Investigation on the role of nodule cytokinins in regulation of nitrate reductase activity ofPhaseolus mungo (L.)

The effects of some nodular cytokinis, zeatin (Z), zeatin riboside (ZR), N⁶ (²-isopentenyl) adenine (IPA), and N⁶ (²-isopentenyI) adenosine (IPAS) on nitrate reductase (E.C 1.9.6.1) activity of root nodules ofPhaseolus mungo were investigated. The cytokinis were also tested for their effect on nitrate uptake by nodules. The results show that IPAS is the most effective of all the four cytokinins tested. Z and IPA, which caused higherin vivo activity than ZR and IPAS, stimulated uptake of nitrate by nodules. The other two (ZR and IPAS) while inhibiting uptake showed greaterin vitro activity than Z and IPA. It may be concluded that some cytokinins, in addition to their direct effects on the enzyme, may increase the substrate availability to it, whereas others may have only an direct effect on the enzyme activation or degradation.Deceased.     

Cytokinins in maturing and germinatingLupinus luteus L. seeds

³H-labelled zeatin riboside (ZR) was applied to pod walls of intactLupinus luteus L. plants. Metabolites present in mature, dry seeds were zeatin nucleotide (ZNT), zeatin riboside (ZR) and zeatin (Z), zeatin O-glucosides and lupinic acid (LA), and the corresponding dihydro-derivatives of the cytokinins listed. Endogenous cytokinins were rapidly metabolised in germinating seeds. In seeds labelled with [³H]ZR for 90 min following a 2 h period of imbibition in water, ZR was actively converted to ZNT and dihydro-ZNT but the prevailing CTK was Z in cotyledons and ZR in embryo axes (EA); later LA and dihydro-LA, and O-glucoside metabolites accumulated. When [³H] zeatin was introduced into imbibing seeds, it was converted to dihydro-ZNT, ZNT, dihydro-ZR, ZR and dihydro-Z; in EA of the Z-labelled seeds, dihydro-ZR and ZR were the main cytokinins. After incubation of the Z-labelled seeds for 6 h in water, the ratios of dihydro-ZNT: ZNT and dihydro-ZR: ZR were, respectively, 20: 1 and 3.4: 1 in EA, and 3.5: 1 and 1.4: 1 in cotyledons.     

Further studies on the effects of different auxins and cytokinins on flower formation in thin cell layers of Nicotiana tabacum: The effects of zeatin riboside, dihydrozeatin and dihydrozeatin riboside

Organogenesis in thin cell layers of Nicotiana tabacum L. was studied in relation to the effects of natural and synthetic auxins in combination with various cytokinins. All cytokinins tested, benzyladenine (BA), kinetin, zeatin (Z), zeatin riboside (ZR), N⁶-Δ²-isopentenyl) adenine (IPA), dihydrozeatin [(diH)Z] and dihydrozeatin riboside [(diH)ZR], seem to be active in flower bud formation. In addition to the initiation of flower buds, vegetative buds or roots were also formed on the explants in the presence of BA, Z or IPA as exogenous cytokinins. Only dihydrozeatin and its riboside stimulated the initation of flower buds alone (as is known for kinetin), especially if supplemented with indole-3-acetic acid (IAA) as exogenous auxin. A high number of explants with flower buds was also found with high cytokinin/2,4-D ratios. In these conditions the presence of (diH)Z yielded the higest number of flower buds per explant.     

Influence of Different Cytokinins on the Transpiration and Senescence of Excised Oat Leaves

In order to investigate the possibility that cytokinins control transpiration indirectly through affecting leaf senescence, a direct comparison was made of the effect of different cytokinins on transpiration and senescence of oat leaves (Avena sativa L. cv. Forward). Senescence was assessed by measuring chlorophyll loss. The synthetic cytokinins N⁶ benzyladenine (BA) and kinetin delayed senescence and increased transpiration of oat leaves to a greater extent than did the naturally occurring compounds zeatin, N^b-Δ² isopentenyladenine (i⁶ Ade) and 6-ø-hydroxybenzyladenosine (hyd-BA riboside). During the early stages of the transpiration experiment zeatin showed similar or greater activity than BA. This period was longest when freshly excised leaves were used, was reduced when leaves were used after incubation in distilled water in the dark for 20 h and was eliminated by incubation in cytokinin solution in the dark. After this period the activity of zeatin declined relative to BA. The effect of cytokinins in increasing transpiration occurred only in the light; no effect was observed in the dark. BA showed higher activity than zeatin in senescence tests but both cytokinins were less effective as the tests progressed, this decrease in activity being more rapid when older leaves were used. The results are discussed in relation to the mechanisms by which endogenous cytokinins might control sensecence and transpiration in oat leaves and to the value of the oat leaf senscence and transpiration bioassays as tests for cytokinin activity of plant extracts.     

