Isolation of zeatin riboside from the chicory root

Summary From 250 kg of fresh chicory roots about 2 mg of a crystalline cytokinin were obtained. This substance was identified as ribosylzeatin (trans isomer). From the procedure employed it seems unlikely that the isolated cytokinin comes from the degradation of tRNAs; rather, it may constitute a separate pool of cytokinins.     

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.     

Effects of Root Cutting on Cytokinin Content in the Shoot Apex Cells of Arabidopsis Plants

The dependence of cytokinin accumulation in the shoot apexes of Arabidopsis plants on the delivery of these hormones from the roots was studied. For the estimation of cytokinin content in the cells, the immunohistochemical localization method using antibodies against zeatin riboside was used. Differential conjugation of free cytokinin bases and their ribosides was used to prevent washing of cytokinins during the dehydration process. Root cutting decreased the immunostaining of zeatin in the cells of the shoot apical meristem, thereby supporting the hypothesis about dependence of cytokinin accumulation in these cells on the hormone delivery from the roots. The level of cytokinins in the cells of the shoot apex decreased under the effect of protonophore, indicating the important role of the secondary-active transmembrane transport process of cytokinins in the maintenance of their level in the cells of the shoot apex.     

Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity

Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which cytokinins are limiting and that, therefore, they might regulate. We have engineered transgenic Arabidopsis plants that overexpress individually six different members of the cytokinin oxidase/dehydrogenase (AtCKX) gene family and have undertaken a detailed phenotypic analysis. Transgenic plants had increased cytokinin breakdown (30 to 45% of wild-type cytokinin content) and reduced expression of the cytokinin reporter gene ARR5:GUS (beta-glucuronidase). Cytokinin deficiency resulted in diminished activity of the vegetative and floral shoot apical meristems and leaf primordia, indicating an absolute requirement for the hormone. By contrast, cytokinins are negative regulators of root growth and lateral root formation. We show that the increased growth of the primary root is linked to an enhanced meristematic cell number, suggesting that cytokinins control the exit of cells from the root meristem. Different AtCKX-green fluorescent protein fusion proteins were localized to the vacuoles or the endoplasmic reticulum and possibly to the extracellular space, indicating that subcellular compartmentation plays an important role in cytokinin biology. Analyses of promoter:GUS fusion genes showed differential expression of AtCKX genes during plant development, the activity being confined predominantly to zones of active growth. Our results are consistent with the hypothesis that cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of cytokinin functions.     

Design and synthesis of photolabile caged cytokinin

Cytokinins are phytohormones that regulate diverse developmental processes throughout the life of a plant. trans-Zeatin, kinetin, benzyladenine and dihydrozeatin are adenine-type cytokinins that are perceived by the AHK cytokinin receptors. Endogenous cytokinin levels are critical for regulating plant development. To manipulate intracellular cytokinin levels, caged cytokinins were designed on the basis of the crystal structure of the AHK4 cytokinin receptor. The caged cytokinin was photolyzed to release the cytokinin molecule inside the cells and induce cytokinin-responsive gene expression. The uncaging of intracellular caged cytokinins demonstrated that cytokinin-induced root growth inhibition can be manipulated with photo-irradiation. This caged cytokinin system could be a powerful tool for cytokinin biology.     

Phenylurea cytokinins assayed for induction of shoot buds in the moss Funaria hygrometrica

