Cytokinin Conjugates Research Articles

Systemic strategies for cytokinin biosynthesis and catabolism in Arabidopsis roots and leaves under prolonged ammonium nutrition

Cytokinins are growth-regulating plant hormones that are considered to adjust plant development under environmental stresses. During sole ammonium nutrition, a condition known to induce growth retardation of plants, altered cytokinin content can contribute to the characteristic ammonium toxicity syndrome. To understand the metabolic changes in cytokinin pools, cytokinin biosynthesis and degradation were analyzed in the leaves and roots of mature Arabidopsis plants. We found that in leaves of ammonium-grown plants, despite induction of biosynthesis on the expression level, there was no active cytokinin build-up because they were effectively routed toward their downstream catabolites. In roots, cytokinin conjugation was also induced, together with low expression of major synthetic enzymes, resulting in a decreased content of the trans-zeatin form under ammonium conditions. Based on these results, we hypothesized that in leaves and roots, cytokinin turnover is the major regulator of the cytokinin pool and does not allow active cytokinins to accumulate. A potent negative-regulator of root development is trans-zeatin, therefore its low level in mature root tissues of ammonium-grown plants may be responsible for occurrence of a wide root system. Additionally, specific cytokinin enhancement in apical root tips may evoke a short root phenotype in plants under ammonium conditions. The ability to flexibly regulate cytokinin metabolism and distribution in root and shoot tissues can contribute to adjusting plant development in response to ammonium stress.

Phytohormone profiling in an evolutionary framework

The genomes of charophyte green algae, close relatives of land plants, typically do not show signs of developmental regulation by phytohormones. However, scattered reports of endogenous phytohormone production in these organisms exist. We performed a comprehensive analysis of multiple phytohormones in Viridiplantae, focusing mainly on charophytes. We show that auxin, salicylic acid, ethylene and tRNA-derived cytokinins including cis-zeatin are found ubiquitously in Viridiplantae. By contrast, land plants but not green algae contain the trans-zeatin type cytokinins as well as auxin and cytokinin conjugates. Charophytes occasionally produce jasmonates and abscisic acid, whereas the latter is detected consistently in land plants. Several phytohormones are excreted into the culture medium, including auxin by charophytes and cytokinins and salicylic acid by Viridiplantae in general. We note that the conservation of phytohormone biosynthesis and signaling pathways known from angiosperms does not match the capacity for phytohormone biosynthesis in Viridiplantae. Our phylogenetically guided analysis of established algal cultures provides an important insight into phytohormone biosynthesis and metabolism across Streptophyta.

Mini-review: modern N9-substituted sugar cytokinin conjugates – synthesis, bioactivity, and use in tissue cultures

Mini-review: modern N9-substituted sugar cytokinin conjugates – synthesis, bioactivity, and use in tissue cultures

Limited light intensity and low temperature: Can plants survive freezing in light conditions that more accurately replicate the cold season in temperate regions?

Plants in temperate regions have evolved mechanisms that enable them to survive sudden temperature drops. Experiments with plants grown in long-day conditions, in which they are most sensitive to freezing stress, indicate that the cold acclimation mechanism is light-dependent and does not fully operate under low light intensity. However, winter annuals like Arabidopsis thaliana Col-0 germinate in the fall, overwinter as rosettes, and thus must acclimate under short photoperiods and low irradiance. Thus, we have analysed effects of variations in light intensity in plants grown under short-day photoperiod at the 1.14 growth stage (14 rosette leaves). Plants were acclimated at 4 °C for seven days under control and limited-light conditions: 100 and 20 μmol m-2s-1 photosynthetic photon flux density (PPFD), respectively. All cold-acclimated plants accumulated molecular markers reportedly associated with acquired freezing tolerance, including proline, sucrose, cold-responsive gene transcripts, dehydrins and low temperature-induced proteins. Observed changes (and similarity of freezing stress survival rates of plants in both light conditions) indicate that low PPFD did not inhibit the cold acclimation process. The molecular analysis identified distinct PPFD-specific adaptation mechanisms manifested in contrasting contents of anthocyanins, cytokinin conjugates, photosystem proteins, and enzymes involved in protein, energy, and reactive oxygen species metabolism. Finally, the results identify putative proteins and metabolite markers correlating with susceptibility to freezing stress of non-acclimated plants grown under low PPFD. Our data show that Arabidopsis plants grown under short-day photoperiods can be fully cold-acclimated under limited light conditions, employing standard and PPFD-specific pathways.

