Cytokinin oxidase activity and cytokinin content in roots of sunflower under water stress

Contents of trans-zeatin riboside (ZR), dihydrozeatin riboside (DZR) and N6-(delta2-isopentenyl) adenosine (iPA) was quantified by an indirect ELISA using polyclonal antibodies, in the roots, xylem sap and leaves of pot grown sunflower plants subjected to water stress (RWC of leaves approximately 65 per cent). The delivery rates of all three cytokinins decreased significantly under stress. Cytokinin levels also decreased in roots and in leaves of stressed plants. Three-fold increase in cytokinin oxidase activity was observed in stressed roots after polymin P-ammonium sulphate fractionation. Further purification using Con A agarose resulted in elution of protein with cytokinin oxidase activity and was found to be 30 kDa protein on SDS-PAGE.     

Cytokinin biochemistry in relation to leaf senescence. VII. Endogenous cytokinin levels and exogenous applications of cytokinins in relation to sequential leaf senescence of tobacco

The cytokinin complex in tobacco leaves of various maturities was characterized by radioimmunoassay and mass spectrometry. Zeatin was the major base, whereas zeatin riboside was identified as the main riboside. in leaves of all maturities studied. Relative to upper younger leaves, the basal yellow leaves had reduced levels of both cytokinin bases and ribosides. Exogenous applications of dihydrozeatin and zeatin to detached tobacco leaves in amounts sufficient to delay senescence, elevated cytokinin base and riboside levels 2–5 fold. Presenescent and senescent leaves of intact plants showed quantitatively similar changes in cytokinin content. which therefore appear to be of significance in control of senescence. When supplied exogenously, the principal cytokinin bases found to occur in tobacco leaves (zeatin and dihydrozeatin) were markedly more effective than auxins and gibberellic acid in retarding senescence. Localised application of cytokinins to leaf blades of detopped plants was much less effective than application to intact plants. The cytokinin induced senescence retardation in tobacco leaves was independent of effects on directed metabolite transport. Evidence that endogenous levels of active cytokinins in intact tobacco leaves are involved in control of sequential leaf senescence is discussed.     

Peach Leaf Senescence Delayed by Criconemella xenoplax

Fall annual leaf senescence of peach was delayed in the field and in microplots in the presence of Criconemella xenoplax. Soil from the rhizosphere of orchard trees with greener leaves had ca. 2.5 × more nematodes than soil around trees in a more advanced state of fall senescence. In microplots, monoclonal antibody enzyme immunoassay (EIA) of leaf cytokinins indicated that concentration of zeatin riboside-like substances and chlorophyll content were greater in leaves of trees growing in nematode-infested soil than in trees in uninfested soil. EIA also indicated the presence of substances resembling trans-zeatin, zeatin riboside, dihydrozeatin, and dihydrozeatin riboside-like substances in whole body homogenates of C. xenoplax. Levels of zeatin-like substances were present in the nematode in greater levels than the other related substances.     

Cytokinin biochemistry in relation to leaf senescence. VIII. Translocation, metabolism and biosynthesis of cytokinins in relation to sequential leaf senescence of tobacco

Cytokinin bases (zeatin and dihydrozeatin) and ribosides (zeatin riboside and dihydrozeatin riboside) were identified as major cytokinins in tobacco xylem sap by radioimmunoassay. When ³H-labelled zeatin riboside or dihydrozeatin riboside were supplied to tobacco plants via the xylem, leaves of differing maturity did not differ appreciably in level of radioactivity or in metabolism of the cytokinin. The major metabolites of zeatin riboside in leaves were adenine, adenosine and adenine nucleotides, whereas that of dihydrozeatin riboside was dihydrozeatin 7-glucoside. Incorporation of [¹⁴C]adenine into zeatin was evident in upper green leaves. indicating that young leaves have the capacity to synthesize cytokinins in situ. In contrast, fully expanded green leaves and senescing tobacco leaves exhibited little or no incorporation of [¹⁴C]adenine into cytokinins. This difference in cytokinin biosynthetic capacity may contribute to the differing cytokinin levels in leaves of different matirity, and may participate in control of sequential leaf senescence in tobacco.     