The induction of shoot buds from the filamentous protonema of moss is a classic bioassay for cytokinin. While a large literature documents this response in many species of moss and for a wide range of natural and synthetic cytokinins, to date only substituted adenine cytokinins have been examined in detail. This paper shows that at least some of the novel phenylurea cytokinins will induce bud formation in mosses. Funaria responds to thidiazuron much as it responds to benzyladenine. Exposure to either substance results in log-linear dose-dependent increases in bud number that reach similar maximal numbers of buds at the optimal concentration of compound. The related compound chloro-pyridyl-phenylurea (CPPU) is slightly less active, but induces buds over a wider range of concentration. Carbanilide (diphenylurea or DPU), an active cytokinin in other systems, induces very few buds in Funaria, but does so over a wide range of concentration. Bioassay of mixtures of benzyladenine and DPU finds no evidence of competition for cytokinin receptors. That result could support suggestions that the phenylurea cytokinins act indirectly, by altering endogenous cytokinin metabolism, but we favor another interpretation. Unlike other cytokinin-responsive systems, the induction of buds from moss protonema involves two cytokinin-mediated events. The number of buds is controlled by the second cytokinin-mediated event. If DPU has little or no affinity for the receptor triggering this second event, DPU treatments will produce few to no buds, and kinetic analysis using bud number would find no evidence for competition with benzyladenine. Our results would support the hypothesis that bud induction in Funaria involves two chemically distinct cytokinin receptors.     

The role of cytokinins in relation to flower-bud blasting in Iris cv. Ideal: Cytokinin determination by an improved enzyme-linked immunosorbent assay

The effect of dark and light treatment on endogenous cytokinins in internodes and buds of Iris was determined. Plant material was purified by chromatographic methods and cytokinins were assayed by an immunoassay.An indirect competitive enzyme immunoassay for the determination of zeatin- and isopentenyl-adenine cytokinins was developed. This assay, which is not dependent on the titre of the antibodies raised against zeatin riboside and isopentenyl-adenosine appeared to be specific, highly sensitive and more reproducible compared to a direct competitive enzyme immunoassay for cytokinins.Isopentenyl-adenosine was the most abundant cytokinin found, followed by zeatin: the latter counteracts bud blast when injected into dark-treated plants. Smaller amounts of isopentenyl-adenine and zeatin riboside were found. Results are in agreement with the hypothesis that deficiency of growth substances like cytokinins plays an important role in the occurrence of flower-bud blasting.A possible role for the major endogenous cytokinin, isopentenyl-adenosine, which earlier was found not to be effective in counteracting bud blast when injected into buds of dark-treated plants, is discussed.     

Mechanisms of cytokinin action

Cytokinins are involved in the control of numerous and important processes associated with plant growthand development. They take part in the control of cell division, chloroplast development, bud differentiation, shoot initiationand growth or leaf senescence. In contrast to the wide knowledge of cytokinin effects, the mechanisms of cytokinin actionremain largely unknown. Therefore, it is still difficult to explain how a group of molecules can control so many different biological responses. In this review, we propose some arguments in order to solve this question. In a first part, we underline that cytokinins act in concert with other signals for the control of biological responses. Therefore, the effects observed in responses to cytokinins would be not only related to cytokinins but would also be dependent on all other environmental and hormonal signals perceived by plant cells. In a second part, we present the different actors which could be involved in the signalling pathways of cytokinins. It is very likely that these different elements could be implicated in different cytokinin signalling pathways. Therefore, we propose that the diversity of the cytokinin responses could be also correlated with a diversity of cytokinin signalling pathways.     

Cytokinins and Senescence in Lemon Leaves

The effect of leaf age on cytokinin level was studied in Citrus limon (L.) Burm. f. cv. Eureca. It was found that mature 1-year-old leaves and senescing 2-year-old leaves contained considerably more cytokinins than young 3-month-old leaves. No consistent distinct decrease in cytokinin activity was revealed when the leaf passed from the mature to the senescing state. It was concluded that the results cannot be regarded as supporting the hypothesis that leaf senescence is brought about, at least partially, by a decrease in the level of its cytokinins.     

