Rapid modulation of nitrate reductase in leaves and roots: Indirect evidence for the involvement of protein phosphorylation/dephosphorylation

Nitrate reductase activity (NRA; NADH-nitrate reductase, E. C. 1.6.6.1) has been measured in extracts from leaves of spinach ( Spinacia oleracea L.) in response to rapid changes in illumination, or supply of CO₂ or oxygen. Measured in buffers containing magnesium, NRA from leaves decreased in the dark and increased again upon illumination. It decreased also, when CO₂ was removed in continuous light, and was reactivated when CO₂ was added. Nitrate reductase (NR) from roots of pea ( Pisum sativum L.) was also rapidly modulated in vivo. It increased under anaerobiosis and decreased in air or pure oxygen. The half time for inactivation or reactivation in roots and leaves was 5 to 30 min.
When spinach leaves were harvested during a normal day/night cycle, extractable NRA was low during the night, and high during daytime. However, at any point of the diurnal cycle, NR could be brought to a similar maximum activity by preincubation of the desalted leaf extract with AMP and/or EDTA. Thus, the observed diurnal changes appeared to be mainly a consequence of enzyme modulation, not of protein turnover. In vivo, the reactivation of the inactivated enzyme from both leaves and roots was prevented by okadaic acid, and inhibitor of certain protein phosphatases. Artificial lowering of the ATP-levels in leaf or root tissues by anaerobiosis (dark), mannose or the uncoupler carbonyl cyanide m -chlorophenyl hydrazon (CCCP), always brought about full activation of NR.
By preincubating crude leaf or root extracts with MgATP, NR was inactivated in vitro. Partial purification from spinach leaves of two enzymes with molecular masses in the 67 kD and 100 kD range, respectively, is reported. Both participate in the ATP-dependent inactivation of NR.
Alltogether these data indicate that NR can be rapidly modulated by reversible protein phosphorylation/dephosphorylation, both in shoots and in roots.     

Changes in cytokinin levels of Phalaenopsis leaves at high temperature

The effect of high temperatures on cytokinin levels in Phalaenopsis hybrida leaves was investigated. Endogenous cytokinins were identified and quantified in Phalaenopsis leaves grown under high temperature conditions (30/25 °C day/night) using high performance liquid chromatography, bioassay and gas chromatography-selected ion monitoring-mass spectrometry. After 5 and 20 d of low temperature (25/20 °C day/night), zeatin, zeatin riboside and dihydrozeatin levels in the leaves were higher than that in leaves subjected to high temperature treatments. When Phalaenopsis leaves were exposed to low temperatures, about 76 % of the free cytokinins detected were of the zeatin-type. Glucoside cytokinins in the leaves increased significantly 5 d following high temperatures, and the rate of increase in glucoside cytokinins corresponded to the duration of high temperatures. At the same time, zeatin riboside and dihydrozeatin declined significantly following high temperature application. A significant accumulation of glucoside cytokinins, zeatin-9-glucoside, zeatin-O-glucoside, zeatin riboside-O-glucoside, and dihydrozeatin-O-glucoside was observed 20 d following high temperatures. These results suggest that high temperatures lead to an accumulation of glucoside cytokinins and a reduction of free base and riboside cytokinins.     

Mixture screening and mixture-amount designs to determine plant growth regulator effects on shoot regeneration from grapefruit (<Emphasis Type="Italic">Citrus paradisi</Emphasis> macf.) epicotyls

The objective of this study was to improve shoot regeneration from grapefruit. Because many commercially grown citrus types are apomictic, important in vitro applications such as Agrobacterium-mediated transformation commonly use epicotyl explants from in vitro seedlings; thus, adequate adventitious shoot production is an important prerequisite for efficient use of these applications. Eight plant growth regulators were studied—six cytokinins (6-benzylaminopurine, kinetin, zeatin trans-isomer, 6-[γ,γ-dimethylallylamino] purine, zeatin riboside trans-isomer and meta-topolin) and two auxins (α-naphthalene acetic acid and indole-3-acetic acid). An iterative design strategy was followed that included mixture and mixture-amount experimental designs suitable for resolving proportional and concentration effects; in vitro effects of cytokinins and auxins are affected by both proportion and concentration. One-centimeter-long explants were excised from the epicotyl of etiolated, in vitro-grown seedlings. Explants were placed onto experimental formulations and cultured in growth cabinets at 27°C over 6 wk, which included 2 wk in the dark followed by 4 wk in the light. The results indicated that (1) 6-benzylaminopurine or zeatin riboside were the most effective cytokinins for inducing shoot regeneration in citrus; (2) zeatin riboside singly or in combination with indole-3-acetic acid resulted in the highest quality, the greatest number of explants with buds/shoots, and the greatest shoot number; and (3) 6-benzylaminopurine and indole-3-acetic acid improved shoot regeneration vs. 6-benzylaminopurine at a considerably lesser cost than zeatin riboside and indole-3-acetic acid.     

