Main Cytokinin Research Articles

Development, Chlorophyll Content, and Nutrient Accumulation in In Vitro Shoots of Melaleuca alternifolia under Light Wavelengths and 6-BAP.

In vitro cultivation of Melaleuca could contribute to the cloning of superior genotypes. Studies of factors affecting micropropagation are needed, such as the interaction with light-emitting diodes (LEDs) and plant growth regulators added to the culture media. This study aimed at better understanding the effects of spectra on the development and physiology of melaleuca cultivated in vitro, as well as the interaction of LEDs with the main cytokinin used in micropropagation, N6-Benzylaminopurine (6-BAP). 6-BAP, spectra, and their interaction had a significant effect on most of the variables analyzed, altering the in vitro development and chlorophyll concentrations in the plants, as well as changing different variables in the culture medium, such as pH, EC, and levels of Ca2+, Mg2+, and P, and nutrient accumulation in the shoots. The results demonstrate that the main effects of adding BAP to the in vitro cultivation of melaleuca are an increase in the number of shoots, which resulted in greater fresh and dry masses; a reduction in height and chlorophyll content; complete inhibition of adventitious rooting; higher consumption of Mg, and lower consumption of Ca and P from the culture medium; higher content of Fe, and lower content of P, S, Mn, Cu and B in the in vitro shoot tissues.

Achromobacter xylosoxidans and Enteromorpha intestinalis Extract Improve Tomato Growth under Salt Stress

The effect of seed coating salt-stressed tomato with the bacterium Achromobacter xylosoxidans BOA4 and/or irrigation with an extract of the marine algae Enteromorpha intestinalis (EI) is herein evaluated. The plant shoots and roots were harvested separately on day 50, following extensive saline stress. The addition of BOA4 and/or EI extract resulted in an average increase of 33% in plant shoot DW, but an averaged decrease of 44% in the root to shoot biomass ratio. Anthocyanin content increased by over 34% and 44% with EI and BOA4 plus EI treatments, respectively. Since enhanced protein tyrosine nitration (PTN) is a known plant response to salt stress, the PTN level was inspected through 3-nitrotyrosine content determination. This was drastically increased by salt stress; however, BOA4, EI or both caused an averaged PTN decrease of 30% in stressed roots or shoots. This PTN response could be associated with tomato phenotypic characteristics and is postulated to be inversely correlated to cytokinin contents in stressed plants, namely cis-zeatin-type-cis-zeatin (cZ) plus cis-zeatin riboside (cZR), and isopentenyladenine (iP). The latter showed a drastic average increase by 3.6-fold following BOA4 and/or EI treatments of salinized tomato. This increment could be related to cytokinin biosynthesis induced by the applied bio-stimulants; IP and derivatives are the main cytokinins in seaweeds, and Achromobacter xylosoxidans BOA4 was shown to produce up to 17.5 pmol mL−1 of isopentenyladenine. This work is the first report on the influence of bio-stimulants, used to improve salt stress tolerance, on plant PTN levels; BOA4 and/or EI treatments decreased PTN, while increasing cis-zeatin-type and iP cytokinins in tomato, the latter showed an enhanced tolerance to salt stress.

3D Domain Swapping Dimerization of the Receiver Domain of Cytokinin Receptor CRE1 From Arabidopsis thaliana and Medicago truncatula.

Cytokinins are phytohormones regulating many biological processes that are vital to plants. CYTOKININ RESPONSE1 (CRE1), the main cytokinin receptor, has a modular architecture composed of a cytokinin-binding CHASE (Cyclases/Histidine kinases Associated Sensory Extracellular) domain, followed by a transmembrane fragment, an intracellular histidine kinase (HK) domain, and a receiver domain (REC). Perception of cytokinin signaling involves (i) a hormone molecule binding to the CHASE domain, (ii) CRE1 autophosphorylation at a conserved His residue in the HK domain, followed by a phosphorelay to (iii) a conserved Asp residue in the REC domain, (iv) a histidine-containing phosphotransfer protein (HPt), and (v) a response regulator (RR). This work focuses on the crystal structures of the REC domain of CRE1 from the model plant Arabidopsis thaliana and from the model legume Medicago truncatula. Both REC domains form tight 3D-domain-swapped dimers. Dimerization of the REC domain agrees with the quaternary assembly of the entire CRE1 but is incompatible with a model of its complex with HPt, suggesting that a considerable conformational change should occur to enable the signal transduction. Indeed, phosphorylation of the REC domain can change the HPt-binding properties of CRE1, as shown by functional studies.