The effect of aluminum on cytokinins in the mycelia of Amanita muscaria

High performance liquid chromatography analysis of immunoaffinity-purified extracts of mycelia of Amanita muscaria, and the Amaranthus bioassay of the eluted fractions, revealed the following seven cytokinins: zeatin, zeatin riboside, zeatin N-9-glucoside, dihydrozeatin, dihydrozeatin riboside, isopentenyl adenine, and isopentenyl adenosine. The decreased growth of aluminum-treated mycelia correlated with a 35% decrease in the total amount of the cytokinins. Among individual cytokinins, zeatin was the most affected, exhibiting a reduction of about 90%. The results are compared with previous investigations of aluminum effects on cytokinins in the mycelia of Lactarius piperatus, whose growth is stimulated by aluminum.Abbreviations ZR zeatin riboside - iPA isopentenyl adenosine - Z zeatin - DHZ dihydrozeatin - iP isopentenyl adenine - DHZR dihydrozeatin riboside - Z-9G zeatin N-9-glucoside - iP-9G isopentenyl N-9-glucoside - HPLC high performance liquid chromatography - DHZRMP dihydrozeatin riboside monophosphate - ZRMP zeatin riboside monophosphate     

Cytokinin production by fluorescent Pseudomonas in the presence of rice root exudates

Free-living bacteria may trigger the plant growth through production of phytohormones viz. gibberellins, auxins and cytokinins. A total 50 isolates of fluorescent Pseudomonas were screened for their ability to produce cytokinins such as isopentenyladenosine (IPA), dihydroxyzeatin riboside (DHZR) and zeatin riboside (ZR) as plant growth-promoting activity. Pseudomonas fluorescens AK1 and Pseudomonas aeruginosa AK2 were found higher phytohormones producing strains. Of the three cytokinins, IPA was the major cytokinin produced by both isolates in pure culture (5.5 and 2.9 pmol/ml, respectively) and with rice root exudates (5.9 and 3.4 pmol/ml, respectively). Production of ZR and DHZR for both organisms was found after 48 and 72 h. Amount of ZR and DHZR increased with time for both isolates in pure culture conditions. In presence of rice, production of ZR was increased 0.8 and 0.6 pmol/ml for P. fluorescence AK1 and P. aeruginosa AK2, respectively, in comparison with controls. There was no significant difference in the production of DHZR with rice exudates.     

Induction of chitinase activity by exogenous cytokinins in excised dark-grown cucumber cotyledons: involvement of Ca and staurosporine-sensitive protein kinase(s) in cytokinin signaling

Cotyledons were excised from 7-day-old dark-grown cucumber seedlings and treated with water, benzyladenine (BA), kinetin (K), zeatin (Z), or zeatin riboside (ZR) in dark after endogenous cytokinin depletion. We have compared changes in chitinase (EC. 3.2.1.14) activity induced by these cytokinins. We find that the activities of chitinase and its isoforms increase by approximately 3- to 6-fold following BA, Z, and ZR treatments. Among these treatments, Z was more effective. K was totally ineffective in inducing chitinase activity. Immunoblot analysis suggests that the cytokinin Z-induction of enzyme activity is due to the induction of higher chitinase protein levels and not the activation of existing enzyme. Furthermore, the Z-induced chitinase activity and its protein accumulation were completely inhibited by the protein kinase inhibitor staurosporine, whereas the protein phosphatase inhibitor sodium fluoride was not effective in such inhibitions. Treatment of cotyledons with extemal CaCl₂ and calcium ionophore increased the basal chitinase activity by 6- and 5-fold, respectively. Moreover, the effects of staurosporine, sodium fluoride, and Ca²⁺ on Z-induced chitinase activity correlate with their effects on chitinase protein levels. Taken together, our data suggests Ca²⁺ and staurosporine-sensitive protein kinase(s) as components of the cytokinin transduction machinery involving induction of chitinase in cucumber.     