New Insights Into the Metabolism and Role of Cytokinin N-Glucosides in Plants.

Cytokinin (CK) N-glucosides are the most abundant group of CK metabolites in many species; however, their physiological role in planta was for a long time perceived as irreversible storage CK forms only. Recently, a comprehensive screen showed that only vascular plants form CK N-glucosides in contrast to mosses, algae, and fungi. The formation of CK N-glucosides as biologically inactive CK conjugates thus represents an evolutionarily young mechanism for deactivation of CK bases. Even though CK N-glucosides are not biologically active themselves due to their inability to activate the CK perception system, new data on CK N-glucoside metabolism show that trans-zeatin (tZ) N7- and N9-glucosides are metabolized in vivo, efficiently releasing free CK bases that are most probably responsible for the biological activities observed in a number of bioassays. Moreover, CK N-glucosides’ subcellular localization as well as their abundance in xylem both point to their possible plasma membrane transport and indicate a role also as CK transport forms. Identification of the enzyme(s) responsible for the hydrolysis of tZ N7- and N9-glucosides, as well as the discovery of putative CK N-glucoside plasma membrane transporter, would unveil important parts of the overall picture of CK metabolic interconversions and their physiological importance.

Naturally Occurring and Artificial N9-Cytokinin Conjugates: From Synthesis to Biological Activity and Back.

Cytokinins and their sugar or non-sugar conjugates are very active growth-promoting factors in plants, although they occur at very low concentrations. These compounds have been identified in numerous plant species. This review predominantly focuses on 9-substituted adenine-based cytokinin conjugates, both artificial and endogenous, sugar and non-sugar, and their roles in plants. Acquired information about their biological activities, interconversions, and metabolism improves understanding of their mechanisms of action and functions in planta. Although a number of 9-substituted cytokinins occur endogenously, many have also been prepared in laboratories to facilitate the clarification of their physiological roles and the determination of their biological properties. Here, we chart advances in knowledge of 9-substituted cytokinin conjugates from their discovery to current understanding and reciprocal interactions between biological properties and associated structural motifs. Current organic chemistry enables preparation of derivatives with better biological properties, such as improved anti-senescence, strong cell division stimulation, shoot forming, or more persistent stress tolerance compared to endogenous or canonical cytokinins. Many artificial cytokinin conjugates stimulate higher mass production than naturally occurring cytokinins, improve rooting, or simply have high stability or bioavailability. Thus, knowledge of the biosynthesis, metabolism, and activity of 9-substituted cytokinins in various plant species extends the scope for exploiting both natural and artificially prepared cytokinins in plant biotechnology, tissue culture, and agriculture.

Regulatory Cross-Talks and Cascades in Rice Hormone Biosynthesis Pathways Contribute to Stress Signaling.