Topolins and Red Light Improve the Micropropagation Efficiency of Passion Fruit (Passiflora edulis Sims) ‘Tainung No. 1’

Passion fruit is a commercial crop of economic importance worldwide, with recent increases in demand for high-quality plants for commercial production. Plant tissue culture is widely used for the mass propagation of many commercial crops, however its application on passion fruit is challenged by the problem of low reproducibility, leaf chlorosis, and growth retardation resulted from in vitro culture. The aim of this study was to evaluate the effects of cytokinins and light quality on in vitro culture of nodal segments of passion fruit ‘Tainung No. 1’. Three aromatic cytokinins were tested in a modified MS basal medium. The bud proliferation rates of segments initiated on a media containing 1 mg·L−1 meta-topolin riboside (mTR) or benzyladenine (BA) were not significantly different at the same concentration. Buds cultured on medium supplemented with mTR grew and elongated for 4 weeks, while buds on a medium containing BA formed rosettes. After transfer to a medium without plant growth regulators (PGRs), shoots rooted spontaneously within 8 weeks. Furthermore, the effects of continuous propagation under a high proportion of red light affected the subsequent plant growth. Red LED induced an increase in the chlorophyll content (2.71 mg·g−1) compared with other light qualities (1.05–2.63 mg·g−1) and improved plantlet quality. Acclimated plants were grown in the field, and the flower morphology and fruit set were of commercial quality. Findings showed that replacing BA with mTR as the main cytokinin and using a high proportion of red light during the tissue culture induction period produced high-quality plantlets in 3 months. This system is economical and will be further developed for the commercial propagation of passion fruit vines in the future.

Cytokinin oxidase/dehydrogenase overexpression modifies antioxidant defense against heat, drought and their combination in Nicotiana tabacum plants

Cytokinins (CKs) as well as the antioxidant enzyme system (AES) play important roles in plant stress responses. The expression and activity of antioxidant enzymes (AE) were determined in drought, heat and combination of both stresses, comparing the response of tobacco plants overexpressing the main cytokinin degrading enzyme, cytokinin oxidase/dehydrogenase, under the control of root-specific WRKY6 promoter (W6:CKX1 plants) or constitutive promoter (35S:CKX1 plants) and the corresponding wild-type (WT). Expression levels as well as activities of cytosolic ascorbate peroxidase, catalase 3, and cytosolic superoxide dismutase were low under optimal conditions and increased after heat and combined stress in all genotypes. Unlike catalase 3, two other peroxisomal enzymes, catalase 1 and catalase 2, were transcribed extensively under control conditions. Heat stress, in contrast to drought or combined stress, increased catalase 1 and reduced catalase 2 expression in WT and W6:CKX1 plants. In 35S:CKX1, catalase 1 expression was enhanced by heat or drought, but not under combined stress conditions. Mitochondrial superoxide dismutase expression was generally higher in 35S:CKX1 plants than in WT. Genes encoding for chloroplastic AEs, stromatal ascorbate peroxidase, thylakoidal ascorbate peroxidase and chloroplastic superoxide dismutase, were strongly transcribed under control conditions. All stresses down-regulated their expression in WT and W6:CKX1, whereas more stress-tolerant 35S:CKX1 plants maintained high expression during drought and heat. The achieved data show that the effect of down-regulation of CK levels on AES may be mediated by altered habit, resulting in improved stress tolerance, which is associated with diminished stress impact on photosynthesis, and changes in source/sink relations.

Auxin and cytokinin relationships in 24 microalgal strains(1).