CYTOKININ INDEPENDENT-1 regulates levels of different forms of cytokinin in Arabidopsis and mediates response to nutrient stress

The gene CYTOKININ INDEPENDENT-1 (CKI-1), previously isolated by enhancer trap screening, has been hypothesised to play a role in cytokinin perception. Alternative hypotheses suggest that it is required for the production of cytokinins or that it has no direct role in cytokinin signalling but simply interferes with the pathway when overexpressed. These hypotheses were investigated by producing transgenic Arabidopsis plants expressing CKI-1 cDNA in antisense orientation. In standard conditions, the phenotype of the plants was similar to wild type. Significantly higher amounts of the free base and riboside forms of cytokinin and lower amounts of membrane-impermeable cytokinins were found in the antisense lines. This supports the hypothesis that CKI-1 is involved in cytokinin perception and demonstrates the existence of a feedback loop altering cytokinin metabolism in response to the level of receptor abundance. An elevation in the content of free bases and ribosides of zeatin and isopentenyladenine, along with a reduction in the content of ribotide forms, suggests that a cytokinin ribotide 5'-ribonucleotidase may be a site at which CKI-1 exerts feedback control. When seed homozygous for the transgene was germinated on medium with reduced total mineral nutrient levels, the cotyledons of seedlings with reduced levels of CKI-1 failed to expand and green, and vegetative growth was inhibited. A similar phenotype was observed on low-phosphate media, suggesting that this failure resulted from an interaction between phosphate and cytokinins.     

Cytokinin biosynthesis and interconversion

To maintain hormone homeostasis, the rate of cytokinin biosynthesis, interconversion, and degradation is regulated by enzymes in plant cells. Cytokinins can be synthesized via direct (de novo) or indirect (tRNA) pathways. In the de novo pathway, a cytokinin nucleotide is synthesized from 5'-AMP and isopentenyl pyrophosphate; a key enzyme which catalyzes this synthesis has been isolated from plant tissues, slime mold, and some microorganisms. Studies on the in vitro synthesis of the isopentenyl side chain of cytokinin in tRNA demonstrated that the isopentenyl group was derived from mevalonate, and turnover of the cytokinin-containing tRNA may serve as a minor source of free cytokinins in plant cells. The interconversion of cytokinin bases, nucleosides and nucleotides is a major feature of cytokinin metabolism; and enzymes that regulate the interconversion have been identified. The N⁶-side chain and purine moiety of cytokinins are often modified and some of the enzymes involved in the modifications have been isolated. Most of the cytokinin metabolites have been characterized but very few enzymes regulating their metabolism have been purified to homogeneity. It remains a significant challenge to isolate plant genes involved in the regulation of cytokinin biosynthesis, interconversion and degradation.     

Growth-Regulating Substances and the Growth of Tiller Buds in Barley; Effects of Cytokinins

An hypothesis was set up from which it was predicted that applicationof cytokinin to barley seedlings grown without mineral nutrientswould lead to rapid growth of the coleoptile and first leaftiller buds. Application of cytokinins to the leaves was ineffective,but supplying a number of known cytokinins by steeping the rootsof 4 d old seedlings in solution for 4 h led to significantgrowth of the coleoptile bud. Adenine and cytokinin analogueshad no effect. Supplying cytokinins through the roots also furtherenhanced the growth of buds of plants given mineral nutrients.Cytokinin treatment reduced root dry matter, with small reductionsin mean axis length and number of lateral roots. For plantsnot given mineral nutrients reduction in root weight was compensatedby an increase in weight of the aerial parts; however, for plantssupplied with mineral nutrients this was not so and the lowerroot weight resulted in a smaller total plant dry weight. An interpretation of tiller bud growth in terms of control byinteracting effects of mineral nutrition, assimilate supply,and cytokinin availability is proposed.     

Does Cytokinin Transport from Root-To-Shoot in the Xylem Sap Regulate Leaf Responses to Root Hypoxia?