Cytokinin-induced changes in the chlorophyll content and fluorescence of in vitro apple leaves

Cytokinins (CKs) are one of the main regulators of in vitro growth and development and might affect the developmental state and function of the photosynthetic apparatus of in vitro shoots. Effects of different cytokinin regimes including different types of aromatic cytokinins, such as benzyl-adenine, benzyl-adenine riboside and 3-hydroxy-benzyladenine alone or in combination were studied on the capacity of the photosynthetic apparatus and the pigment content of in vitro apple leaves after 3 weeks of culture. We found that the type of cytokinins affected both chlorophyll a and b contents and its ratio. Chlorophyll content of in vitro apple leaves was the highest when benzyl-adenine was applied as a single source of cytokinin in the medium (1846–2176 μg/1 g fresh weight (FW) of the leaf). Increasing the concentration of benzyl-adenine riboside significantly decreased the chlorophyll content of the leaves (from 1923 to 1183 μg/1 g FW). The highest chl a/chl b ratio was detected after application of meta-topolin (TOP) at concentrations of 2.0 and 6.0 μM (2.706 and 2.804). Chlorophyll fluorescence was measured both in dark-adapted (F_v/F_m test) and in light-adapted leaf samples (Yield test; Y(II)). The maximum quantum yield and efficiency of leaves depended on the cytokinin source of the medium varied between 0.683 and 0.861 (F_v/F_m) indicating a well-developed and functional photosynthetic apparatus. Our results indicate that the type and concentration of aromatic cytokinins applied in the medium affect the chlorophyll content of the leaves in in vitro apple shoots. Performance of the photosynthetic apparatus measured by chlorophyll fluorescence in the leaves was also modified by the cytokinin supply. This is the first ever study on the relationship between the cytokinin supply and the functionability of photosystem II in plant tissue culture and our findings might help to increase plantlet survival after transfer to ex vitro conditions.     

Enhanced cytokinin degradation in leaf primordia of transgenic Arabidopsis plants reduces leaf size and shoot organ primordia formation

The plant hormone cytokinin is a key morphogenic factor controlling cell division and differentiation, and thus the formation and growth rate of organs during a plant's life cycle. In order to explore the relevance of cytokinin during the initial phase of leaf primordia formation and its impact on subsequent leaf development, we increased cytokinin degradation in young shoot organ primordia of Arabidopsis thaliana by expressing a cytokinin oxidase/dehydrogenase (CKX) gene under control of the AINTEGUMENTA (ANT) promoter. The final leaf size in ANT:CKX3 plants was reduced to ∼27% of the wild-type size and the number of epidermal cells was reduced to ∼12% of the wild type. Kinematic analysis revealed that cell proliferation ceased earlier and cell expansion was accelerated in ANT:CKX3 leaves, demonstrating that cytokinin controls the duration of the proliferation phase by delaying the onset of cell differentiation. The reduction of the cell number was partially compensated by an increased cell expansion. Interestingly, ANT:CKX3 leaf cells became about 60% larger than those of 35S:CKX3 leaves, indicating that cytokinin has an important function during cell expansion as well. Furthermore, ANT:CKX3 expression significantly reduced the capacity of both the vegetative as well as the generative shoot apical meristem to initiate the formation of new leaves and flowers, respectively. We therefore hypothesize that the cytokinin content in organ primordia is important for regulating the activity of the shoot meristem in a non-autonomous fashion.     

tRNA is the source of low-level trans-zeatin production in Methylobacterium spp

Pink-pigmented facultatively methylotrophic bacteria (PPFMs), classified as Methylobacterium spp., are persistent colonizers of plant leaf surfaces. Reports of PPFM-plant dialogue led us to examine cytokinin production by PPFMs. Using immunoaffinity and high-performance liquid chromatography (HPLC) purification, we obtained 22 to 111 ng of trans-zeatin per liter from culture filtrates of four PPFM leaf isolates (from Arabidopsis, barley, maize, and soybean) and of a Methylobacterium extorquens type culture originally recovered as a soil isolate. We identified the zeatin isolated as the trans isomer by HPLC and by a radioimmunoassay in which monoclonal antibodies specific for trans-hydroxylated cytokinins were used. Smaller and variable amounts of trans-zeatin riboside were also recovered. trans-Zeatin was recovered from tRNA hydrolysates in addition to the culture filtrates, suggesting that secreted trans-zeatin resulted from tRNA turnover rather than from de novo synthesis. The product of the miaA gene is responsible for isopentenylation of a specific adenine in some tRNAs. To confirm that the secreted zeatin originated from tRNA, we mutated the miaA gene of M. extorquens by single exchange of an internal miaA fragment into the chromosomal gene. Mutant exconjugants, confirmed by PCR, did not contain zeatin in their tRNAs and did not secrete zeatin into the medium, findings which are consistent with the hypothesis that all zeatin is tRNA derived rather than synthesized de novo. In germination studies performed with heat-treated soybean seeds, cytokinin-null (miaA) mutants stimulated germination as well as wild-type bacteria. While cytokinin production may play a role in the plant-PPFM interaction, it is not responsible for stimulation of germination by PPFMs.     