Endogenous auxins and cytokinins were quantitated in 24 axenic microalgal strains from the Chlorophyceae, Trebouxiophyceae, Ulvophyceae, and Charophyceae. These strains were in an exponential growth phase, being harvested on day 4. Acutodesmus acuminatus Mosonmagyaróvár Algal Culture Collection-41 (MACC) produced the highest biomass and Chlorococcum ellipsoideum MACC-712 the lowest biomass. The auxins, indole-3-acetic acid (IAA) and indole-3-acetamide (IAM) were present in all microalgal strains. No other auxin conjugates were detected. IAA and IAM concentrations varied greatly, ranging from 0.50 to 71.49nmol IAA·g(-1) DW and 0.18 to 99.83nmol IAM·g(-1) DW, respectively. In 19 strains, IAA occurred in higher concentrations than IAM. Nineteen cytokinins were identified in the microalgal strains. Total cytokinin concentrations varied, ranging from 0.29nmol·g(-1) DW in Klebsormidium flaccidum MACC-692 to 21.40nmol·g(-1) DW in Stigeoclonium nanum MACC-790. The general trend was that cis-zeatin types were the predominant cytokinins; isopentenyladenine-type cytokinins were present in moderate concentrations, while low levels of trans-zeatin-type and very low levels of dihydrozeatin-type cytokinins were detected. Ribotides were generally the main cytokinin conjugate forms present with the cytokinin free bases and ribosides present in similar but moderate levels. The levels of O-glucosides were low. Only one N-glucoside was detected, being present in nine strains in very low concentrations. In 15 strains, the auxin content was 2- to 4-fold higher than the cytokinin content.

Comparison of salinity and drought stress effects on abscisic acid metabolites activity of cytokinin oxidase/dehydrogenase and chlorophyll levels in radish audtabacco

Stronger negative impact of salinity (100 mM NaCl for 4 days) on radish plants in comparison with tobacco ones, correlated with more profound decrease of bioactive cytokinins and higher elevation of activity of the main cytokinin degrading enzyme cytokinin oxidase/dehydrogenase and abscisic acid in the former species.

Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling.

Cytokinins are plant hormones involved in regulation of diverse developmental and physiological processes in plants whose molecular mechanisms of action are being intensely researched. However, most rapid responses to cytokinin signals at the proteomic and phosphoproteomic levels are unknown. Early cytokinin responses were investigated through proteome-wide expression profiling based on image and mass spectrometric analysis of two-dimensionally separated proteins and phosphoproteins. The effects of 15 min treatments of 7-day-old Arabidopsis thaliana seedlings with four main cytokinins representing hydroxyisopentenyl, isopentenyl, aromatic, and urea-derived type cytokinins were compared to help elucidate their common and specific function(s) in regulating plant development. In proteome and phosphoproteome maps, significant differences were reproducibly observed for 53 and 31 protein spots, respectively. In these spots, 96 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), providing a snapshot of early links in cytokinin-regulated signalling circuits and cellular processes, including light signalling and photosynthesis, nitrogen metabolism, the CLAVATA pathway, and protein and gene expression regulation, in accordance with previously described cytokinin functions. Furthermore, they indicate novel links between temperature and cytokinin signalling, and an involvement of calcium ions in cytokinin signalling. Most of the differentially regulated proteins and phosphoproteins are located in chloroplasts, suggesting an as yet uncharacterized direct signalling chain responsible for cytokinin action in chloroplasts. Finally, first insights into the degree of specificity of cytokinin receptors on phosphoproteomic effects were obtained from analyses of cytokinin action in a set of cytokinin receptor double mutants.