Postharvest changes in endogenous cytokinins and cytokinin efficacy in potato tubers in relation to bud endodormancy

Using an indirect enzyme‐linked immunosorbent assay (ELISA), the effects of postharvest storage duration and temperature on endogenous cytokinins in potato ( Solanum tuberosum L. cv. Russet Burbank) tuber apical bud tissues in relation to endodormancy status were determined. Following fractionation by HPLC, a total of eight cytokinins were detected and these were: zeatin riboside‐5'‐monophosphate (ZRMP), zeatin‐ O ‐glucoside (ZOG), zeatin (Z), zeatin riboside (ZR), isopentenyl adenosine‐5'‐monophosphate (IPMP), isopentenyl adenine‐9‐glucoside (IP‐9‐G), isopentenyl adenine (IP) and isopentenyl adenosine (IPA). Regardless of postharvest storage temperature or endodormancy status, IP‐9‐G was the most abundant cytokinin detected while ZRMP and ZOG were the least abundant ones. In tubers preincubated at a growth‐permissive temperature (20°C) prior to extraction, the loss of endodormancy was preceded by significant increases in the endogenous levels of Z, ZR, IPMP and IP‐9‐G. When stored continuously at a growth‐inhibiting temperature (3°C), significant increases in ZR, IP‐9‐G and IP + IPA were observed. The total content of cytokinins increased by over 7‐fold during postharvest storage and this increase was a result of de novo biosynthesis. Dose‐response studies using IPA and ZR demonstrated a time‐dependent increase in apparent cytokinin sensitivity during postharvest storage. With the exception of IP‐9‐G, injection of any of these endogenous cytokinins resulted in the rapid and complete termination of tuber endodormancy. The significance of these results with respect to endodormancy regulation and the possible mechanisms controlling cytokinin levels in potato tubers are discussed.     

Involvement of cis-Zeatin, Dihydrozeatin, and Aromatic Cytokinins in Germination and Seedling Establishment of Maize, Oats, and Lucerne

The aims of this study were to monitor endogenous cytokinin levels during germination and early seedling establishment in oats, maize, and lucerne to determine which cytokinin forms are involved in these processes; to quantify the transfer ribonucleic acid (tRNA)-bound cytokinins; and to measure cytokinin oxidase/dehydrogenase (CKX) activity. Cytokinins were identified using UPLC-MS/MS. The predominant free cytokinins present in the dry seeds were dihydrozeatin-type (DHZ) in lucerne and maize and cZ-type (cis-zeatin) in oats. Upon imbibition, there was a large increase in cZ-type cytokinins in lucerne although the cZ-type cytokinins remained at high levels in oats. In maize, the high concentrations of DHZ-type cytokinins decreased prior to radicle emergence. Four tRNA-bound cytokinins [cis-zeatin riboside (cZR)>N ⁶-(2-isopentenyl)adenosine (iPR), dihydrozeatin riboside (DHZR), trans-zeatin riboside (tZR)] were detected in low concentrations in all three species investigated. CKX activity was measured using an in vitro radioisotope assay. The order of substrate preference was N ⁶-(2-isopentenyl)adenine (iP)>trans-zeatin (tZ)>cZ in all three species, with activity fluctuating as germination proceeded. There was a negative correlation between CKX activity and iP concentrations and a positive correlation between CKX activity and O-glucoside levels. As O-glucosides are less resistant to CKX degradation, they may provide a readily available source of cytokinins that can be converted to physiologically active cytokinins required during germination. Aromatic cytokinins made a very small contribution to the total cytokinin pool and increased only slightly during seedling establishment, suggesting that they do not play a major role in germination.     

