The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.     

Antagonistic Effects of Triazolo[4,5-d]pyrimidine and Pyridylurea Derivatives on Cytokinin-Induced Cytokinin Oxidase/Dehydrogenase Activity in Young Pea Plants

The effect of strong and weak cytokinin antagonists, belonging to the groups of triazolo[4,5-d]pyrimidines (TP), and pyridyl-phenylurea derivatives (PU), on cytokinin oxidase/dehydrogenase activity (CKX) in the tissues of young pea plants was studied. Tested anticytokinins, with the exception of the most efficient one – PU-1, were able to promote increased CKX activity in roots, when applied alone, but they had no significant influence on the enzymatic activity in leaves. N⁶-benzyladenine (BA) and 1-(2-chloropyridin-4-yl)-3-phenylurea (CPPU) provoked strong increase in CKX activity in roots, while in leaves considerable inhibition of enzymatic activity was observed. Different types of anticytokinins exhibited diverse preference towards taking off the action of purine and phenylurea cytokinins over CKX activity.     

Analysis of Cytokinin Metabolism in ipt Transgenic Tobacco by Liquid Chromatography-Tandem Mass Spectrometry

The endogenous levels of the major, naturally occurring cytokinins in Pisum sativum ribulose-1,5-bisphosphate carboxylase small subunit promoter-isopentenyl transferase gene (Pssu-ipt)-transformed tobacco (Nicotiana tabacum L.) callus were quantified using electrospray-liquid chromatography-tandem mass spectrometry during a 6-week subcultivation period. An ipt gene was expressed under control of a tetracycline-inducible promoter for a more detailed study of cytokinin accumulation and metabolism. Activation of the ipt in both expression systems resulted in the production of mainly zeatin-type cytokinins. No accumulation of isopentenyladenine or isopentenyladenosine was observed. In Pssu-ipt-transformed calli, as well as in the tetracycline-inducible ipt leaves, metabolic inactivation occurred through O-glucoside conjugation. No significant elevation of cytokinin N-glucosides levels was observed. Side-chain reduction to dihydrozeatin-type cytokinins was observed in both systems. The levels of the endogenous cytokinins varied in time and were subject to homeostatic regulatory mechanisms. Feeding experiments of ipt transgenic callus with [3H]isopentenyladenine and [3H]isopentenyladenosine mainly led to labeled adenine-like compounds, which are degradation products from cytokininoxidase activity. Incorporation of radioactivity in zeatin riboside was observed, although to a much lesser extent.     

Effects of tetracycline on wild-type and inducible P35So IPT-5/TETR transgenic tobacco plants

Introduction of the Agrobacterium ipt gene, coding for isopentenyl transferase, under control of a tetracycline (Tc)-inducible promoter results in a very specific system in which cytokinin levels can be changed. Because Tc belongs to the group of antibiotics that affect 70S ribosomes, it is important to study the effects of Tc on untransformed plants. Although 1 mg l⁻¹ Tc was previously reported to have no physiological effects, this study revealed several changes in hydroponically grown wild-type Nicotiana tabacum L. cv. Wisconsin. Therefore, lower Tc concentrations (0.1 and 0.2 mg l⁻¹ Tc) were used to induce ipt -transgenic (tr) plants. Upon induction, real-time PCR analysis showed that the ipt gene was expressed several times higher in roots of tr plants, but not in leaves. Consequently, cytokinin levels were also elevated to a large extent in roots. This resulted in a disturbance of the cytokinin to auxin ratio, leading to an obstructed root growth. In leaves, no significant increase in cytokinins was observed. However, phenotypic and physiological effects, which could be attributed to cytokinin, were apparent in leaves of ipt -induced trs: chlorophyll and carotenoid content were elevated and grana stacking increased. Our study demonstrates that caution has to be taken to determine the 'safe' concentration of inducers when using inducible gene-expression systems.     

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.     