Comparison of hormonal responses to heat, drought and combined stress in tobacco plants with elevated proline content

In order to test the possibility of improving tolerance to heat and drought (alone and in combination) by elevation of the osmoprotectant proline (Pro) content, stress responses were compared in tobacco plants constitutively over-expressing a gene for the Pro biosynthetic enzyme Δ 2-pyrroline-5-carboxylate synthetase ( P5CSF129A; EC 2.7.2.11/1.2.1.41) and in the corresponding wild-type. Significantly enhanced Pro production in the transformant coincided with a more negative leaf osmotic potential (both at control conditions and following stress) and enhanced production of protective xanthophyll cycle pigments. Heat stress (40 °C) caused a transient increase in the level of bioactive cytokinins, predominantly N 6-(2-isopentenyl)adenosine, accompanied by down-regulation of the activity of the main cytokinin degrading enzyme cytokinin oxidase/dehydrogenase (EC 1.4.3.18/1.5.99.12). No significant differences were found between the tested genotypes. In parallel, a transient decrease of abscisic acid was observed. Following drought stress, bioactive cytokinin levels decreased in the whole plants, remaining relatively higher in preferentially protected upper leaves and in roots. Cytokinin suppression was less pronounced in Pro transformants. Exposure to heat stress (40 °C for 2 h) at the end of 10-d drought period strongly enhanced the severity of the water stress, as indicated by a drop in leaf water potential. The activity of cytokinin oxidase/dehydrogenase was strongly stimulated in upper leaves and roots of stressed plants, coinciding with strong down-regulation of bioactive cytokinins in whole plants. Combined heat and drought stress resulted in a minor decrease in abscisic acid, but only in non-wilty upper leaves. Both stresses as well as their combination were associated with elevation of free auxin, indolylacetic acid, in lower leaves and/or in roots. Auxin increase was dependent on the stress strength. After rehydration, a marked elevation of bioactive cytokinins in leaves was observed. A greater increase in cytokinin content in Pro transformants indicated a mild elevation of their stress tolerance.

Comparison of salinity and drought stress effects on abscisic acid metabolites activity of cytokinin oxidase/dehydrogenase and chlorophyll levels in radish audtabacco

Stronger negative impact of salinity (100 mM NaCl for 4 days) on radish plants in comparison with tobacco ones, correlated with more profound decrease of bioactive cytokinins and higher elevation of activity of the main cytokinin degrading enzyme cytokinin oxidase/dehydrogenase and abscisic acid in the former species.

Endogenous cytokinins, auxins and abscisic acid in Ulva fasciata (Chlorophyta) and Dictyota humifusa (Phaeophyta): towards understanding their biosynthesis and homoeostasis

Hormones are present in seaweeds, but little is known about their biosynthesis and regulation. The aim of this study was to investigate variation in concentration and composition of endogenous cytokinins, auxins and abscisic acid in two seaweeds collected over a 1 year period to gain a more complete picture of the types of hormones present during a range of environmental and developmental conditions. Ulva fasciata (Ulvales, Chlorophyta) and Dictyota humifusa (Dictyotales, Phaeophyta) were collected bimonthly from the inter-tidal zone at Rocky Bay, South Africa. Ethanol extracts of the samples containing a mixture of internal standards were purified using a combined DEAE-Sephadex-octadecylsilica column, followed by immuno-affinity chromatography and analysed by high-performance liquid chromatography–mass spectrometry to quantify different cytokinins, auxins and abscisic acid. cis-Zeatin, isopentenyladenine and their ribotide and riboside conjugates were the main cytokinins present in both U. fasciata and D. humifusa with low concentrations of trans-zeatin, dihydrozeatin and aromatic cytokinins. Very low concentrations of O-glucosides and riboside-O-glucosides and no N-glucosides were detected in any of the samples. Based on the cytokinins detected, we propose that as in higher plants, the ribotides are also the first intermediates formed during de novo biosynthesis in seaweeds and are subsequently converted to free-bases and ribosides. The only indole compounds detected in both species were free indole-3-acetic acid and indole-3-acetamide (IAM), suggesting that IAM is the intermediate in auxin biosynthesis. Endogenous abscisic acid was detected in most samples with levels in U. fasciata generally being higher than those measured in D. humifusa.

