Cytokinin hormones are important regulators of development and environmental responses of plants that execute their action via the molecular machinery of signal perception and transduction. The limiting step of the whole process is the availability of the hormone in suitable concentrations in the right place and at the right time to interact with the specific receptor. Hence, the hormone concentrations in individual tissues, cells, and organelles must be properly maintained by biosynthetic and metabolic enzymes. Although there are merely two active cytokinins, isopentenyladenine and its hydroxylated derivative zeatin, a variety of conjugates they may form and the number of enzymes/isozymes with varying substrate specificity involved in their biosynthesis and conversion gives the plant a variety of tools for fine tuning of the hormone level. Recent genome-wide studies revealed the existence of the respective coding genes and gene families in plants and in some bacteria. This review summarizes present knowledge on the enzymes that synthesize cytokinins, form cytokinin conjugates, and carry out irreversible elimination of the hormones, including their phylogenetic analysis and possible variations in different organisms.

Cytokinins (CKs) are plant hormones affecting numerous developmental processes. Zeatin and its derivatives are the most important group of isoprenoid CKs. Zeatin occurs as two isomers: while trans-zeatin (transZ) was found to be a bioactive substance, cis-zeatin (cisZ) was reported to have a weak biological impact. Even though cisZ derivatives are abundant in various plant materials their biological role is still unknown. The comprehensive screen of land plants presented here suggests that cisZ-type CKs occur ubiquitously in the plant kingdom but their abundance might correlate with a strategy of life rather than with evolutionary complexity. Changing levels of transZ and cisZ during Arabidopsis ontogenesis show that levels of the two zeatin isomers can differ significantly during the life span of the plant, with cisZ-type CKs prevalent in the developmental stages associated with limited growth. A survey of the bioassays employed illustrates mild activity of cisZ and its derivatives. No cis↔trans isomerization, which would account for the effects of cisZ, was observed in tobacco cells and oat leaves. Differences in uptake between the two isomers resulting in distinct bioactivity have not been detected. In contrast, cisZ and transZ have a different metabolic fate in oat and tobacco. Analysis of a CK-degrading enzyme, cytokinin oxidase/dehydrogenase (CKX), reveals that Arabidopsis possesses two isoforms, AtCKX1 expressed in stages of active growth, and AtCKX7, both of which have the highest affinity for the cisZ isomer. Based on the present results, the conceivable function of cisZ-type CKs as delicate regulators of CK responses in plants under growth-limiting conditions is hypothesized.

Decades ago, the importance of cytokinins (CKs) during Rhodococcus fascians pathology had been acknowledged, and an isopentenyltransferase gene had been characterized in the fas operon of the linear virulence plasmid, but hitherto, no specific CK(s) could be associated with virulence. We show that the CK receptors AHK3 and AHK4 of Arabidopsis thaliana are essential for symptom development, and that the CK perception machinery is induced upon infection, underlining its central role in the symptomatology. Three classical CKs [isopentenyladenine, trans-zeatin, and cis-zeatin (cZ)] and their 2-methylthio (2MeS)-derivatives were identified by CK profiling of both the pathogenic R. fascians strain D188 and its nonpathogenic derivative D188 -5. However, the much higher CK levels in strain D188 suggest that the linear plasmid is responsible for the virulenceassociated production. All R. fascians CKs were recognized by AHK3 and AHK4, and, although they individually provoked typical CK responses in several bioassays, the mixture of bacterial CKs exhibited clear synergistic effects. The cis-and 2MeS-derivatives were poor substrates of the apoplastic CK oxidase/dehydrogenase enzymes and the latter were not cytotoxic at high concentrations. Consequently, the accumulating 2MeScZ (and cZ) in infected Arabidopsis tissue contribute to the continuous stimulation of tissue proliferation. Based on these results, we postulate that the R. fascians pathology is based on the local and persistent secretion of an array of CKs.phytopathogen ͉ actinomycete ͉ phytohormone T he fine-tuned balance of plant regulators has a key role in the growth and development of plants. Many plant-associated bacteria can influence their hosts by either modulating the phytohormone production or producing the phytohormones themselves. The main advantages for the bacteria are increased nutrient release, suppression of defense, and/or disease establishment (1, 2). Hyperplasia-inducing bacteria, such as Pantoea agglomerans and Pseudomonas savastanoi, secrete high amounts of cytokinins (CKs) and auxins to facilitate or initiate gall development (3, 4), and Agrobacterium tumefaciens genetically transforms plant cells to convert them into CK and auxin (and opine) factories (5).In contrast to the undifferentiated galls induced by the bacteria mentioned above, the Actinomycete Rhodococcus fascians that shares persistence strategies with the closely related human pathogen Mycobacterium tuberculosis (6) provokes the formation of differentiated leafy galls, consisting of numerous shoot primordia whose further outgrowth is inhibited (7). The shooty symptoms can be partially mimicked by exogenous addition of CKs (8, 9), and analyses of culture supernatants of different nonisogenic virulent and avirulent R. fascians strains grown under rich culture conditions identified 11 different CKs: methylaminopurine, 2-methylthioisopentenyladenine (2MeSiP), iP, cis-zeatin (cZ), trans-zeatin (tZ), dihydrozeatin (DZ), 2MeScZ, and their respective ribosides (10-14). Except for iP, the p...