We have examined the hypothesis that cytokinins transportedfrom roots to shoots affects leaf growth, stomatal conductance,and cytokinin concentration of leaves of Phaseolus and a hybridpoplar (Populus trichocarpa x Populus deltoides) with hypoxicroots. Because cytokinins may interact with other substances,potassium and calcium concentrations were determined in xylemsap of Populus plants with hypoxic and aerated roots while gibberellin(GA) concentrations were measured in shoot tissues. Root hypoxiadecreased leaf growth and closed stomata in both species. Inboth species, fluxes of cytokinins out of the roots were reduced,but no differences in bulk leaf concentrations were measuredbetween the hypoxic and aerated plants. Shoots with aeratedroots contained slightly higher concentrations of GA₁ and GA₃than shoots from hypoxic plants. There were no differences incalcium or potassium concentrations in xylem sap between aerationtreatments. Exogenously applied cytokinins did not alleviatethe growth or stomatal responses caused by root hypoxia. Informationon the site(s) and mechanism(s) of cytokinin action and theways in which cytokinins are compartmentalized within plantcells will be required to understand the physiological significanceof cytokinin transport in the transpirational stream. Key words: Cytokinins, hypoxia, Populus, Phaseolus     

Extracellular invertase is an essential component of cytokinin-mediated delay of senescence

Leaf senescence is the final stage of leaf development in which the nutrients invested in the leaf are remobilized to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, exogenous application of cytokinins or an increase of the endogenous concentration delays senescence and causes nutrient mobilization. The finding that extracellular invertase and hexose transporters, as the functionally linked enzymes of an apolasmic phloem unloading pathway, are coinduced by cytokinins suggested that delay of senescence is mediated via an effect on source-sink relations. This hypothesis was further substantiated in this study by the finding that delay of senescence in transgenic tobacco (Nicotiana tabacum) plants with autoregulated cytokinin production correlates with an elevated extracellular invertase activity. The finding that the expression of an extracellular invertase under control of the senescence-induced SAG12 promoter results in a delay of senescence demonstrates that effect of cytokinins may be substituted by these metabolic enzymes. The observation that an increase in extracellular invertase is sufficient to delay leaf senescence was further verified by a complementing functional approach. Localized induction of an extracellular invertase under control of a chemically inducible promoter resulted in ectopic delay of senescence, resembling the naturally occurring green islands in autumn leaves. To establish a causal relationship between cytokinins and extracellular invertase for the delay of senescence, transgenic plants were generated that allowed inhibition of extracellular invertase in the presence of cytokinins. For this purpose, an invertase inhibitor was expressed under control of a cytokinin-inducible promoter. It has been shown that senescence is not any more delayed by cytokinin when the expression of the invertase inhibitor is elevated. This finding demonstrates that extracellular invertase is required for the delay of senescence by cytokinins and that it is a key element of the underlying molecular mechanism.     

The binding of kinetin to plant ribosomes

The synthetic cytokinins kinetin and 6-benzylaminopurine exhibit equilibrium-type binding to purified chinese-cabbage leaf ribosomes. At 23mum and 4 degrees C one molecule of kinetin and 1.34 molecules of 6-benzylaminopurine are bound per ribosome. Adenine and adenine derivatives that are inactive as cytokinins showed much less affinity for ribosomes. Pretreatment of ribosomes with 0.5m-ammonium chloride or Triton X-100 did not decrease the extent of cytokinin binding. Binding appeared to be to the 83S ribosome species. A positive correlation between the extent of binding and the biological effect of various cytokinin analogues was demonstrated. These results are discussed in terms of cytokinin control of growth processes at the ribosomal level.     