Cytokinins in the Bryophyte Physcomitrella patens: Analyses of Activity, Distribution, and Cytokinin Oxidase/Dehydrogenase Overexpression Reveal the Role of Extracellular Cytokinins

Ultra-performance liquid chromatography-tandem mass spectrometry was used to establish the cytokinin profile of the bryophyte Physcomitrella patens (Hedw.) B.S.G.; of 40 analyzed cytokinins, 20 were detected. cis-Zeatin-riboside-O-glucoside, N(6)-(Delta(2)-isopentenyl)adenosine-5'-monophosphate (iPRMP), and trans-zeatin-riboside-O-glucoside were the most abundant intracellular cytokinins. In addition, the aromatic cytokinins N(6)-benzyladenosine (BAR), N(6)-benzyladenine, meta-, and ortho-topolin were detected. Unexpectedly, the most abundant extracellular cytokinin was the nucleotide iPRMP, and its identity was confirmed by quadrupole time-of-flight mass spectrometry. The effects of overexpressing a heterologous cytokinin oxidase/dehydrogenase (CKX; EC 1.4.3.18/1.5.99.12) gene (AtCKX2 from Arabidopsis [Arabidopsis thaliana]) on the intracellular and extracellular distribution of cytokinins was assessed. In cultures of CKX-transformed plants, ultra-performance liquid chromatography-tandem mass spectrometry measurements showed that there were pronounced reductions in the extracellular concentrations of N(6)-(Delta(2)-isopentenyl)adenine (iP) and N(6)-(Delta(2)-isopentenyl)adenosine (iPR), but their intracellular cytokinin concentrations were only slightly affected. In vitro and in vivo measured CKX activity was shown to be strongly increased in the transformants. Major phenotypic changes observed in the CKX-overexpressing plants included reduced and retarded budding, absence of sexual reproduction, and abnormal protonema cells. In bud-induction bioassays with wild-type Physcomitrella, the nucleotides iPRMP, trans-zeatin-riboside-5'-monophosphate, BAR monophosphate, and the cis-zeatin forms cZ and cZR had no detectable effects, while the activities displayed by other selected cytokinins were in the following order: iP > tZ > N(6)-benzyladenine > BAR > iPR > tZR > meta-topolin > dihydrozeatin > ortho-topolin. The results on wild type and CKX transgenics suggest that extracellular iP and iPR are the main cytokinins responsible for inducing buds in the bryophyte Physcomitrella. Cytokinin profile is discussed regarding the evolution of cytokinin biosynthetic pathways.

Ability of bacterium Bacillus subtilis to produce cytokinins and to influence the growth and endogenous hormone content of lettuce plants

Hormone production by micro-organisms selected as antagonists of pathogenic fungi and the effect of their introduction into soil on hormone content and growth of lettuce plants were studied. Hormones in bacterial cultural media and in plant extracts were immunopurified and assayed using specific antibodies to indolyl-3-acetic acid (IAA), abscisic acid (ABA), and different cytokinins (zeatin riboside (ZR), dihydrozeatinriboside (DHZR) and isopentenyladenosine (iPA)). ZR was shown to be the main cytokinin present in bacterial cultural media as a complex with a high molecular weight component. Inoculation of lettuce plants with bacteria increased the cytokinin content of both shoots and roots. Accumulation of zeatin and its riboside was greatest in roots shortly 2 days after inoculation, when their content was 10 times higher than in control. Changes in the content of other hormones (ABA and IAA) were observed at the end of experiments only. Accumulation of cytokinins in inoculated lettuce plants was associated with an increase in plant shoot and root weight of approximately 30% over 8 days.

Alteration of endogenous cytokinins in axillary buds of conventionally grown greenhouse rose plants