Crosstalk among different hormone signaling pathways play an important role in modulating plant response to both biotic and abiotic stress. Hormone activity is controlled by its bio-availability, which is again influenced by its biosynthesis. Thus, independent hormone biosynthesis pathways must be regulated and co-ordinated to mount an integrated response. One of the possibilities is to use cis-regulatory elements to orchestrate expression of hormone biosynthesis genes. Analysis of CREs, associated with differentially expressed hormone biosynthesis related genes in rice leaf under Magnaporthe oryzae attack and drought stress enabled us to obtain insights about cross-talk among hormone biosynthesis pathways at the transcriptional level. We identified some master transcription regulators that co-ordinate different hormone biosynthesis pathways under stress. We found that Abscisic acid and Brassinosteroid regulate Cytokinin conjugation; conversely Brassinosteroid biosynthesis is affected by both Abscisic acid and Cytokinin. Jasmonic acid and Ethylene biosynthesis may be modulated by Abscisic acid through DREB transcription factors. Jasmonic acid or Salicylic acid biosynthesis pathways are co-regulated but they are unlikely to influence each others production directly. Thus, multiple hormones may modulate hormone biosynthesis pathways through a complex regulatory network, where biosynthesis of one hormone is affected by several other contributing hormones.

Integration of Hormonal and Nutritional Cues Orchestrates Progressive Corolla Opening.

Flower opening is essential for pollination and thus successful sexual reproduction; however, the underlying mechanisms of its timing control remain largely elusive. We identify a unique cucumber (Cucumis sativus) line '6457' that produces normal ovaries when nutrients are under-supplied, and super ovaries (87%) with delayed corolla opening when nutrients are oversupplied. Corolla opening in both normal and super ovaries is divided into four distinct phases, namely the green bud, green-yellow bud, yellow bud, and flowering stages, along with progressive color transition, cytological tuning, and differential expression of 14,282 genes. In the super ovary, cell division and cell expansion persisted for a significantly longer period of time; the expressions of genes related to photosynthesis, protein degradation, and signaling kinases were dramatically up-regulated, whereas the activities of most transcription factors and stress-related genes were significantly down-regulated; concentrations of cytokinins (CKs) and gibberellins were higher in accordance with reduced cytokinin conjugation and degradation and increased expression of gibberellin biosynthesis genes. Exogenous CK application was sufficient for the genesis of super ovaries, suggesting a decisive role of CKs in controlling the timing of corolla opening. Furthermore, 194 out of 11,127 differentially expressed genes identified in pairwise comparisons, including critical developmental, signaling, and cytological regulators, contained all three types of cis-elements for CK, nitrate, and phosphorus responses in their promoter regions, indicating that the integration of hormone modulation and nutritional regulation orchestrated the precise control of corolla opening in cucumber. Our findings provide a valuable framework for dissecting the regulatory pathways for flower opening in plants.

Cytokinin dynamics in differently senescing laminae of Phragmites australis plants grown in different habitats

Common reed is a cosmopolitan species, which occurs also in South Africa. At a bay of the Baltic Sea stands of tall, large-leafed reed (TR) and stands of short, small-leafed reed (SR) were found. While TR grew where deep soil with much humus and phosphate was available, SR was found growing on shallow soil, which was often of relatively high salinity. As changes in coloration and chlorophyll analyses showed, SR laminae senesced earlier than did TR laminae. The differences in growth and senescence were eliminated by transplanting TR and SR into garden earth. As literature data from other species show, soil components like phosphate and NaCl can influence levels of the antisenescence hormones cytokinins. Our aim was to find correlations between cytokinin levels and the vitality in reed laminae from natural sites. We used ELISAs for cytokinin analysis and completed the investigations by LC-MS determination of 17 cytokinins and cytokinin conjugates in laminae of HR and SR grown near Prague. From July to autumn the content of trans-zeatin, dihydrozeatin and their 9-ribosides decreased in third laminae under panicles at TR on an average to 1.3nmol trans-zeatin-9-riboside equivalents kg−1 fresh mass and at SR to 0.7 (8 sample pairs). In autumn SR laminae had also lower levels of N6-(Δ2-isopentenyl)adenine and its 9-riboside and probably also of trans-zeatin-O-glucoside and cis-zeatin-9-riboside-O-glucoside compared to TR laminae. The putatively inactive or weakly active cis-zeatin compound increased during the summer to very high quantities. After isolation of laminae, the content of cytokinins declined and cytokinin application delayed yellowing. Contrary to some other examples, our results are in concordance with the rule that endogenous cytokinins retard leaf senescence and help thus to explain the senescence differences in TR and SR.