Stable RNA interference-based technology was used to silence the expression of the HvCKX1 gene in barley and the TaCKX1 gene in wheat and triticale. The silencing cassettes containing the fragments of these genes in the sense and antisense orientations were cloned into the pMCG161 binary vector and used for Agrobacterium-based transformation. Out of the five cultivars representing the three studied species, transgenic plants were obtained from one barley cultivar Golden Promise, one wheat cultivar Kontesa, and one triticale cultivar Wanad. Almost 80% of 52 regenerated lines of Golden Promise exhibited significantly decreased cytokinin oxidase/dehydrogenase (CKX) enzyme activity in bulked samples of their T(1) roots. There was a positive correlation between the enzyme activity and the plant productivity, expressed as the yield, the number of seeds per plant, and the 1000 grain weight. Additionally, these traits were associated with a greater root mass. Lower CKX activity led to a higher plant yield and root weight. This higher plant productivity and altered plant architecture were maintained in a population of segregating T(1) plants. The levels of HvCKX1 transcript accumulation were measured in various tissues of Golden Promise and Scarlett non-transgenic barley plants in order to choose the most appropriate plant organs to study the expression and/or silencing of the gene in those transgenic lines. The highest levels of the HvCKX1 transcript were detected in spikes 0 days after pollination (0 DAP), 7 DAP, and 14 DAP, and in the seedling roots. The analysis of HvCKX1 gene expression and CKX enzyme activity and the evaluation of the phenotype were performed in the progeny of seven selected transgenic T(1) lines. The relative expression of HvCKX1 measured in the spikes 0 DAP and 14 DAP, respectively, ranged from 0.52+/-0.04 to 1.15+/-0.26 and from 0.47+/-0.07 to 0.89+/-0.15. The lowest relative values were obtained for the enzyme activity in the spikes at 0 DAP, which ranged from 0.15+/-0.02 to 1.05+/-0.14 per single progeny plant. Based on these three values, the coefficient of HvCKX1 silencing in the spikes was estimated. Possible mechanisms leading to higher plant productivity via the silencing of HvCKX1 and a decrease in CKX enzyme activity are discussed.

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Plant hormones, cytokinins (CKs), have been for a long time considered to be involved in plant responses to stress. However, their exact roles in processes linked to stress signalization and acclimatization to adverse environmental conditions are unknown. In this study, expression profiles of the entire gene families of CK biosynthetic and degradation genes in maize (Zea mays) during development and stress responses are described. Transcript abundance of particular genes is discussed in relation to the levels of different CK metabolites. Salt and osmotic stresses induce expression of some CK biosynthetic genes in seedlings of maize, leading to a moderate increase of active forms of CKs lasting several days during acclimatization to stress. A direct effect of CKs to mediate activation of stress responses does not seem to be possible due to the slow changes in metabolite levels. However, expression of genes involved in cytokinin signal transduction is uniformly down-regulated within 0.5 h of stress induction by an unknown mechanism. cis-Zeatin and its derivatives were found to be the most abundant CKs in young maize seedlings. We demonstrate that levels of this zeatin isomer are significantly enhanced during early stress response and that it originates independently from de novo biosynthesis in stressed tissues, possibly by elevated specific RNA degradation. By enhancing their CK levels, plants could perhaps undergo a reduction of growth rates maintained by abscisic acid accumulation in stressed tissues. A second role for cytokinin receptors in sensing turgor response is hypothesized besides their documented function in CK signaling.