Content and structure of cytokinins were measured in axillary buds along shoots of rose plants ( Rosa hybrida cv. Texas). Three different shoot sections could be distinguished beneath the flower peduncle. The upper section with tri- and monofoliate leaves is characterized by lateral shoots from non-inhibited buds growing concomitantly with the growth of terminal flower. The second section from the top is characterized by leaves with five or more leaflets. The third section is characterized by few five-leaflet leaves followed downward by less developed leaves. Isopentenyl cytokinins were the main cytokinins in axillary buds of three-leaflet leaves in the uppermost section and in the buds of the upper five-leaflet leaves in the second section of non-decapitated shoots. Zeatin and zeatin riboside were the main cytokinins in the lowest buds in axils of five-leaflet leaves of the third section. Three days after decapitation of the shoots above the uppermost five-leaflet leaf (80–90 cm long stems) or above the lowest five-leaflet leaf (10–15 cm long stems) a relatively very high content of zeatin and zeatin riboside was present in the uppermost remaining axillary buds. On the other hand, 1 week after decapitation the isopentenyl cytokinins were the main cytokinins in the two top axillary buds on the long shoots. The role of isopentenyl cytokinins in ready to sprout, to grow and to flower axillary buds as well as accumulation of root synthesized cytokinins in the lowest buds are discussed.

Hormonal Control of Tumor Formation in Radish

The role of phytohormones in genetic tumor formation on radish crop-roots was investigated using the collection of inbred Raphanus sativus lines as a model system. The genetic analysis showed that the trait > was recessive and monogenic in some crossings. The spectrum of main phytohormones in tumor and non-tumor radish lines has shown that at the initiation of tumor formation (30 days old plants) the amounts of main cytokinins in the lower part of plants from the tumor line were dramatically increased. The transformation of the non-tumor line by the ipt gene of Agrobacterium tumefaciens resulted in tumor formation in plants of the T1 progeny. We propose that increasing the cytokinin/auxin ratio may lead to tumor formation on radish crop roots.

Changes in cytokinin and auxin concentrations in seaweed concentrates when stored at an elevated temperature

Two seaweed concentrates were made from the kelps Ecklonia maxima and Macrocystis pyrifera using a cell burst method. Cytokinin- and auxin-like activities were measured using the soybean callus and mungbean bioassays, respectively. Cytokinin-like activity was detected in both seaweed concentrates, being equivalent to approximately 50 μg L−1 kinetin. Auxin-like activity was also detected in both concentrates with the E. maxima derived concentrate having higher biological activity, equivalent to 10−5–10−4 M indole-butyric acid. Two replicates of each concentrate were stored at 54 °C for 14 days to accelerate the effects of storage. Both fresh and stored samples of the two seaweed concentrates were analysed for their endogenous cytokinin and auxin content. The samples were purified using a combined DEAE-Sephadex octadecylsilica column and immunoaffinity chromatography based on wide-range cytokinin and IAA specific monoclonal antibodies. These extracts were analysed by HPLC linked to a Micromass single quadrupole mass spectrophotometer. Eighteen and nineteen different cytokinins were detected, respectively, in the two concentrates, with trans-zeatin-O-glucoside being the main cytokinin present. Accelerated storage of the concentrates caused an increase in the total cytokinin concentration with a large increase in the aromatic meta-topolin. Indole-3-acetic acid was the main auxin in both seaweed concentrates. Indole conjugates, including amino acid conjugates, were also quantified. The total auxin concentration decreased with accelerated storage for both concentrates.

Hormonal relationships in xylem sap of grafted and ungrafted Prunus rootstocks

Three Prunus species, characterised by different levels of growth vigour, were used as rootstocks for peach [ Prunus persica (L.) Batsch] grafting and compared for their hormone transport rates in the sap extracted from both the grafted scions and the ungrafted rootstocks. Two of these species were hybrid rootstocks widely used in peach orchards, while the third was a commercial cultivar, which was used both as rootstock and as scion. The xylem exudates were analysed for the concentrations of indoleacetic acid and for the main cytokinins, namely zeatin, zeatin riboside, isopentenyl-adenine and isopentenyl-adenosine. In ungrafted rootstocks the growth potential was positively correlated with both indoleacetic acid and zeatin riboside transport rates. In the xylem sap of peach scions grafted on the same rootstocks, the growth potential and the zeatin riboside levels were also positively correlated, while a negative correlation was found with indoleacetic acid levels. The results are discussed in view of the most widely accepted hypothesis concerning the control exerted by rootstocks on scion growth.

Changes in cytokinin distribution in the ovule and ovary of Taraxacum officinale Web. in early stages of embryogenesis.