Evolution of cytokinin biosynthesis and degradation

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.

Recalcitrant effects associated with the development of basal callus-like tissue on caulogenesis and rhizogenesis in Sclerocarya birrea

In the micropropagation of woody plant species, adventitious root and shoot formation remain some of the major bottlenecks due to their recalcitrance to in vitro manipulation. Some plant growth regulators may ameliorate these recalcitrant effects and improve in vitro caulogenic and rhizogenic processes. Shoot induction on shoot meristems, hypocotyls and epicotyls was evaluated using equimolar concentrations of benzyladenine (BA), meta-topolin (mT), meta-topolin riboside (mTR), and meta-methoxytopolin riboside (MemTR). Three auxins, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA) were used in the induction of adventitious roots. Moderately high shoot formation (62.7%) was achieved at a concentration of 8.0 μM mT after 8 weeks in culture. The highest number of adventitious shoots per explant (2.4 ± 0.3) and the longest shoots (23.5 ± 3.16 mm) were recorded on 8.0 μM mT, though not significantly different from BA treatments. Most shoots progressively produced brown basal callus, which is a potential sink for cytokinin conjugates that are inhibitory to further proliferation of adventitious shoots. Good adventitious shoot formation occurred in 55% of hypocotyl explants on 8.0 μM mT. The highest rooting (91.6%) was achieved with IBA-treated shoots at a concentration of 4.0 μM. The use of mT and IBA provide an efficient micropropagation method for S. birrea, though further research is required especially in overcoming ex vitro plantlet survival challenges.

A 40-bp A/T-rich repressor element involved in organ-dependent transcriptional regulation of ZmGLU1

Maize β-glucosidase (ZmGLU1) hydrolyzes cytokinin conjugates to release free cytokinins, and regulation of its expression is essential to control cytokinin homeostasis during plant growth and development. Although the promoter of ZmGLU1 gene was found to confer its organ-dependent expression, the underlying mechanism remained to be uncovered. In this study, by promoter deletion assay, a 40-bp A/T-rich sequence in ZmGLU1 promoter region ranging from −798 to −758 (T40) was identified as mediating the repression of promoter activity in seeds and shoot but not in root. Additional fusion of T40 upstream of the constitutive cauliflower mosaic virus 35S promoter (35S) suppressed 35S-driven gene expression in shoot and seeds. Electrophoretic mobility shift assay further revealed a specific interaction between T40 and an unknown binding protein in seeds and shoot but not in root. The negative correlation between ZmGLU1 gene expression/promoter activity and the intensity of T40–protein interaction suggests that T40 acts as a repressor cis-element involved in ZmGLU1 organ-dependent expression. Furthermore, this result provides a new A/T-rich sequence exhibiting transcriptional repression in higher plant.

Discoveries and Dilemmas Concerning Cytokinin Metabolism.

Natural cytokinin compounds that function as plant hormones are adenine molecules connected to an N6 side chain. Metabolic pathways produce various structural modifications to the adenine moiety and/or the side chain that affect cytokinin function, activity, stability, and transport in plants. This review focuses on recent research that is elucidating the genes and biochemical pathways involved in cytokinin conjugation, deconjugation, and interconversion. One area of new discovery concerns the process of O-glucoside conjugate formation. Genes have been identified for O-glucosyltransferase enzymes in Phaseolus (ZOG1 and ZOX1) and maize (cisZOG1) and have been used to study enzyme activity, structure, and function in plant development. New information about the genetic basis of the adenine salvage pathway and its relationship to cytokinin interconversion and regulation has also become available. However, various dilemmas about cytokinin metabolism remain and need to be resolved using a combination of research approaches.