Development of Agrobacterium tumefaciens C58-induced plant tumors and impact on host shoots are controlled by a cascade of jasmonic acid,auxin, cytokinin,ethylene and abscisic acid

The development of Agrobacterium tumefaciens-induced plant tumors primarily depends on the excessive production of auxin and cytokinin by enzymes encoded on T-DNA genes integrated into the plant genome. The aim of the present study was to investigate the involvement of additional phytohormone signals in the vascularization required for rapid tumor proliferation. In stem tumors of Ricinus communis L., free auxin and zeatin riboside concentrations increased within 2 weeks to 15-fold the concentrations in control stem tissue. Auxin and cytokinin immunolocalization revealed the highest concentrations within and around tumor vascular bundles with concentration gradients. The time-course of changes in free auxin concentration in roots was inversely correlated with that in the tumors. The high ethylene emission induced by increased auxin- and cytokinin correlated with a 36-fold accumulation of abscisic acid in tumors. Ethylene emitted from tumors and exogenously applied ethylene caused an increase in abscisic acid concentrations also in the host leaves, with a diminution in leaf water vapor conductance. Jasmonic acid concentration reached a maximum already within the first week of bacterial infection. A wound effect could be excluded. The results demonstrate the concerted interaction of a cascade of transiently induced, non-T-DNA-encoded phytohormones jasmonic acid, ethylene and abscisic acid with T-DNA-encoded auxin and zeatin riboside plus trans-zeatin, all of which are required for successful plant tumor vascularization and development together with inhibition of host plant growth.     

Over-expression of SOB5 suggests the involvement of a novel plant protein in cytokinin-mediated development

Cytokinins are a class of phytohormones that play a critical role in plant growth and development. sob5-D, an activation-tagging mutant, shows phenotypes typical of transgenic plants expressing the Agrobacterium tumefaciens isopentenyltransferase (ipt) gene that encodes the enzyme catalyzing the first step of cytokinin biosynthesis. The sob5-D mutant phenotypes are caused by over-expression of a novel gene, SOB5. Sequence analysis places SOB5 in a previously uncharacterized family of plant-specific proteins. A translational fusion between SOB5 and the green fluorescent protein reporter was localized in the cytoplasm as well as associated with the plasma membrane when transiently expressed in onion epidermal cells. Analysis of transgenic plants harboring an SOB5:SOB5-beta-glucuronidase (GUS) translational fusion under the control of the SOB5 promoter region showed GUS activity in vegetative tissues (hydathodes and trichomes of leaves, shoot meristems and roots) as well as in floral tissues (pistil tips, developing anthers and sepal vasculature). Cytokinin quantification analysis revealed that adult sob5-D plants accumulated higher levels of trans-zeatin riboside, trans-zeatin riboside monophosphate and isopentenyladenine 9-glucoside when compared to the wild-type. Consistent with this result, AtIPT3 and AtIPT7 were found to be up-regulated in a tissue-specific manner in sob5-D mutants. Physiological analysis of the sob5-D mutant demonstrated reduced responsiveness to exogenous cytokinin in both root-elongation and callus-formation assays. Taken together, our data suggest a role for the novel gene SOB5 in cytokinin-mediated plant development.     

Evidence for Cytokinin Involvement in Rhizobium (IC3342)-Induced Leaf Curl Syndrome of Pigeonpea (Cajanus cajan Millsp.)