The Involvement of Cytokinin Oxidase/Dehydrogenase and Zeatin Reductase in Regulation of Cytokinin Levels in Pea (Pisum sativum L.) Leaves

Cytokinin metabolism in plants is very complex. More than 20 cytokinins bearing isoprenoid and aromatic side chains were identified by high performance liquid chromatography-mass spectrometry (HPLC-MS) in pea (Pisum sativum L. cv. Gotik) leaves, indicating diverse metabolic conversions of primary products of cytokinin biosynthesis. To determine the potential involvement of two enzymes metabolizing cytokinins, cytokinin oxidase/dehydrogenase (CKX, EC 1.5.99.12) and zeatin reductase (ZRED, EC 1.3.1.69), in the control of endogenous cytokinin levels, their in vitro activities were investigated in relation to the uptake and metabolism of [2−³H]trans-zeatin ([2−³H]Z) in shoot explants of pea. Trans-zeatin 9-riboside, trans-zeatin 9-riboside-5′-monophosphate and cytokinin degradation products adenine and adenosine were detected as predominant [2−³H]Z metabolites during 2, 5, 8, and 24 h incubation. Increasing formation of adenine and adenosine indicated extensive degradation of [2−³H]Z by CKX. High CKX activity was confirmed in protein preparations from pea leaves, stems, and roots by in vitro assays. Inhibition of CKX by dithiothreitol (15 mM) in the enzyme assays revealed relatively high activity of ZRED catalyzing conversion of Z to dihydrozeatin (DHZ) and evidently competing for the same substrate cytokinin (Z) in protein preparations from pea leaves, but not from pea roots and stems. The conversion of Z to DHZ by pea leaf enzyme was NADPH dependent and was significantly inhibited or completely suppressed in vitro by diethyldithiocarbamic acid (DIECA; 10 mM). Relations of CKX and ZRED in the control of cytokinin levels in pea leaves with respect to their potential role in establishment and maintenance of cytokinin homeostasis in plants are discussed.     

Comparison of endogenous cytokinins and cytokinin oxidase/dehydrogenase activity in germinating and thermoinhibited Tagetes minuta achenes

Tagetes minuta L. achenes are thermoinhibited at temperatures above 35°C and have accelerated radicle emergence (germination) when subsequently transferred to an optimal temperature (25°C). Endogenous cytokinins and cytokinin oxidase/dehydrogenase (CKX) activity were compared in normally germinating (25°C) and thermoinhibited (72h at 36°C then transferred to 25°C) T. minuta achenes. Following imbibition, endogenous cytokinin concentrations changed in normally germinating T. minuta achenes, with a gradual decrease in dihydrozeatin-type (DHZ) cytokinins, a large increase in cis-zeatin-type (cZ) cytokinins, a smaller increase in N?-(2-isopentenyl)adenine-type (iP) cytokinins and a peak of trans-zeatin-type (tZ) cytokinins at 13 h. These changes in the isoprenoid cytokinin profile were similar in the thermoinhibited achenes imbibed at 36°C, despite the thermal block preventing radicle emergence. The exception was the iP-type cytokinins that only increased when transferred to 25°C. Profiles of the physiologically active free bases showed an increase in tZ prior to radical emergence in both normally germinating (13 h) and thermoinhibited achenes. A large transient peak in aromatic cytokinins [N?-benzyladenine-type (BA)] occurred during early seedling establishment in normally germinating achenes (40 h) while a transient maximum in BA-type cytokinins was found prior to radicle emergence in the thermoinhibited achenes (24 h). The CKX activity was enhanced in normally germinating achenes as the cytokinin concentration increased following imbibition. In thermoinhibited achenes, an elevated temperature negatively affected the CKX activity that only increased when the achenes were transferred to 25°C, corresponding to an increase in iP-type cytokinins. However, the favored cytokinin deactivation pathway in T. minuta appears to be 9-glycosylation, as 9-glucosides accounted for over 50% of the total cytokinin pool in both normal and thermoinhibited achenes.     

Overexpression of the cytosolic cytokinin oxidase/dehydrogenase (CKX7) from Arabidopsis causes specific changes in root growth and xylem differentiation