Induction and Turnover of Nitrate Reductase in Zea mays (Influence of Light)

Both light and NO3- are necessary for the appearance of nitrate reductase (NR) activity (NRA) in photosynthetic tissues. To define the light effect more precisely, we examined the response to light/dark transitions on NRA, NR protein (NRP), and NR mRNA in 6-d-old maize (Zea mays cv W64A x W182E) seedlings that had been grown in a light/dark regime for 5 d and then induced with 5 mM KNO3 for 24 h. The decay of NRA and NR mRNA in the shoot was immediate, but there were only minor changes in NRP during the initial 4 h in the dark. In root tissues, in contrast, there was a 4-h delay in the loss of NRA, NRP, and NR mRNA after transfer to the dark. When the seedlings were returned to light after a 2-h interval in the dark, shoot NRA reached 92% of the initial levels within 30 min of illumination. These results indicate that in the shoots (a) NR message production requires light and (b) the NRP that appears with light treatment and that is active is inactivated in the dark. The NRP can be reactivated when the light is turned on after short periods of darkness (2 h). Root tissues, on the other hand, probably respond to the supply of photosynthetically produced metabolites rather than to immediate products of the light reactions of photosynthesis.     

Hormones in Plants Bearing Nitrogen-fixing Root Nodules: Cytokinin Transport from the Root Nodules of Alnus glutinosa (L.) Gaertn.

The movement and metabolism of [8-¹⁴C]zeatin applied to theroot nodules of Alnus glutinosa (L.) Gaertn, was investigated.Twenty-four hours after the start of uptake, zeatin and a numberof its metabolites were detected in all parts of the plant.The major radioactive compounds present in a cationic fractionof different plant parts at this time co-chromatographed onSephadex LH20 with zeatin (in nodules, stems, and leaves) andwith zeatin riboside (in roots, stems, and buds). In the roots,in addition to the peak co-chromatographing with zeatin riboside,there was also a prominent unidentified polar peak. The presence of zeatin and zeatin riboside in the stems andleaves was indicated also by chromatographic behaviour in othersystems, effects of permanganate oxidation, and cocrystallisationwith the authentic unlabelled compounds. Biological activitywas exhibited by both peaks in the soybean callus bioassay.Other metabolites in the shoot, possibly active as cytokinins,had the characteristics of dihydrozeatin, zeatin or dihydrozeatin-5'-nucleotide(s),and zeatin or dihydrozeatin glucosides. The gradual disappearancewith time of zeatin and its riboside from the shoot was accompaniedby an increase in the proportion of more polar metabolites. These results are discussed in relation to the possible exportof endogenous cytokinins by the nodules.     

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.     

Changes in the level of endogenous cytokinin-like substances in Acer pseudoplatanus embryos during stratification and germination

The bioassay used to detect and quantify cytokinin activity was the Amaranthus test. Free cytokinin-like substances in embryos of Acer pseudoplatanus L. fruits increased during the first 20 d of fruit stratification at 5°C, but subsequently fell rapidly to values well below the amounts present in the embryos of freshly harvested fruits. These lower levels persisted throughout the remainder of a 60 d stratification period. Bound cytokinins fell during stratification from the highest detected levels present in freshly harvested material to values which were lower by about one third. No peaks of bound cytokinin activity were detected at any stage during stratification. In embryos from fruits stored at 17°C and unable to germinate, both free and bound cytokinins remained at a very low level throughout the 60 d period. Embryos from fruits previously stratified for 60 d showed increases in both free and bound cytokinins during the first 24 h of their incubation at 20°C in light, but after longer incubation periods up to 72 h, cytokinin concentrations decreased again to levels similar to those present at the commencement of the incubation period. Determinations conducted in 1979 and 1980 showed quantitative differences, but similar qualitative changes were observed in the two years. Most of the cytokinin activity was associated with compound(s) that co-chromatographed with zeatin and zeatin riboside.     