Exogenous zeatin accumulation in wheat root cells shows its role in regulation of cytokinin transport

Here we have shown that 24 hours after addition of zeatin to the nutrient solution the cytokinin content in xylem sap of wheat plants appears to be about 10 times lower that in the nutrient solution. Cytokinins accumulated mostly in roots and not in shoots of treated plants. These data demonstrate the existence of some barrier on cytokinin pathway from the nutrient solution to the plant shoot. With the help of Sudan III an increase in lignin and suberin deposition in the endodermis could be detected, being stronger with the increase in the distance from the root tip. The increase in deposition of suberin and lignin coincided with the decrease in cytokinin immunolabeling in root cells revealed with the help of monoclonal cytokinin antibodies and the second gold-labelled antibodies. Simultaneously exogenous cytokinins accumulated in root stele cells showing that the Casparian band was not only barrier on cytokinin pathway to plant shoot. It is concluded that high cytokinin immunolabe ling in the stele parenchyma cells around the stele vessels demonstrated accumulation of cytokinins by these cells, which could be important in regulation of cytokinin loading to the xylem vessels during there transport to the shoot. The role of cytokinin transporters is discussed.     

The role of adenine in the synthesis of cytokinins in tomato plants and in cell-free root extracts

The incorporation of labelled adenine into cytokinin-like compounds was investigated in intact tomato plants, decapitated tomato roots and cell-free root extracts. In all three cases evidence was found for the incorporation of adenine into endogenous cytokinins. In intact plants and decapitated root systems no evidence was found for the incorporation into cytokinin nucleotides. Cytokinin bases and nucleosides were however, labelled. In the cell-free root extract there was some evidence for the incorporation of labelled adenine into cytokinin nucleotides. This suggests that the biosynthetic process may be strictly compartmentalized. The present results provide no evidence for the relative importance of cytokinin nucleotides in the biosynthetic process. What is clear is that the rate of adenine incorporation into cytokinins is extremely low and that only a small proportion of the cytokinins which became labelled were exported to the shoot via the root exudate.The financial support of the CSIR/Israel Collaborative Programme is gratefully acknowledged.     

Cytokinin biosynthesis and perception

Cytokinin has been considered to be a master regulator of plant growth and development, but only in the past several years has substantial progress been made uncovering the roles of cytokinins at various developmental stages. Recent studies on key metabolic enzymes and signaling components have contributed to understanding the basic mechanism of biosynthesis and perception of cytokinin within a whole plant body. The initial products of de novo cytokinin biosynthesis in higher plants and Agrobacterium are different, and the regulatory systems in biosynthesis and homeostasis are finely controlled and appear to be important in communicating nutrient signals to morphogenetic responses. The cytokinin receptors have largely overlapping, but still specific, functions in diverse cytokinin responses. In this review, we will specifically emphasize the biosynthesis of isoprenoid cytokinins and perception of cytokinin signals in Arabidopsis.     

Characterization of cytokinin and adenine transport in Arabidopsis cell cultures

Cytokinins are distributed through the vascular system and trigger responses of target cells via receptor-mediated signal transduction. Perception and transduction of the signal can occur at the plasma membrane or in the cytosol. The signal is terminated by the action of extra- or intracellular cytokinin oxidases. While radiotracer studies have been used to study transport and metabolism of cytokinins in plants, little is known about the kinetic properties of cytokinin transport. To provide a reference dataset, radiolabeled trans-zeatin (tZ) was used for uptake studies in Arabidopsis (Arabidopsis thaliana) cell culture. Uptake kinetics of tZ are multiphasic, indicating the presence of both low- and high-affinity transport systems. The protonophore carbonyl cyanide m-chlorophenylhydrazone is an effective inhibitor of cytokinin uptake, consistent with H(+)-mediated uptake. Other physiological cytokinins, such as isopentenyl adenine and benzylaminopurine, are effective competitors of tZ uptake, whereas allantoin has no inhibitory effect. Adenine competes for zeatin uptake, indicating that the degradation product of cytokinin oxidases is transported by the same systems. Comparison of adenine and tZ uptake in Arabidopsis seedlings reveals similar uptake kinetics. Kinetic properties, as well as substrate specificity determined in cell cultures, are compatible with the hypothesis that members of the plant-specific purine permease family play a role in adenine transport for scavenging extracellular adenine and may, in addition, be involved in low-affinity cytokinin uptake.