Species of the Fusarium fujikuroi species complex (FFC) cause a wide spectrum of often devastating diseases on diverse agricultural crops, including coffee, fig, mango, maize, rice, and sugarcane. Although species within the FFC are difficult to distinguish by morphology, and their genes often share 90% sequence similarity, they can differ in host plant specificity and life style. FFC species can also produce structurally diverse secondary metabolites (SMs), including the mycotoxins fumonisins, fusarins, fusaric acid, and beauvericin, and the phytohormones gibberellins, auxins, and cytokinins. The spectrum of SMs produced can differ among closely related species, suggesting that SMs might be determinants of host specificity. To date, genomes of only a limited number of FFC species have been sequenced. Here, we provide draft genome sequences of three more members of the FFC: a single isolate of F. mangiferae, the cause of mango malformation, and two isolates of F. proliferatum, one a pathogen of maize and the other an orchid endophyte. We compared these genomes to publicly available genome sequences of three other FFC species. The comparisons revealed species-specific and isolate-specific differences in the composition and expression (in vitro and in planta) of genes involved in SM production including those for phytohormome biosynthesis. Such differences have the potential to impact host specificity and, as in the case of F. proliferatum, the pathogenic versus endophytic life style.

The phytopathogenic actinomycete Rhodococcus fascians D188 relies mainly on the linear plasmid-encoded fas operon for its virulence. The bacteria secrete six cytokinin bases that synergistically redirect the developmental program of the plant to stimulate proliferation of young shoot tissue, thus establishing a leafy gall as a niche. A yeast-based cytokinin bioassay combined with cytokinin profiling of bacterial mutants revealed that the fas operon is essential for the enhanced production of isopentenyladenine, trans-zeatin, cis-zeatin, and the 2-methylthio derivatives of the zeatins. Cytokinin metabolite data and the demonstration of the enzymatic activities of FasD (isopentenyltransferase), FasE (cytokinin oxidase/dehydrogenase), and FasF (phosphoribohydrolase) led us to propose a pathway for the production of the cytokinin spectrum. Further evaluation of the pathogenicity of different fas mutants and of fas gene expression and cytokinin signal transduction upon infection implied that the secretion of the cytokinin mix is a highly dynamic process, with the consecutive production of a tom initiation wave followed by a maintenance flow.Rhodococcus fascians is a phytopathogenic actinomycete with a very broad host range that causes important commercial losses in the ornamentals industry because it triggers severe malformations of shoots, referred to as leafy galls (Depuydt et al. 2008b). In strain D188, the virulence determinants are encoded by a large conjugative linear plasmid, pFiD188 , and the pathology is induced by the secretion of a mix of six synergistically acting cytokinins: isopentenyladenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and their 2-methylthio (2MeS) derivatives (Pertry et al. 2009). In Arabidopsis thaliana, these cytokinins are perceived by the receptors AHK3 and CRE1/AHK4 (Pertry et al. 2009), activating a signaling cascade that stimulates cell proliferation and meristematic gene expression and, ultimately, results in the establishment of a specific niche (Depuydt et al. 2008a.Comparison of the cytokinin profiles of two near-isogenic strains, D188 and its plasmid-free derivative, D188-5, has shown that a basal level of the six cytokinins is produced by a chromosomally encoded pathway. However, the much higher levels of iP, cZ, tZ, 2MeScZ, and 2MeStZ secreted by strain D188 strongly suggest an additional linear plasmid-encoded de novo biosynthetic pathway (Pertry et al. 2009). By sequence analysis of pFiD188, the fas operon was identified, consisting of six genes putatively involved in cytokinin biosynthesis and essential for virulence (Fig. 1). FasA is similar to P450-type cytochrome monooxygenases. The N-terminal region of FasB corresponds to 4Fe-3S-type ferredoxins of Actinomycetes, whereas its carboxy-terminus is homologous to the α subunit of pyruvate dehydrogenase. FasC is similar to the β subunit of the latter enzyme. Both FasB and FasC have a binding site for the cofactor thiamine pyrophosphate (Crespi et al. 1994). FasD is an isopentenyltransferase (Ipt) protein that mediates th...