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.     

Effect of light/dark cycles on expression of nitrate assimilatory genes in maize shoots and roots

The level of nitrate reductase (NR) and nitrite reductase (NiR) varied in both shoot and root tissue from nitrate-fed Zea mays L. grown under a 16-hour light/8-hour dark regime over a 10-day period postgermination, with peak activity occurring in days 5 to 6. To study the effect of different light regimes on NR and NiR enzyme activity and mRNA levels, 6-day-old plants were grown in the presence of continuous KNO₃ (10 millimolar). Both shoot NRA and mRNA varied considerably, peaking 4 to 8 hours into the light period. Upon transferring plants to continuous light, the amplitude of the peaks increased, and the peaks moved closer together. In continuous darkness, no NR mRNA or NR enzyme activity could be detected by 8 hours and 12 hours, respectively. In either a light/dark or continuous light regime, root NRA and mRNA did not vary substantially. However, when plants were placed in continuous darkness, both declined steadily in the roots, although some remained after 48 hours. Although there was no obvious cycling of NiR enzyme activity in shoot tissue, changes in mRNA mimicked those seen for NR mRNA. The expression of NR and NiR genes is affected by the light regime adopted, but light does not have a direct effect on the expression of these genes.     

Cytokinins of dryZea mays seed: Quantification by radioimmunoassay and gas chromatography-mass spectrometry

The endogenous cytokinins present in dryZea mays seed were determined using both radioimmunoassay and gas chromatography—mass spectrometry. Similar values for bases and ribosides were obtained by the two methods. The cytokinins present in embryo and endosperm were estimated separately using radioimmunoassay; similar levels of cytokinins were found in these two tissues. The major cytokinins detected on a whole-seed basis were dihydrozeatin riboside, O-glucosyldihydrozeatin riboside, zeatin 9-glucoside, zeatin, and the nucleotides of zeatin, dihydrozeatin, and isopentenyladenine. Cytokinin levels in the mature dry seed were considerably lower than cytokinin levels published in the literature for immature seed. Unexpected activity in the radioimmunoassays was detected in the wash from the DEAE cellulose column chromatography step. The compound(s) responsible for this activity did not have the solvent partitioning characteristics of a cytokinin base or riboside. They eluted as a single fraction following high-performance liquid chromatography on a Zorbax C8 column; this fraction showed no activity in theAmaranthus bioassay for cytokinins, but inhibited the activity of authentic zeatin riboside present at an optimal concentration.     

Endogenous cytokinin accumulation and cytokinin oxidase activity during shoot organogenesis of Petunia hybrida

Changes in endogenous cytokinin content and cytokinin oxidase activity were characterized in leaf explants from two Petunia hybrida Vilm. genetic lines which differed in their shoot organogenic response to exogenous N⁶-benzyladenine (BA). Endogenous cytokinin content in leaf explants of the highly shoot organogenic line, St40, increased 1.7-fold during the shoot induction phase (days 6–10) and had an additional 2.6-fold cytokinin increase correlated with the shift from induction to the shoot development phase. The cytokinins isopentenyl adenine (iP) and isopentenyl adenosine (iPAR) increased, while the cytokinins zeatin, zeatin riboside and dihydrozeatin remained at consistently low levels. In contrast, isoprenoid cytokinins did not accumulate in petunia TLV1 leaf explants which were incapable of shoot induction during 12 days of culture with BA. Cytokinin oxidase activity continuously increased in leaf explants of both petunia genotypes in response to BA, with a larger increase in St40. These results suggest that the differences in organogenic response in the two petunia genotypes may be the result of differences in BA uptake and metabolism which subsequently affects the accumulation of isoprenoid cytokinins and the activity of cytokinin oxidase in the early stages of shoot development.     