Effect of applied and internal hormones on vitrification and apical necrosis of different plants cultured in vitro

Development of vitrification and apical necrosis was followed in Camellia sinensis, Gerbera jamesonii, Malus domestica and hybrid Populus tremula x P. alba shoots cultured in vitro on Murashige & Skoog (MS) medium with different concentrations of growth regulators. High humidity in the culture vessels and excess of BA in the medium were found to be the major factors influencing vitrification. Lack of exogenous cytokinin in the medium during successive subcultures induced apical necrosis in poor-rooting species (Malus domestica, Camellia sinensis). The level of internal phytohormones (ABA, IAA, IPA, 2iP, Z, ZR) was determined in the apple shoots by means of ELISA. The content of internal cytokinins in the vitrified apple shoots was several times greater than in normal ones, which supports the hypothesis that excess of cytokinins, inducing rapid divisions of cells in meristems in the atmosphere with high humidity, is responsible for vitrification. Apical necrosis of the plantlets that appeared after cultivation on cytokinin-free medium is the result of deficiency in endogenous hormones in apple shoots and this being confirmed by analysis of endogenous hormones in apple shoots.Abbreviations BA benzyladenine - BHT butylated hydroxy-toluene - ABA abscisic acid - IAA indole-3-acetic acid - ELISA enzyme-linked immunosorbent assay - IPA isopentenyladenosine - 2iP isopentenyladenine - NAA naphthyl-3-acetic acid - TBS trishydroxymethylaminomethane buffered saline - TLC thin layer chromatography - Z zeatin - ZR zeatin riboside     

Correlation of continuous ryegrass regrowth with cytokinin induced by root nitrate absorption

This study investigated the separate and combined effects of nitrate (NO₃ ^?) and cytokinin additions on continuous ryegrass regrowth after defoliation and the underlying mechanisms. Our results showed that frequent defoliation reduced the biomass of newly grown leaves and roots, the root soluble carbohydrate contents, the root vitality (an indicator of root absorption capacity), and the leaf contents of NO₃ ^?, zeatin and zeatin riboside (Z + ZR), and isopentenyl adenine and isopentenyl adenosine (IP + IPA). NO₃ ^?addition to the roots or leaves increased the biomass of newly grown leaves as well as the leaf contents of NO₃ ^?, Z + ZR, and IP + IPA without increasing the root-to-shoot delivery of endogenous cytokinin. Interestingly, cytokinin directly added to the leaves also increased the biomass of newly grown leaves and their Z + ZR and IP + IPA contents, suggesting that nitrate-induced leaf cytokinin production mediates the growth-promoting effects of nitrate. We also found that cytokinin had a direct whereas NO₃ ^? had an indirect effect on the biomass of newly grown leaves. Taken together, our results indicate that leaf cytokinin production induced by NO₃ ^? absorbed through the roots plays a key role in continuous ryegrass regrowth after defoliation.     

Involvement of cytokinins in the germination of chick-pea seeds

Eight cytokinins detected in germinated chick-pea (Cicer arietinum L. var. Castellana) seeds were first present in the embryonic axes but appeared in the cotyledons after 12h of germination. The cytokinins detected in the cotyledons originate in the embryonic axes, but no passage of these substances from the cotyledons to the axes was detected, except when the seeds were treated with red light.It is concluded that the role played by the embryonic axis in mobilizating the main reserves of the cotyledons is mainly effected through these cytokinins. Both natural and synthetic cytokinins exert an important regulatory role in the hydrolysis of reserve proteins and calcium could be involved as an intermediate.Abbreviations BA benzyladenine - cot. cotyledon - (diH)Z dihydrozeatin - (diH)ZR dihydrozeatin riboside - GZR glycosyl zeatin riboside - 2iP 277-1 - iPA 277-2 riboside - Kin kinetin - Z zeatin - ZG zeatin glucoside - ZR zeatin riboside     

Cytokinin metabolism and the modulation of cytokinin activity in radish

The metabolism of zeatin, N⁶-(Δ²-isopentenyl) adenine, dihydrozeatin, zeatin-O-glucoside and dihydrozeatin-O-glucoside has been studied using derooted radish seedlings. The metabolites were identified by UV and GC/MS. The patterns of metabolism are compared and provide evidence that the O-glucosyl conjugates may be storage forms of the cytokinins.     