Quantitative changes in the hormonal status of the ovules and ovaries were first studied in Taraxacum officinale Web. at the early stages of embryogenesis. The plant material was analyzed by ELISA using labeled anti-rabbit antibodies. A new procedure for differential and quantitative determination of the main endogenous cytokinins based on the estimation of the effective zeatin and zeatin riboside concentrations from calibration curves constructed using zeatin and zeatin riboside as standard antigens was developed. It was shown that, at the three initial stages of embryogenesis examined, the concentration of zeatin uniformly increased in T. officinale ovules. The concentration of zeatin riboside, conversely, uniformly decreased. However, their total concentration changed insignificantly. A gradual increase in the concentration of the active and storage hormone forms from the ovary to the ovule was shown.

Cytokinins in Rosa hybrida in relation to bud break

To assess the role of endogenous cytokinins in growth and development of Rosa hybrida, their concentrations in bleeding sap and in roots, stem, leaves, axillary shoots and bottom breaks in three stages of development were quantified. Cytokinins were purified by means of immunoaffinity chromatography and HPLC, and identified by retention time, UV spectrum and GC‐MS. The major translocation form in the xylem was zeatin riboside (ZR). In all mature tissues, cytokinins of the zeatin‐type were predominant, amounting to 80–90% of the total cytokinin concentration. The stems contained high concentrations of cytokinins, probably caused by lateral movement of ZR from the xylem to adjacent stem tissue and the ability of the stem to metabolize cytokinins. In young leaves the contribution of isopentenyl adenine (iP)‐type cytokinins to the total cytokinin pool was about 50%, indicating that these leaves might be capable of de novo synthesis of cytokinins. In older leaves, the concentration of an unidentified cytokinin‐like compound increased to more than 50% of total cytokinins. This compound, which was also found in the roots, might be a storage form of cytokinins. In young axillary shoots, about 50% of the cytokinins are iP‐compounds, suggesting either import of iP‐type cytokinins via the phloem or de novo synthesis of cytokinins. In buds forming bottom breaks, ZR and zeatin riboside monophosphate (ZRMP) are the main cytokinins, indicating that these buds receive their cytokinins from the roots.

Photoperiodic control of cytokinin transport and metabolism in Chenopodium rubrum

Cytokinin (CK) levels in the short‐day plant Chenopodium rubrum L. are known to fluctuate diurnally. The aim of this work was to investigate if the diurnal changes are brought about by changes in transport and/or metabolism of CKs. The effect of photo‐period on cytokinin transport was studied by analysing CK concentrations in root, leaf and apical exudates, respectively, under constant light (CL), a 12‐h photoperiod (DL) inductive for flowering, DL in which darkness was interrupted at the end of hour 6 by 15 min red light (R), or by 15 min R followed by 30 min far‐red irradiation (R/FR). The concentrations of cytokinins (zeatin, zeatin riboside, isopentenyladenine, isopen‐tenyladenosine) in all three types of exudates were significantly higher in the first 12‐h period after the end of 12 h darkness than in CL. The R break almost fully negated the effect of darkness and its effect was reversed by FR, showing the involvement of phytochrome in the regulation of CK transport. In the next 12‐h interval, i.e. 12–24 h after the end of darkness, the CK level remained high in the leaf exudate only, but to a much lower extent than in the previous 12 h. The highest CK concentration (increase by 108%) was observed in apical exudates during inductive darkness. A comparison of the CKs present in the individual exudates indicates that those arriving at the apical part are derived mostly from leaves with varying contributions by the xylem. The metabolism of applied [3H]‐zeatin riboside (ZR) was studied using HPLC separation of the metabolites. Metabolism was found to be very rapid and different glucosides, adenine and adenosine were the main metabolites after 12 h incubation with labelled ZR in all regimes tested. The only metabolite that seems to be under photoperiodic control is ZR‐5′‐monophosphate. It is as yet not clear if photoperiod controls the phosphorylation or dephosphorylation reaction. The activity of the main cytokinin degradative enzyme, cytokinin oxidase, did not change during the photoperiodic regimes tested.