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     

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     

The capacity of antioxidant protection during modulated ageing of bean (Phaseolus vulgaris L.) cotyledons. 2. The low-molecular weight antioxidants

The aim of the present work was to evaluate non-enzymic antioxidants during natural and artificially modulated senescence. Senescence of bean (Phaseolus vulgaris L. cv. Jantar) cotyledons was modulated by UV C irradiation or by the decapitation of plants apices. The content of beta-carotene and zeaxanthin decreased in control and decapitated plants but in UV C irradiated plants these contents increased. The degree of de-epoxidation increased in all cultivations with age. The content of total glutathione (sum of reduced and oxidized) sharply decreased in bean cotyledons grown in all conditions. Interestingly, the content of total ascorbate increased at the end of cotyledon life span of control and decapitated plants but decreased in UV plants. Decrease of reduced/oxidized ratio of ascorbate and glutathione during cotyledon ageing confirmed increasing oxidative stress during senescence in all cultivations.     

Endogenous cytokinins in the vegetative and reproductive phases of development in the dioecious plant Rumex acetosella L.

The content of endogenous cytokinins has been analysed in leaf and inflorescence extracts of male and female R. acetosella plants, using gas chromatography. Plant parts were extracted at four stages of development: leaves of juvenile plants, leaves of adult plants at the time of flower initiation and in full bloom, and upper internodes of the inflorescence stalks. Cytokinins with characteristics similar to isopentenyl adenine and adenosin, zeatin, zeatin riboside, and a bound form of zeatin, were all found in the extracts. The total amount of cytokinins was higher in female than in male plants during all these stages.Reprint requests     

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.     

Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric Isopentenyl Transferase Gene

Cytokinins, a class of phytohormones, appear to play an important role in the processes of plant development. We genetically engineered the Agrobacterium tumefaciens isopentenyl transferase gene, placing it under control of a heat-inducible promoter (maize hsp70). The chimeric hsp70 isopentenyl transferase gene was transferred to tobacco and Arabidopsis plants. Heat induction of transgenic plants caused the isopentenyl transferase mRNA to accumulate and increased the level of zeatin 52-fold, zeatin riboside 23-fold, and zeatin riboside 5[prime]-monophosphate twofold. At the control temperature zeatin riboside and zeatin riboside 5[prime]-monophosphate in transgenic plants accumulated to levels 3 and 7 times, respectively, over levels in wild-type plants. This uninduced cytokinin increase affected various aspects of development. In tobacco, these effects included release of axillary buds, reduced stem and leaf area, and an underdeveloped root system. In Arabidopsis, reduction of root growth was also found. However, neither tobacco nor Arabidopsis transgenic plants showed any differences relative to wild-type plants in time of flowering. Unexpectedly, heat induction of cytokinins in transgenic plants produced no changes beyond those seen in the uninduced state. The lack of effect from heat-induced increases could be a result of the transient increases in cytokinin levels, direct or indirect induction of negating factor(s), or lack of a corresponding level of competent cellular factors. Overall, the effects of the increased levels of endogenous cytokinins in non-heat-shocked transgenic plants seemed to be confined to aspects of growth rather than differentiation. Since no alterations in the programmed differentiation pattern were found with increased cytokinin levels, this process may be controlled by components other than absolute cytokinin levels.     

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.     

Regulators of cell division in plant tissues. XXX. Cytokinin metabolism in relation to radish cotyledon expansion and senescence

Kinetic studies of formation of glucosides of 6-benzylaminopurine (BAP) in excised radish cotyledons indicated that the 3-, 7-, and 9-glucosides (N-glucosides) were each formed directly from BAP. The 7- and 9-glucosides of BAP and the 7-glucoside of zeatin exhibited great stability in the cotyledons, but the 3-glucoside was converted to free BAP and to the 7- and 9-glucosides of BAP. When³H-labeled zeatin was supplied to developed cotyledons, at high concentrations (100 μM), 7-glucosylzeatin was the principal metabolite, but an appreciable proportion of the extracted³H was due to O-glucosylzeatin. In immature cotyledons, as used in the radish cotyledon cytokinin bioassay, this O-glucoside was shown to be converted into zeatin 7-glucoside probably via free zeatin. Metabolism of BAP and zeatin in radish cotyledons was studied in relation to cytokinin-induced cotyledon expansion. Cytokinin N-glucosides were not metabolites responsible for the observed cytokinin-induced expansion, and were not detoxification products, or deactivation products formation of which was coupled with cytokinin action. However, the free base, its riboside, and nucleotide were possible active forms of BAP associated with cotyledon expansion. The possible significance of cytokinin N-glucosides is discussed. Senescent and nonsenescent cotyledons differed in their metabolism of BAP, zeatin, and zeatin riboside. Senescence was associated principally with a reduction in ability to form 7-glucosylzeatin, enhanced metabolism to adenine derivatives, and an inability to form appreciable amounts of 3-glucosyl-BAP. A two-dimensional thin layer chromatography (TLC) system, based on adjoining layers of cellulose and silica gel, for separating zeatin metabolites is described. This does not completely separate zeatin and zeatin riboside from the corresponding dihydro-compounds. A reversed phase TLC method for achieving these separations is also reported.     