Mass-spectrometric quantification of cytokinin nucleotides and glycosides in tobacco crown-gall tissue

the cytokinins of tobacco crown-gall tissue have been analysed by quantitative mass spectrometry using ²H₂-labelled cytokinin riboside 5-monophosphates and ¹⁵N₄-labelled cytokinin glycosides as internal standards. The principal endogenous cytokinin of this tissue is zeatin riboside 5-monophosphate. The biologically inactive 7-glucoside of zeatin is the most abundant basic cytokinin in the tissue. These findings expose the limitations of previously reported analyses of similar tissues, which were restricted to biologically active basic cytokinins. The present study demonstrates that the endogenous cytokinins of tobacco crowngall tissue show a clear correspondence to the range of metabolites formed when exogenous cytokinins are supplied to nontumorous tobacco cells.Abbreviations DHZ dihydrozeatin - DHZ7G dihydrozeatin 7-glucoside - DHZMP dihydrozeatin 9-riboside 5-monophosphate - DHZR dihydrozeatin 9-riboside - GC-MS coupled gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - Z7G zeatin 7-glucoside - Z9G zeatin 9-glucoside - ZOG zeatin O-glucoside - ZMP zeatin 9-riboside 5-monophosphate - ZR zeatin 9-riboside - ZROG zeatin 9-riboside O-glucoside     

On the biogenesis of cytokinins in roots of Phaseolus vulgaris

Roots of intact bean plants were supplied with [¹⁴C]adenine by pulse-chase experiments. The rate of incorporation of radioactivity into tRNA and oligonucleotides of roots as well as the content of radioactive labeled cytokinin nucleotides in these RNA fractions were determined. On the average, 1/70 of the radioactivity incorporated into tRNA was localized in N⁶(²isopentenyl)adenosine. The half life of tRNA was estimated to be 65–70 h. Shortly after the pulse period, oligonucleotides contained zeatin riboside at a ratio of 1:800, on the basis of radioactivity. The half life of these oligonucleotides was determined to be about 8 h. The main free radioactive cytokinin of roots and leaves was zeatin. Comparing the rate of degradation of ¹⁴C-labeled tRNA and the oligonucleotides of roots and the rate of appearance of radioactive cytokinins in roots and leaves, we found strong indications for their dependency. The results contradict the hypothesis of de novo synthesis of cytokinins in roots of intact bean plants.Abbreviations AMP adenosine monophosphate - IPA N⁶(²isopentenyl)adenosine - IPAde N⁶(²isopentenyl)adenosine - Z zeatin - ZR zeatinriboside - TLC thin-layer chromatography - HPLC high performance liquid chromatography Part of the doctoral thesis, Bonn 1980     

Application of a Rapid and Sensitive Method for Hormonal and Vitamin E Profiling Reveals Crucial Regulatory Mechanisms in Flower Senescence and Fruit Ripening

Knowledge of ripeness and regulation of postharvest processes is an important tool to prevent loss of commercial value in both fruit and cut flower markets. The joint analysis of hormones and vitamin E levels can reveal complex interactions between hormones and oxidative stress as key regulators of postharvest processes. Profiling of both groups of metabolic compounds was performed during the ripening of non-climacteric fruits (red raspberry, Rubus idaeus L.) and senescence of ethylene-insensitive flowers (Dutch Iris, Iris x hollandica L.). After an initial extraction of the sample, without further purification steps, the hormonal profile was analyzed by UPLC-MS/MS and vitamin E levels were measured by HPLC. This methodological approach was very fast and had enough sensitivity for the analysis of small samples. Raspberry fruit maturation was characterized by a decline of cytokinin levels [zeatin, zeatin riboside, 2-isopentenyl adenine, and isopentenyl adenosine (Z, ZR, 2-iP, and IPA, respectively)] and gibberellins (GA₁ in particular). Exogenous application of ABA prevented δ-tocopherol loss during fruit ripening. Iris floral senescence was also under strict hormonal control, also mediated by cytokinins and gibberellins. Z, ZR, 2-iP, GA₉, and GA₂₄ levels decreased in inner tepals, whereas the level of IPA decreased in style-merged-to-stigma tissues, thus suggesting tissue-specific roles for different hormones. α-Tocopherol levels decreased during senescence of inner tepals, hence suggesting enhanced oxidative stress. In conclusion, the rapid and sensitive hormonal and vitamin E profiling presented here can help in understanding the key physiological processes underlying fruit ripening and floral senescence.     