Degradation of the plant hormone cytokinin is catalyzed by cytokinin oxidase/dehydrogenase (CKX) enzymes. The Arabidopsis thaliana genome encodes seven CKX proteins which differ in subcellular localization and substrate specificity. Here we analyze the CKX7 gene, which to the best of our knowledge has not yet been studied. pCKX7:GUS expression was detected in the vasculature, the transmitting tissue and the mature embryo sac. A CKX7–GFP fusion protein localized to the cytosol, which is unique among all CKX family members. 35S:CKX7‐expressing plants developed short, early terminating primary roots with smaller apical meristems, contrasting with plants overexpressing other CKX genes. The vascular bundles of 35S:CKX7 primary roots contained only protoxylem elements, thus resembling the wol mutant of the CRE1/AHK4 receptor gene. We show that CRE1/AHK4 activity is required to establish the CKX7 overexpression phenotype. Several cytokinin metabolites, in particular cis‐zeatin (cZ) and N‐glucoside cytokinins, were depleted stronger in 35S:CKX7 plants compared with plants overexpressing other CKX genes. Interestingly, enhanced protoxylem formation together with reduced primary root growth was also found in the cZ‐deficient tRNA isopentenyltransferase mutant ipt2,9. However, different cytokinins were similarly efficient in suppressing 35S:CKX7 and ipt2,9 vascular phenotypes. Therefore, we hypothesize that the pool of cytosolic cytokinins is particularly relevant in the root procambium where it mediates the differentiation of vascular tissues through CRE1/AHK4. Taken together, the distinct consequences of CKX7 overexpression indicate that the cellular compartmentalization of cytokinin degradation and substrate preference of CKX isoforms are relevant parameters that define the activities of the hormone.     

Inhibitory effects of elevated endogenous cytokinins on nitrate reductase in ipt-expressing tobacco are eliminated by short-term exposure to benzyladenine

Using a novel system for expressing ipt gene from Agrobacterium tumefaciens in tobacco ( Nicotiana tabacum L., cv. Petit Havana SR1), we were able to grow seedlings and teratoma-like tissue with increased content of cytokinins. This material enabled us to investigate new regulatory aspects of nitrate reduction. We grew control plants and plants with elevated cytokinins on MS media, with or without nitrate and benzyladenine (BA). We determined in vitro nitrate reductase (EC 1.6.6.1) activity (NRA) in this plant material. Initially, we found that ipt -expressing plants always displayed lowered levels of NRA when compared to wild-type SR1 plants. We determined that long-term exposure of tobacco plants and tissue to cytokinins caused up to 60% decrease in NRA. Exposure to 40 m M nitrate was able to induce the activity in such plants 3-fold, increasing the activity in SR1 plants more than 5-fold. We were able to restore wild-type levels of NRA in ipt -expressing plants by simultaneous induction of NR with BA and nitrate. Our results suggest that regulation of NR by nitrate and cytokinin is a result of overlaying cytokinin-driven regulatory processes, with those acting in the short-term having a positive effect on NRA, and those acting over extended periods of time having inhibitory effects on NRA.     

Developmental stage as a possible factor affecting cytokinin content and cytokinin dehydrogenase activity in <Emphasis Type="Italic">Pinus sylvestris</Emphasis>

In the present study cytokinin dehydrogenase (CKX) activity was for the first time found in a conifer species, Pinus sylvestris. The activities were correlated with the endogenous cytokinin contents. Several enzyme substrates and two different electron acceptors were used to search for the enzyme activity in the extract from seeds, seedlings and plantlets. The highest specific activity was found in one-year-old plantlets with isopentenyladenine as the substrate and 2,6-dichlorophenolindophenol as the electron acceptor, at pH 8. An enhancement in the CKX specific activity corresponded to increasing contents of cytokinins, mainly isopentenyladenine and isopentenyladenosine, indicating that the enzyme activity is affected by the endogenous supply of cytokinins. CKX affinity for the ribosylated form of isopentenyladenine was dependent on the developmental stage, being higher in seeds than in seedlings, and not detectable in plantlets. The results are indicative of the presence of different isoenzymes throughout the development.     

Regenerative Capacity of Cacti Schlumbergera and Rhipsalidopsis in Relation to Endogenous Phytohormones, Cytokinin Oxidase/Dehydrogenase, and Peroxidase Activities

The recalcitrant nature and increased regenerative capacity in relation to in vitro subcultures in two cactus genera Rhipsalidopsis (Easter cactus) and Schlumbergera (Christmas cactus) were studied by examining the endogenous concentrations of several endogenous phytohormones and enzyme activities. Leaf tissue from greenhouse-grown mother plants, in vitro subcultures 1 and 3, and callus tissues were analyzed and correlated with regenerative ability. The cytokinins present in the two cacti genera were mainly isopentenyl-type derivatives. The total content of isopentenyl-type cytokinins in greenhouse-grown leaves of Rhipsalidopsis was more than twice the amount found in greenhouse-grown leaves of Schlumbergera. The total cytokinin content decreased during subculturing. Cytokinin oxidase/dehydrogenase (CKX, EC 1.4.3.18/1.5.99.12) activity increased during subculturing. In Schlumbergera there is no effect of subculturing on CKX and related cytokinin homeostasis. The total peroxidase (POX, EC 1.11.1.7) activity in greenhouse-grown leaves of both genera was low, and the activity increased significantly during subculturing, more specifically in the tissue of Rhipsalidopsis. The results clearly indicated that an enhanced auxin metabolism (biosynthesis, conjugation/deconjugation, and POX activity), in combination with an enhanced CKX activity, shifts the auxin and cytokinin pool, favoring adventitious shoot formation in Rhipsalidopsis, whereas the low level of POX activity, together with auxin autotrophy/conjugation, makes Schlumbergera more recalcitrant. S. S. and E. P. contributed equally to this work     