A uniquely abnormal shoot development (shoot tip-bending, leaf curling, release from apical dominance, and stunted growth) in pigeonpea (Cajanus cajan Millsp) induced by a nodulating Rhizobium strain, IC3342, is thought to be due to a hormonal imbalance. Amaranthus betacyanin bioassay indicated that xylem exudate and leaf extracts from pigeonpea plants with Rhizobium-induced leaf curl symptoms contained high concentrations of cytokinin relative to those in normal plants. Radioimmunoassay (RIA) of samples purified with high performance liquid chromatography revealed that zeatin riboside (ZR) and dihydrozeatin riboside (DZR) concentrations in xylem sap from plants with leaf curl symptoms were 7 to 9 times higher than those in the sap from symptomless, nodulated plants. The sap from symptomless plants nodulated by a Curl⁻ mutant had ZR and DZR concentrations comparable to those in the normal plant sap. RIA indicated that the respective concentrations of zeatin and N⁶-isopenteny-ladenine in culture filtrates of the curl-inducing strain IC3342 were 26 and 8 times higher than those in filtrates of a related normal nodulating strain (ANU240). Gas chromatographic-mass spectrometric analyses revealed similar differences. Gene-specific hybridization and sequence comparisons failed to detect any homology of IC3342 DNA to Agrobacterium tumefaciens or Pseudomonas savastanoi genetic loci encoding enzymes involved in cytokinin biosynthesis.     

Molecular characterization of cytokinin-responsive histidine kinases in maize. Differential ligand preferences and response to cis-zeatin

Genes for cytokinin-responsive His-protein kinases (ZmHK1, ZmHK2, and ZmHK3a) were isolated from maize (Zea mays). Heterologous expression of each of the ZmHKs in Escherichia coli having the DeltarcsC and cpslacZ genetic background conferred cytokinin-inducibility of lacZ expression on the bacteria. In the recombinant E. coli system, ZmHK1 and ZmHK3a were more sensitive to free-base cytokinins than to the corresponding nucleosides; isopentenyladenine was most effective for ZmHK1, while ZmHK2 tended to be most sensitive to trans-zeatin and the riboside. In contrast to a known cytokinin receptor of Arabidopsis (AHK4/CRE1/WOL), all ZmHKs responded to cis-zeatin (cZ), which generally is believed to be inactive or only weakly active. In cultured maize cells, expression of ZmRR1, a cytokinin-inducible response regulator, was induced by cZ as well as by trans-zeatin. These results strongly suggest that maize cytokinin receptors differ in ligand preference, and that cZ is an active cytokinin at least in maize.     

Transport and accumulation rates of abscisic acid and aldehyde oxidase activity in Pisum sativum L. in response to suboptimal growth conditions.

Pea plants (Pisum sativum L.) grown initially in nutrient solutions with adequate nitrogen supply (4 mM NO3-) were transferred to solutions containing salt (50 or 100 mM NaCl), ammonium (4 mM) or a low nitrogen supply (0.4 mM NO3-). No changes of abscisic acid (ABA) content were found in roots of stressed pea plants 9 d after the beginning of the treatments; however, accumulation of ABA in the leaves was observed. Old leaves accumulated ABA to a higher extent than young leaves. Accumulation of ABA in leaves of ammonium-fed plants and plants grown under low nitrogen supply occurred in the absence of both increased ABA xylem loading rate and enhanced aldehyde oxidase (AO, EC 1.2.3.1) activity in roots. Enhanced leaf AO activity was observed in all treatments, with the highest increase in old leaves. Among the three AO isoforms (AO-1, AO-2 and AO-3) detected in extracts of pea leaves, the lowest one AO-3 (highest mobility in the gel) correlated with ABA production and showed the highest increment in response to the treatments. The increase of AO activity detected in leaves after 2 weeks of stress application was less prominent than after 9 d, suggesting a transient enhancement of ABA production following the onset of stress. An increase of ABA xylem loading rate as well as AO root activity 4 d and 9 d after application of the treatments was observed only in salt-treated plants followed by a decrease after 14 d in 100 mM NaCl. Decreased cytokinin (trans-zeatin riboside) delivery rate into the xylem sap was observed in all treatments. The role of abscisic acid and cytokinins as positive and negative growth signals, as well as the involvement of root-generated ABA on ABA accumulation in leaves is discussed.     

The Effect of Ringing on Cytokinin Distribution in Salix baylonica

Although quantitative differences were observed in the cytokinin content of mature leaves and bark of Salix babylonica it would appear as if these tissues contained the same cytokinin complement. Ringing resulted in a decrease in the level of cytokinins in the leaves and an increase in the bark, both above and below the girdle. In the leaves the decrease was due mainly to a drop in the level of those compounds that co-chromatographed with the cytokinin glucosides. These compounds were also almost undetectable in the bark above the girdle, where callus was formed. The observed increase in the cytokinin content of the bark above the girdle was due to higher activity in those parts of the chromatograms where zeatin and zeatin riboside occurred. Ringing stimulated the growth of lateral buds below the girdle. These developing buds as well as the bark below the girdle contained very high levels of cytokinins that cochromatographed with zeatin and zeatin riboside.     