The influence of cytokinins in nitrate regulation of nitrate reductase activity and expression in barley

The responses of nitrate reductase (NR) activity and levels of NR-mRNA to environmental nitrate and exogenous cytokinins are characterised in roots and shoots of barley ( Hordeum vulgare L., cv. Golf), using a chemostate-like culture system for controlling nitrate nutrition. Experiments were mainly performed with split root cultures where nitrate-N was supplied at a constant relative addition rate of 0.09 day⁻¹, and distributed between the subroots in a ratio of 20%:80%. The subroot NR-mRNA level and NR activity, as well as the endogenous level of zeatin riboside (ZR), increased when the local nitrate supply to one of the subroots was increased 4-fold by reversing the nitrate addition ratio (i.e. from 20%:80% to 80%:20%). Also shoot levels of ZR, NR-mRNA and NR activity increased in response to this treatment, even though the total nitrate supply remained unaltered. External supply of ZR at 0.1 μ M caused an approximately 3-fold increase in root ZR levels within 6 h. which is comparable to the nitrate-induced increase in root ZR. External application of ZR. zeatin. isopentenyl adenine or isopentenyl adenosine at 0.1 μ M caused from insignificant to 25% increases in NR-mRNA and activity in roots and up to 100% stimulation in shoots, whereas adenine or adenosine had no effect. No synergistic effects of perturbed nitrate supply and cytokinin application were detected in either roots or shoots. The translocation of nitrate from the root to the shoot was unaffected by application of ZR or switching the nitrate distribution ratio between subroots. The data give arguments for a physiological role of cytokinins in the response of root and shoot NR to environmental nitrate availability. The nature and limitations of the physiological role of cytokinins are discussed.     

Zeatin-9-glucoside,a major endogenous cytokinin of Vinca rosea L. crown gall tissue

Cultured crown gall tissue of Vinca rosea L. was found to contain, in addition to the previously reported cytokinins zeatin, zeatin riboside, and the 0-glucosides of these two compounds, relatively high levels of zeatin-9-D-glucopyranoside. This is the first conclusive identification of an endogenous cytokinin 9-glucoside.Abbreviations GC gas chromatography - HPLC high-performance liquid chromatography - I.D. internal diameter - RFE rotary film evaporation - TLC thin layer chromatography - TMS trimethylsilyl - UV ultraviolet - Z zeatin - Z7G zeatin-7-glucoside - Z9G zeatin-9-glucoside - Z0G zeatin-0-glucoside - ZR zeatin riboside - ZR0G zeatin riboside-0-glucoside     

Mass spectrometric measurement of zeatin glycoside levels in Vinca rosea L. crown gall tissue

The range of zeatin glycosides found in crown gall tissue of Vinca rosea L. has been quantified using a mass spectrometric isotope dilution procedure. Problems in the quantitative analysis of cytokinins in plant extracts are discussed.Abbreviations GC/MS coupled gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - Me methyl - Z zeatin - Z9G zeatin 9-glucoside - ZOG zeatin O-glucoside - ZR zeatin 9-riboside - ZROG zeatin 9-riboside O-glucoside     

Changes in Cytokinins before and during Early Flower Bud Differentiation in Lychee (Litchi chinensis Sonn.)

Lychee (Litchi chinensis) has been analyzed for cytokinins in buds before and after flower bud differentiation, using reversephase high performance liquid chromatography in combination with Amaranthus bioassay and gas chromatography-mass spectrometry-selected ion monitoring. Four cytokinins, zeatin, zeatin riboside, N⁶-(δ²-isopentenyl)adenine, and N⁶-(δ⁶-isopentenyl) adenine riboside, were detected in buds. There was an increase of cytokinin activity in the buds during flower bud differentiation. In dormant buds, the endogenous cytokinin content was low, and the buds did not respond to exogenous cytokinin application. Application of kinetin promotes flower bud differentiation significantly after bud dormancy. These results are interpreted as an indication that the increase in endogenous cytokinin levels during flower bud differentiation may be correlative rather than the cause of flower bud initiation.