Cytokinin conjugation : recent advances and patterns in plant evolution

Cytokinin (CK) conjugates are important in plant development because they regulate active CK concentrations, CK transport, storage, and irreversible inactivation. While numerous CK conjugates have been identified in higher plants, the biological functions of these compounds, their location within cells and tissues, and the enzymes and genes involved in their regulation are not clearly understood. In this paper, recent advances are reported which have occurred through the study of transgenic plants containing the ipt or rolC genes, the identification of new regulatory enzymes affecting CKs, and the characterization of new CK conjugates. In addition, a survey of the literature is presented which examines the pattern of CK conjugates found in different plant taxa. Based on current knowledge, it appears that green algae, mosses, and ferns contain relatively few CK conjugates of isopentenyl adenine (iP) and zeatin (Z). In contrast, higher land plants, such as gymnosperms and angiosperms, contain a more complex set of CKs, primarily conjugates of Z and dihydrozeatin (DHZ). This suggests that the pattern of CK conjugation has become more complex in parallel with the increasing complexity of higher plants.

Expression of the Agrobacterium rhizogenes rolC Gene in a Deciduous Forest Tree Alters Growth and Development and Leads to Stem Fasciation.

We have altered the growth and development of a deciduous forest tree by transforming hybrid aspen (Populus tremula x Populus tremuloides) with the Agrobacterium rhizogenes rolC gene expressed under the strong cauliflower mosaic virus 35S promoter. We demonstrate that the genetically manipulated perennial plants, after a period of dormancy, maintain the induced phenotypical changes during the second growing period. Furthermore, mass-spectrometrical quantifications of the free and conjugated forms of indole-3-acetic acid and cytokinins and several gibberellins on one transgenic line correlate the induced developmental alterations such as stem fasciation to changes in plant hormone metabolism. We also show that the presence of the RolC protein increases the levels of the free cytokinins, but not by a process involving hydrolysis of the inactive cytokinin conjugates.

Chemically induced expression of the rolC‐encoded β‐glucosidase in transgenic tobacco plants and analysis of cytokinin metabolism: rolC does not hydrolyze endogenous cytokinin glucosides in planta

SummaryThe rolC gene of Agrobacterium rhizogenes T‐DNA plays an essential role in the establishment of hairy root disease and its overexpression in transgenic plants causes pleiotropic developmental alterations. This study investigated whether the biological activity of the rolC β‐glucosidase is due to an alteration of the cytokinin balance in planta. HPLC radiocounting assays of [3H]‐labeled cytokinin glucosides fed exogenously to tobacco leaf disks, to rolC expressing Escherichia coli cells or cell‐free extracts showed that cytokinin N3‐ and O‐glucosides are the preferred substrate of the rolC protein. Hydrolysis of N7‐ and N9‐glucosides was not detected at substrate concentrations close to physiological levels. Furthermore, these conjugates were also not active as cytokinins in biotests when fed to rolC‐expressing tissues. For analysis of the rolC activity on endogenous cytokinin conjugates the gene was expressed under the transcriptional control of a modified tetracycline‐inducible 35S promoter. This was done to avoid possible interference with secondary effects or plant homeostatic mechanisms which could mask primary in planta events when transgenes are expressed constitutively. No changes in the endogenous pool of different cytokinin glucosides, as determined by a newly developed electrospray tandem mass spectroscopy directly coupled to high performance liquid chromatography, were found following chemical induction of the rolC gene. Also the levels of free cytokinins remained unchanged after gene induction. Hybrid tobacco plants expressing the cytokinin‐synthezising ipt gene and the rolC gene showed added phenotypes indicating that the rolC phenotype is mediated on a signalling pathway different from those of cytokinins. RolC/ipt hybrids also accumulated high levels of cytokinin O‐glucosides. It is concluded that the phenotypic alterations caused by the rolC gene product are not due to a release of free cytokinins from inactive conjugates, most likely because of subcellular compartmentation of the putative substrate.

Identification of a benzyladenine disaccharide conjugate produced during shoot organogenesis in Petunia leaf explants.

