the phloem structure.     

Biochemical Characterization of Cytokinin Oxidases/Dehydrogenases from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Expressed in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.

Transgenic tobacco plants overexpressing single Arabidopsis thaliana cytokinin dehydrogenase (CKX, EC 1.5.99.12) genes AtCKX1, AtCKX2, AtCKX3, AtCKX4, AtCKX5, AtCKX6, and AtCKX7 under the control of a constitutive 35S promoter were tested for CKX-enzymatic activity with varying pH, electron acceptors, and substrates. This comparative analysis showed that out of these, only AtCKX2 and AtCKX4 were highly active enzymes in reaction with isoprenoid cytokinins (N ⁶ -(2-isopentenyl)adenine (iP), zeatin (Z)) and their ribosides using the artificial electron acceptors 2,6-dichlorophenol indophenol (DCPIP) or 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q₀). Turnover rates of these cytokinins by four other AtCKX isoforms (AtCKX1, AtCKX3, AtCKX5, and AtCKX7) were substantially lower, whereas activity of AtCKX6 was almost undetectable. The isoenzymes AtCKX1 and AtCKX7 showed significant preference for cytokinin glycosides, especially N ⁶ -(2-isopentenyl)adenine 9-glucoside, under weakly acidic conditions. All enzymes preferentially cleave isoprenoid cytokinins in the presence of an electron acceptor, but aromatic cytokinins are not resistant and are degraded with lower reaction rates as well. Cytokinin nucleotides, considered as resistant to CKX attack until now, were found to be potent substrates for some of the CKX isoforms. Substrate specificity of AtCKXs is discussed in this study with respect to the structure of the CKX active site. Further biochemical characterization of the AtCKX1, AtCKX2, AtCKX4 and AtCKX7 enzymes showed pH-dependent activity profiles.     

The possible involvement of cytokinins in the pathogenicity of Helminthosporium maydis

Green islands/infection sites recorded higher cytokinin activity than surrounding tissue as well as non-inoculated tissue. This activity in infected areas increased with time of incubation while in tissue surrounding the green islands and non-inoculated tissue, cytokinin activity decreased with time of incubation. The culture filtrate extracts of H. maydis had cytokinin activity which increased with growth of the fungus. Cytokinin activity of thin-layer Chromatographic fractions from tissue and culture filtrate extracts revealed that a major portion of the activity was confined to Rf zone 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. Presence of zeatin and zeatin riboside in tissue and culture filtrates was confirmed by high performance liquid chromatography. Cytokinin substances, such as zeatin and zeatin riboside, increase at infection sites with growth of the pathogen suggesting they may be involved in the pathogenicity of H. maydis on maize.     

Cytokinin oxidase/dehydrogenase in Pisum sativum plants during vegetative development. Influence of UV-B irradiation and high temperature on enzymatic activity

Cytokinin oxidase/dehydrogenase (EC 1.5.99.12) specific activity was determined in leaves and roots of two P. sativum cultivars (cv. Scinado and cv. Manuela) during vegetative development and the effect of UV-B irradiation or elevated temperature was assessed. The measurement of CKX activity during development showed localisation of this enzyme to roots. The reduction in CKX activity in leaves after UV-B irradiation and the increased levels of the enzyme in high temperature-treated plants suggests that the enzymes from the CKX gene family have a different expression during stress responses provoked by different factors and probably are tissue specific. Differences regarding cytokinin oxidase/dehydrogenase activity stress response were observed between the two pea cultivars.     

Rate enhancement of cytokinin oxidase/dehydrogenase using 2,6-dichloroindophenol as an electron acceptor

Cytokinin oxidase/dehydrogenase degrades cytokinins by dehydrogenating the N6-C1 bond of cytokinins. The resulting imine is then hydrolyzed. For example, isopentenyl-adenine is cleaved into 3-methyl-2-butenal (isopentenyl-aldehyde) and adenine . The reducing equivalents from dehydrogenation are transferred to an unknown sink, in vivo. It has been hypothesized that the enzyme requires oxygen , possibly resulting in the formation of hydrogen peroxide. 2,6-dichloroindophenol (DCPIP) can function as an acceptor of reducing equivalents for in vitro cytokinin oxidase/dehydrogenase reactions. For the predominant cytokinin oxidase/dehydrogenase in maize, ZmCKX1, the addition of DCPIP to in vitro reactions increases the reaction rate to nearly 4000-fold faster than the oxygen-dependent rate. Further, the change in absorbance of DCPIP at 600 nm, as it is reduced, forms the basis for an assay suitable for following biochemical purification of cytokinin oxidase/dehydrogenases , detailed kinetic studies , and rapid measurement of cytokinin oxidase/dehydrogenase activity in large numbers of samples.     