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     

Ontogenetic variation of four cytokinins in soybean root pressure exudate

Cytokinins exported from the root may be involved in the correlative control of plant development. To test this hypothesis in soybean ((Glycine max [L.] Merr. cv. McCall, cv Chippewa 64, and cv Hodgson 78), cytokinins were intercepted en route from the root to the shoot by collecting root pressure exudate from detopped roots. The quantities of four cytokinins in the exudate were studied throughout the development of plants grown in the field and in controlled environment chambers. Zeatin, zeatin riboside, and their dihydro derivatives, dihydrozeatin and dihydrozeatin riboside, were isolated and quantitated using high-performance liquid chromatography.

Cytokinin fluxes (pmoles per plant per hour) were independent of exudate flux (grams per plant per hour). All fluxes are averages for a 6- or 8-h collection period. The ribosides accounted for the majority of the observed cytokinin transport. The fluxes of zeatin riboside and dihydrozeatin riboside increased from low levels during vegetative growth to maxima during late flowering or early pod formation. Before the seeds began rapid dry matter accumulation, zeatin riboside and dihydrozeatin riboside fluxes decreased and remained at low levels through maturation. The fluxes of zeatin and dihydrozeatin were low throughout development.

No correlation was found between cytokinin fluxes and nodule dry weight or specific nodule activity (acetylene reduction).

The timing of distinct peaks in zeatin riboside and dihydrozeatin riboside fluxes during flowering or pod formation suggests that cytokinins exported from the root may function in the regulation of reproductive growth in soybean.

     

Movement to bark and metabolism of xylem cytokinins in stems of Lupinus angustifolius

Following uptake of [(3)H]zeatin riboside and [(3)H]dihydrozeatin riboside by girdled lupin (Lupinus angustifolius L.) stems via the transpiration stream, rapid lateral movement of the radioactivity from xylem to bark was observed. Short-term studies with intact stems, and other studies with excised stem tissues, revealed that the ribosides and/or the corresponding nucleotides were the cytokinin forms which actually moved into the bark tissues. Relative to cytokinin metabolism in xylem plus pith, metabolism in bark was both more rapid and more complex. Riboside cleavage and formation of the O-acetylzeatin and O-acetyldihydrozeatin ribosides and nucleotides were almost completely confined to bark tissues. Exogenous (3)H-labelled O-acetylzeatin riboside was converted to zeatin riboside in bark tissue, but the presence of the acetyl group suppressed degradation to adenine metabolites. The sequestration and modification of xylem cytokinins by stem tissues probably contributes significantly to the cytokinin status of the shoot. New cytokinins identified by mass spectrometry in lupin were: O-acetyldihydrozeatin 9-riboside, a metabolite of exogenous dihydrozeatin riboside in stem bark; O-methylzeatin nucleotide and O-methyldihydrozeatin 9-riboside, metabolites of endogenous cytokinins in stem bark; O-methylzeatin nucleotide and O-methylzeatin 9-riboside, metabolites of exogenous zeatin riboside in excised pod walls.     

Daily changes in uptake, reduction and storage of nitrate in spinach grown at low light intensity

Under poor light conditions, as normally used during winter production of greenhouse vegetables, the nitrate concentration in the shoot of spinach ( Spinacia oleracea L. cv. Vroeg Reuzenblad) showed a diurnal rhythm. This rhythm was mainly caused by a decrease during the day, followed by an increase during the night in the leaf blade nitrate concentration. Nitrate was mainly located in the vacuoles of the leaf blades. A strong correlation was found between net uptake of nitrate by the roots and the nitrate concentration in the leaf blade vacuoles. The nitrate concentration in the leaf blades increased during the initial hours of the night. This increase was caused by a marked increase in the net uptake rate of nitrate by the roots during the first hours of the dark period. During the second part of the night both net uptake rate of nitrate by the roots and the vacuolar nitrate concentration in the leaf blades remained constant.
We conclude that nitrate is taken up for osmotic purposes when light conditions are poor because of a lack of organic solutes. During the night, nitrate influx into the vacuole is needed for replacement of organic solutes, which are metabolized during the night, and possibly also for leaf elongation growth. During the day, vacuolar nitrate may be exchanged for newly synthesized organic solutes and be metabolized in the cytoplasm. A strong diurnal rhythm in nitrate reductase (NR; EC 1.6.6.1.) activity was absent, due to the poor light conditions, and in vitro NR activity was not correlated with nitrate flux from the roots. In vivo NR activity also lacked a strong diurnal rhythm, but it was calculated that in situ nitrate reduction was much lower during the night, so that the major nitrate assimilation took place during the day.     