Prior studies of benzyladenine (BA) metabolism in Petunia hybrida Vilm. leaf explants during shoot organogenesis revealed the presence of an abundant unidentified BA conjugate. The BA conjugate, compound C, made up to 39% of the total pool of BA conjugates in two Petunia lines and was associated with an increased shoot organogenic response when compared with a third Petunia line that did not produce any compound C. Structural analysis of compound C utilizing fast atom bombardment mass spectrometry, two methods of carbohydrate analysis, ultraviolet absorption spectra, and Fourier transform infrared spectra identified it as a new cytokinin conjugate, 6-benzylamino-9-[O-glucopyranosyl-(1-->3)-ribofuranosyl]-purine. Based on our prior biological studies and the similarity of this compound to related cytokinin conjugates, this disaccharide cytokinin conjugate may be part of the interconvertible pool of cytokinins active in Petunia shoot organogenesis.

667 (PS 4) IDENTIFICATION OF A NEW BENZYL ADENINE COMPOUNDPRODUCED DURING PETUNIA SHOOT ORGANOGENESIS

Studies of benzyl adenine (BA) uptake and metabolism in Petunia leaf explants during shoot organogenesis revealed a novel BA conjugate. The new conjugate comprised up to 39% of the total pool of BA conjugates in two petunia lines and was associated with an increased shoot organogenic response when compared with a third petunia line which did not produce the conjugate. To identify the new compound, structural analysis was conducted using FAB mass spectrometry, two methods of carbohydrate analysis, UV absorption spectra and FTIR spectra. Experimental results identified the compound as 6-benzylamino-9-[O-glucopyranosyl-(1→3)-ribofuranosyl]-purine, a disaccharide conjugate of BA. Based on biological studies and similarity to other cytokinin conjugates, this disaccharide cytokinin conjugate may be part of the interconvertible pool of cytokinins active in shoot organogenesis.

Cytokinins in the Leaves of Ginkgo biloba

Cytokinin conjugates of zeatin, ribosylzeatin, and their respective dihydro derivatives tentatively have been identified as the major cytokinins present in mature Ginkgo biloba L. leaves. Ribosylzeatin was present in higher levels than zeatin and dihydrozeatin. No evidence could be found that 6-(2,3,4-trihydroxy-3-methylbutylamino)purine occurs as a metabolite in the mature leaves. From the available evidence, it is concluded that cytokinin conjugates are probably the major metabolites formed in the leaves of this deciduous gymnosperm.

Regulators of cell division in plant tissues XXIX. The activities of cytokinin glucosides and alanine conjugates in cytokinin bioassays

In a number of cytokinin bioassays, the activities of the following compounds were compared: 3-, 7-, and 9-glucosides of 6-benzylaminopurine (BAP); 7- and 9-glucosides of zeatin; O-glucosides of zeatin, dihydrozeatin, and their ribosides; 9-alanine conjugates of zeatin, and BAP. The bioassays included the radish cotyledon, theAmaranthus betacyanin, the oat leaf senescence, and the tobacco pith callus. Cytokinin activity was markedly reduced by 7- and 9-glucosylation in nearly all bioassays, but 3-glucosylation of BAP and O-glucosylation of the zeatin sidechain usually had little effect on activity. However, there were two notable exceptions to this generalization: the activity of O-glucosylzeatin markedly exceeded that of zeatin in the oat leaf senescence assay; 9-glucosyl-BAP and free BAP were similarly active in retarding the senescence of radish leaf discs. The 9-alanine conjugate of zeatin (lupinic acid) and of BAP were markedly less active than zeatin and BAP, respectively, in all bioassays, but the responses evoked by these conjugates at high concentrations in theAmaranthus bioassay approached those caused by the corresponding base. The activities of several new compounds related to the alanine conjugate of BAP were also assessed. To serve as a guide in the selection of the most suitable bioassay for detection of the above-mentioned cytokinin conjugates, the lowest detectable amounts in selected bioassays have been compared.