Response of <Emphasis Type="Italic">Pisum Sativum</Emphasis> Cytokinin Oxidase/Dehydrogenase Expression and Specific Activity to Drought Stress and Herbicide Treatments

The expression of cytokinin oxidase/dehydrogenase (CKX EC: 1.5.99.12) is subject to fine regulation and it provides a rapid turnover of cytokinins, which serves as a signal for triggering developmental events during plant growth. The activity of this enzyme is believed to be responsible for the changes in cytokinin pool under adverse environmental conditions. CKX gene-specific assay to measure the expression in response to different stress treatments in the tissues of Pisum sativum plants was developed. Pea CKX genes were amplified and sequenced using primers designed from the sequences of Medicago truncatula CKX genes. Expression of two P. sativum CKX genes was assessed using relative-quantification in real time two-step RT-PCR, in leaves and roots of drought-, glufosinate- and atrazine-treated cv. Manuela pea plants. Varied CKX responses support the existence of complicated regulating mechanism of cytokinin oxidase/ dehydrogenase gene expression.     

New insights into the biology of cytokinin degradation

A survey of recent results is presented concerning the role of cytokinin degradation in plants, which is catalyzed by cytokinin oxidase/dehydrogenase (CKX) enzymes. An overview of Arabidopsis CKX gene expression suggests that their differential regulation by biotic and abiotic factors contributes significantly to functional specification. Here, we show using reporter gene and semiquantitative RT-PCR analyses regulation of individual CKX genes by cytokinin, auxin, ABA, and phosphate starvation. Partially overlapping expression domains of CKX genes and cytokinin-synthesizing IPT genes in meristematic tissues and endo-reduplicating cells lend support for a locally restricted function of cytokinin. On the other hand, their expression in vascular tissue suggests a function in controlling transported cytokinin. Recent studies led to a model for the biochemical reaction mechanism of CKX-mediated catalysis, which was refined on the basis of the three-dimensional enzyme structure. Last but not least, the developmental functions of CKX enzymes are addressed. The recent identification of the rice OSCKX2 gene as an important novel breeding tool is highlighted. Together the results corroborate the relevance of metabolic control in determining cytokinin activity.     

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.     

Kinetic and chemical analyses of the cytokinin dehydrogenase-catalysed reaction: correlations with the crystal structure

CKX (cytokinin dehydrogenase) is a flavoprotein that cleaves cytokinins to adenine and the corresponding side-chain aldehyde using a quinone-type electron acceptor. In the present study, reactions of maize (Zea mays) CKX with five different substrates (N6-isopentenyladenine, trans-zeatin, kinetin, p-topolin and N-methyl-isopentenyladenine) were studied. By using stopped-flow analysis of the reductive half-reaction, spectral intermediates were observed indicative of the transient formation of a binary enzyme-product complex between the cytokinin imine and the reduced enzyme. The reduction rate was high for isoprenoid cytokinins that showed formation of a charge-transfer complex of reduced enzyme with bound cytokinin imine. For the other cytokinins, flavin reduction was slow and no charge-transfer intermediates were observed. The binary complex of reduced enzyme and imine product intermediate decays relatively slowly to form an unbound product, cytokinin imine, which accumulates in the reaction mixture. The imine product only very slowly hydrolyses to adenine and an aldehyde derived from the cytokinin N6 side-chain. Mixing of the substrate-reduced enzyme with Cu2+/imidazole as an electron acceptor to monitor the oxidative half-reaction revealed a high rate of electron transfer for this type of electron acceptor when using N6-isopentenyladenine. The stability of the cytokinin imine products allowed their fragmentation analysis and structure assessment by Q-TOF (quadrupole-time-of-flight) MS/MS. Correlations of the kinetic data with the known crystal structure are discussed for reactions with different cytokinins.