Comparative studies of the light modulation of nitrate reductase and sucrose-phosphate synthase activities in spinach leaves

We recently obtained evidence that the activity of spinach (Spinacia oleracea L.) leaf nitrate reductase (NR) responds rapidly and reversibly to light/dark transitions by a mechanism that is strongly correlated with protein phosphorylation. Phosphorylation of the NR protein appears to increase sensitivity to Mg²⁺ inhibition, without affecting activity in the absence of Mg²⁺. In the present study, we have compared the light/dark modulation of sucrose-phosphate synthase (SPS), also known to be regulated by protein phosphorylation, and NR activities (assayed with and without Mg²⁺) in spinach leaves. There appears to be a physiological role for both enzymes in mature source leaves (production of sucrose and amino acids for export), whereas NR is also present and activated by light in immature sink leaves. In mature leaves, there are significant diurnal changes in SPS and NR activities (assayed under selective conditions where phosphorylation status affects enzyme activity) during a normal day/night cycle. With both enzymes, activities are highest in the morning and decline as the photoperiod progresses. For SPS, diurnal changes are largely the result of phosphorylation/dephosphorylation, whereas with NR, the covalent modification is super-imposed on changes in the level of NR protein. Accumulation of end products of photosynthesis in excised illuminated leaves increased maximum NR activity, reduced its sensitivity of Mg²⁺ inhibition, and prevented the decline in activity with time in the light seen with attached leaves. In contrast, SPS was rapidly inactivated in excised leaves. Overall, NR and SPS share many common features of control but are not identical in terms of regulation in situ.     

Influence of Different Cytokinins on the Germination of Lettuce (Lactuca sativa) and Celery (Apium graveolens) Seeds

The naturally occurring cytokinins, zeatin, zeatin riboside and dihydrozeatin did not promote the germination of celery (Apium graveolens L.) seeds and 6-Δ²-isopentenyladenine (2iPA) and its riboside were only moderately active. Of the synthetic cytokinins, kinetin, kinetin riboside, and the disubstituted urea, N-phenyl-N′-pyridyl urea (NC5392) were moderately active, and 6-benzyl-aminopurine (BA) and its derivatives BA riboside and 6-benzyl-amino-9(tetrahydropyran-2yl)purine (SD8339) were the most active cytokinins tested. 6-(o-hydroxybenzyl)aminopurine (hyd-BA) and its naturally occurring riboside inhibited germination under normally inductive conditions. All the cytokinins examined were more active in promoting germination of lettuce (Lactuca sativa L.) than celery seeds. BA, BA riboside and SD8339 were again the most active cytokinins. In contrast to the results with celery, zeatin and zeatin riboside were highly active. The other cytokinins also showed high activity with the exception of dihydrozeatin, hyd-BA and hyd-BA riboside which were less active. Cytokinin ribosides were less active than the corresponding free bases during the early period of the lettuce seed incubation but total germination after 90 h was similar.     

Effects of temperature and gibberellin on the activity of endogenous cytokinin-like substances in leaves of Begonia x cheimantha

Changes in activity of endogenous cytokinin-like substances were examined in intact plants and excised leaves of Begonia x chemantha Everett cv. Prinsesse Astrid (Christimas Begonia) by means of the tobacco callus bioassay. Cytokinin activity in the leaves of intact plants was higher in plants grown at 18°C than in those grown at 21° or 24°C. In excised leaves, an increase in cytokinin activity was observed during the first 4 days following leaf detachment. However, after the seventh day cylokinin activity decreased again. This decrease was more profound in leaves exposed to 24°C than in those exposed to 18°C.
Treatment of detached leaves with gibberellic acid (2.8 m M ) caused an increase in measurable cytokinin activity. This increase was more profound in the zones of activity which correspond with zeatin glucosides on paper and Sephadex LH-20 chromatography. Additional zones of activity appeared after Sephadex chromatography. These were of a more slow moving nature with elution volumes corresponding to N^b-(Δ²-isopentenyl)adenine and its derivaties. Water-treated control leaves had higher activity in the regions corresponding to zeatin and zeatin riboside.     

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

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