Inhibition of enzymatic cellulolysis by phenolic compounds

Phenolics derived from lignin and other plant components can pose significant inhibition on enzymatic conversion of cellulosic biomass materials to useful chemicals. Understanding the mechanism of such inhibition is of importance for the development of viable biomass conversion technologies. In native plant cell wall, most of the phenolics and derivatives are found in polymeric lignin. When biomass feedstocks are pretreated (prior to enzymatic hydrolysis), simple or oligomeric phenolics and derivatives are often generated from lignin modification/degradation, which can inhibit biomass-converting enzymes. To further understand how such phenolic substances may affect cellulase reaction, we carried out a comparative study on a series of simple and oligomeric phenolics representing or mimicking the composition of lignin or its degradation products. Consistent to previous studies, we observed that oligomeric phenolics could exert more inhibition on enzymatic cellulolysis than simple phenolics. Oligomeric phenolics could inactivate cellulases by reversibly complexing them. Simple and oligomeric phenolics could also inhibit enzymatic cellulolysis by adsorbing onto cellulose. Individual cellulases showed different susceptibility toward these inhibitions. Polyethylene glycol and tannase could respectively bind and degrade the studied oligomeric phenolics, and by doing so mitigate the oligomeric phenolic's inhibition on cellulolysis.     

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.     

Phloem-transported cytokinin regulates polar auxin transport and maintains vascular pattern in the root meristem

Cytokinin phytohormones regulate a variety of developmental processes in the root such as meristem size, vascular pattern, and root architecture [1-3]. Long-distance transport of cytokinin is supported by the discovery of cytokinins in xylem and phloem sap [4] and by grafting experiments between wild-type and cytokinin biosynthesis mutants [5]. Acropetal transport of cytokinin (toward the shoot apex) has also been implicated in the control of shoot branching [6]. However, neither the mode of transport nor a developmental role has been shown for basipetal transport of cytokinin (toward the root apex). In this paper, we combine the use of a new technology that blocks symplastic connections in the phloem with a novel approach to visualize radiolabeled hormones in planta to examine the basipetal transport of cytokinin. We show that this occurs through symplastic connections in the phloem. The reduction of cytokinin levels in the phloem leads to a destabilization of the root vascular pattern in a manner similar to mutants affected in auxin transport or cytokinin signaling [7]. Together, our results demonstrate a role for long-distance basipetal transport of cytokinin in controlling polar auxin transport and maintaining the vascular pattern in the root meristem.     

Vacuolar and cytosolic cytokinin dehydrogenases of Arabidopsis thaliana: Heterologous expression,purification and properties

The catabolism of cytokinins is a vital component of hormonal regulation, contributing to the control of active forms of cytokinins and their cellular distribution. The enzyme catalyzing the irreversible cleavage of N⁶-side chains from cytokinins is a flavoprotein classified as cytokinin dehydrogenase (CKX, EC 1.5.99.12). CKXs also show low cytokinin oxidase activity, but molecular oxygen is a comparatively poor electron acceptor. The CKX gene family of Arabidopsis thaliana comprises seven members. Four code for proteins secreted to the apoplast, the remainder are not secreted. Two are targeted to the vacuoles and one is restricted to the cytosol. This study presents the purification and characterization of each of these non-secreted CKX enzymes and substrate specificities are discussed with respect to their compartmentation. Vacuolar enzymes AtCKX1 and AtCKX3 were produced in Pichia pastoris and cytosolic enzyme AtCKX7 was expressed in Escherichia coli. The recombinant proteins were purified by column chromatography. All enzymes preferred synthetic electron acceptors over oxygen, namely potassium ferricyanide and 2,3-dimetoxy-5-methyl-1,4-benzoquinone (Q₀). In slightly acidic conditions (pH 5.0), N⁶-(2-isopentenyl)adenine 9-glucoside (iP9G) was the best substrate for AtCKX1 and AtCKX7, whereas AtCKX3 preferentially degraded N⁶-(2-isopentenyl)adenine 9-riboside-5′-monophosphate (iPMP). Moreover, vacuolar AtCKX enzymes in certain conditions degraded N⁶-(2-isopentenyl)adenine di- and triphosphates two to five times more effectively than its monophosphate.     

Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem,hydathodes, and pollen of Arabidopsis

Nucleobases and derivatives like cytokinins and caffeine are translocated in the plant vascular system. Transport studies in cultured Arabidopsis cells indicate that adenine and cytokinin are transported by a common H+-coupled high-affinity purine transport system. Transport properties are similar to that of Arabidopsis purine transporters AtPUP1 and 2. When expressed in yeast, AtPUP1 and 2 mediate energy-dependent high-affinity adenine uptake, whereas AtPUP3 activity was not detectable. Similar to the results from cell cultures, purine permeases (PUP) mediated uptake of adenine can be inhibited by cytokinins, indicating that cytokinins are transport substrates. Direct measurements demonstrate that AtPUP1 is capable of mediating uptake of radiolabeled trans-zeatin. Cytokinin uptake is strongly inhibited by adenine and isopentenyladenine but is poorly inhibited by 6-chloropurine. A number of physiological cytokinins including trans- and cis-zeatin are also efficient competitors for AtPUP2-mediated adenine uptake, suggesting that AtPUP2 is also able to mediate cytokinin transport. Furthermore, AtPUP1 mediates transport of caffeine and ribosylated purine derivatives in yeast. Promoter-reporter gene studies point towards AtPUP1 expression in the epithem of hydathodes and the stigma surface of siliques, suggesting a role in retrieval of cytokinins from xylem sap to prevent loss during guttation. The AtPUP2 promoter drives GUS reporter gene activity in the phloem of Arabidopsis leaves, indicating a role in long-distance transport of adenine and cytokinins. Promoter activity of AtPUP3 was only found in pollen. In summary, three closely related PUPs are differentially expressed in Arabidopsis and at least two PUPs have properties similar to the adenine and cytokinin transport system identified in Arabidopsis cell cultures.     

Cytokinin Activity in Water-stressed Shoots

Water stress applied to the plant shoot through enhanced evaporative demands reduced cytokinin activity in extracts of xylem exudate and leaves. This reduction resembled the changes in cytokinin activity caused by water stress applied to the root. Cytokinin activity in detached wilting leaves decreased rapidly. Recovery took place after several hours in a humid chamber. Experiments with ¹⁴C-kinetin indicated that the mechanism of the inactivation and its reversal involve a chemical transformation of the cytokinin molecule.     

Cytokinin metabolism in Physcomitrella patens – differences and similarities to higher plants

Cytokinins play an important role in plant development and occur informs with different hormonal activity. As the nucleotide forms of cytokininsare considered to have little or no biological activity, the conversion ofcytokinin bases and ribosides to their nucleotides can contribute to the tuningof cytokinin activity in plant cells. Cytokinin metabolism was monitoredin vivo by feeding either radiolabelledisopentenyladenosine (³H-[9R]iP) or isopentenyladenine(³H-iP) to liquid grown chloronema tissue ofPhyscomitrellapatens (Hedw.) B.S.G. wild type. The riboside ³H-[9R]iPwas rapidly converted to ³H-iP, which was released into the culturemedium. The intracellular concentration of the ³H-iP was twice ashigh as extracellular. From the overall amount of ³H-iP about 95%were present in the medium. Cytokinin nucleotides occurred as tritiated mono-,di- and triphosphates of ³H-[9R]iP. When feeding the base³H-iP however, its main metabolic fate was degradation and nosignificant amounts of radiolabelled cytokinin nucleotides were detected. Forthe cytokinin metabolism in P. patens it is concludedthat,in contrast to higher plants nucleotides are mainly formed from ribosidesvia the adenosine kinase pathway and not byribophosphorylation of the cytokinin base via adeninephosphoribosyltransferase.     

Differential decolorization of textile dyes in mixtures and the joint effect of laccase and cellobiose dehydrogenase activities present in extracellular extracts from Funalia trogii

The largest part of the bio-decolorization investigations have been performed to date on a single dye without exploring the behavior in complex mixtures as the real dyeing baths. Therefore, mixtures of dyes belonging to azo and anthraquinonic classes, chosen among the most utilized in textile wool dyeing, were employed for comparative enzymatic decolorization studies using the extracellular extracts from the white rot fungus Funalia trogii, to understand how the concomitant presence of more than one dye could influence their degradation course and yield.Fungal extracts containing laccase activity only were capable to partially decolorize dyes mixtures from the different classes analyzed. The deconvolution of the decolorization with time allowed to monitor the degradation of the single dyes in the mixtures evidencing a time dependent differential decolorization not observed for the singles alone. Some dyes in the blend were in fact decolorized only when the most easily converted dyes were largely transformed. These experiments would allow to help the dyeing factories in the selection of the most readily degraded dyes.Since F. trogii grown on different media and activators shows diverse levels of expression of the redox enzymes laccase and cellobiose dehydrogenase (CDH), the dyes mixtures recalcitrant to decolorization by laccase activity alone, were subjected to the combined action of extracts containing laccase and CDH. The use of CDH, in support to the activity of laccase, resulted in substantial decolorization increases (>84%) for all the refractory dyes mixtures.     

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.