Increased cytokinin levels in transgenic PSAG12–IPT tobacco plants have large direct and indirect effects on leaf senescence, photosynthesis and N partitioning

We studied the impact of delayed leaf senescence on the functioning of plants growing under conditions of nitrogen remobilization. Interactions between cytokinin metabolism, Rubisco and protein levels, photosynthesis and plant nitrogen partitioning were studied in transgenic tobacco (Nicotiana tabacum L.) plants showing delayed leaf senescence through a novel type of enhanced cytokinin syn‐thesis, i.e. targeted to senescing leaves and negatively auto‐regulated (P_SAG12–IPT), thus preventing developmental abnormalities. Plants were grown with growth‐limiting nitrogen supply. Compared to the wild‐type, endogenous levels of free zeatin (Z)‐ and Z riboside (ZR)‐type cytokinins were increased up to 15‐fold (total ZR up to 100‐fold) in senescing leaves, and twofold in younger leaves of P_SAG12–IPT. In these plants, the senescence‐associated declines in N, protein and Rubisco levels and photosynthesis rates were delayed. Senescing leaves accumulated more (¹⁵N‐labelled) N than younger leaves, associated with reduced shoot N accumulation (–60%) and a partially inverted canopy N profile in P_SAG12–IPT plants. While root N accumulation was not affected, N translocation to non‐senescing leaves was progressively reduced. We discuss potential consequences of these modified sink–source relations, associated with delayed leaf senescence, for plant productivity and the efficiency of utilization of light and minerals.     

Isolation of two cytokinin metabolites from the rhizosphere of Norway spruce seedlings (Picea abies L. Karst.)

Roots of young Norway spruce seedlings were incubated under hydroculture conditions in a synthetic nutrient medium containing either ³H-isopentenyladenosine, isopentenyladenosine or zeatin riboside. When feeding with ³H-isopentenyladenosine a new radiaolabelled metabolite was found in the feeding solution as well as in root extracts. Isopentenyladenosine and zeatin riboside were metabolised and for both compounds an unknown metabolite was detected in the feeding solution. The metabolites were purified by solid phase extraction, HPLC and partially characterised. A major characteristic of the metabolites is their reactivity in the presence of NH₄OH, which results in the formation of the cytokinin bases isopentenyladenine or zeatin, respectively. UV-spectra and the chemical characteristics indicate that the new metabolites are closely related. The GC-MS analysis revealed, that the metabolites are true derivatives of isopentenyladenine and zeatin. The biogenesis of the new metabolites is discussed with regard to plant microbial interactions.Abbreviations Ck(s) = cytokinin(s) - GC-MS = gas chromatography-mass spectrometry - iP = isopentenyladenine - [9R]iP = isopentenyladenosine - [9G]iP = isopentenyladenine-9-glucoside - [9R-MP]iP = isopentenyladenosine-5-monophosphate - Z = trans-zeatin - [9R]Z = trans-zeatin riboside     

Effects of temperature and gibberellin on the activity of endogenous cytokinin-like substances in leaves of Begonia x cheimantha

Changes in activity of endogenous cytokinin-like substances were examined in intact plants and excised leaves of Begonia x chemantha Everett cv. Prinsesse Astrid (Christimas Begonia) by means of the tobacco callus bioassay. Cytokinin activity in the leaves of intact plants was higher in plants grown at 18°C than in those grown at 21° or 24°C. In excised leaves, an increase in cytokinin activity was observed during the first 4 days following leaf detachment. However, after the seventh day cylokinin activity decreased again. This decrease was more profound in leaves exposed to 24°C than in those exposed to 18°C.
Treatment of detached leaves with gibberellic acid (2.8 m M ) caused an increase in measurable cytokinin activity. This increase was more profound in the zones of activity which correspond with zeatin glucosides on paper and Sephadex LH-20 chromatography. Additional zones of activity appeared after Sephadex chromatography. These were of a more slow moving nature with elution volumes corresponding to N^b-(Δ²-isopentenyl)adenine and its derivaties. Water-treated control leaves had higher activity in the regions corresponding to zeatin and zeatin riboside.