Gas Chromatography-Flame Thermionic Detector Analysis of Cytokinins in Etiolated Squash Seedlings using 14C-BA as an Internal Standard

Cytokinin contents in cotyledon, hypocotyl and root of etiolatedsquash (Cucurbita maxima Duch.) seedlings were determined byinstrumental analysis using ¹⁴C-benzyladenine (¹⁴C-BA) as aninternal standard. Crude extracts were purified using insolublepolyvinylpyrrolidone, cellulose-phosphate column and SEP-PAKC18 cartridge, then applied to a Sephadex LH-20 column to separatezeatin riboside (ZR), isopentenyl adenosine, isopentenyl adenine,¹⁴C-BA and a mixture of zeatin (Z) and dihydrozeatin (DHZ).The recovery rate for the cytokinin fractions after LH-20 wascorrected by ¹⁴C-BA. Each cytokinin fraction was further purifiedby HPLC which also separated Z and DHZ in the LH-20 fraction.Before permethylation, ¹⁴C-BA was added to each of the cytokininfractions to correct the methylation rates. Each methylatedcytokinin fraction was again purified by HPLC, then subjectedto gas chromatography with a capillary column and flame thermionicdetector. The detection limit of cytokinins by this system was0.1 ng. cis-ZK was the most abundant cytokinin in all tissues of theetiolated squash seedlings. Active cytokinins such as trans-ZRand trans-Z were mostly found in cotyledons with lesser amountsin the roots. DHZ was most abundant in the cotyledon. All cytokininsisolated by this procedure were confirmed by gas chromatographyselectedion monitoring. (Received December 26, 1986; Accepted June 1, 1987)     

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.     

Endogenous hormonal content and somatic embryogenic capacity of Corylus avellana L. cotyledons

Endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and cytokinins [zeatin (Z) zeatin riboside, dihydrozeatin, dihydrozeatin riboside, N⁶-isopentenyl adenine (iP) and N⁶-isopentenyladenine riboside] were evaluated in hazelnut (Corylus avellana L.) cotyledons of different developmental stage and genetic source for their somatic embryogenic capacity. There was an inverse correlation between the embryogenic potential of cotyledons and the degree of maturity of zygotic embryos, the first characteristic being associated with iP-type cytokinins and the second with Z-type cytokinins. Although the differences in total cytokinin, ABA and IAA contents between the cotyledons were small, the IAA/ABA and, mainly, the iP-type/Z-type cytokinin ratios were found to be two good indexes of the embryogenic competence of explants, suggesting that the endogenous hormonal balance is a very important factor defining the in vitro potential of hazelnut cotyledons. Received: 6 January 1997 / Revision received: 3 March 1997 / Accepted 1 April 1997     

Cloning an ipt gene from Agrobacterium tumefaciens: characterisation of cytokinins in derivative transgenic plant tissue

The isopentenyl transferase gene was isolated from Agrobacterium tumefaciens AcH5 using polymerase chain reaction and transformed into Petunia and Kalanchoë using both A. tumefaciens and A. rhizogenes transformation systems. Morphological evidence and elevated endogenous cytokinin levels indicated that the PCR product was an active gene. Accurate quantification of the cytokinins was obtained by radioimmunoassay, following purification and separation of the free bases and ribosides by HPLC. Of the six cytokinins quantified, zeatin riboside and its stabilised dihydro-derivative, dihydrozeatin riboside, showed the greatest increases in the transformed Petunia tissue (up to 600-fold). The importance of measuring changes in individual cytokinins is discussed.     

An improved bioassay for cytokinins using cucumber cotyledons

The cucumber cotyledon greening bioassay is frequently used for detecting cytokinins. Beneficial modifications of the original technique included using 5-day-old cucumber (Cucumus sativus L., cv. National Pickling) cotyledons treated with combinations of 40 millimolar KCl and various concentrations of cytokinins. A dark incubation period of 20 hours was followed by an exposure to light for 3.5 hours. Under these conditions, extremely low (0.0001 milligram per liter) concentrations of N⁶-benzyladenine, zeatin, kinetin, or zeatin riboside can be detected. Of the four cytokinins tested, kinetin appeared to be the least active. With these improvements, the assay is 10 times more sensitive than is the previously described cucumber cotyledon greening bioassay for cytokinins.     

The capacity of antioxidant protection during modulated ageing of bean (Phaseolus vulgaris L.) cotyledons. 1. The antioxidant enzyme activities

Reactive oxygen species are known to increase in plant senescence. We investigated the participation of antioxidative enzymes in initiation of cotyledon senescence. Senescence of bean (Phaseolus vulgaris L.) cotyledons was modulated by UV C irradiation and by the decapitation of plant apices. Senescence was accompanied by a decrease of protein content and by a decrease of photochemical efficiency. A drop in activity of antioxidative enzymes preceded the onset of senescence in control plants. In cotyledons with prolonged life span, the decrease of antioxidant activities and the markers of senescence onset appeared at a similar age as in controls. Thus we presumed that the period from senescence initiation to cotyledon abscission was extended. On the other hand, in UV C irradiated plants we did not observe actual senescence initiation, and antioxidant enzymes although elevated, did not effectively play their role. The decrease of antioxidant enzymes activity and the markers of senescence appeared at a similar age both in control and in decapitated (D) plants, so we can presume that we prolonged mainly the period from senescence onset to cotyledon abscission in D plants. In UV C irradiated plants the antioxidative enzymes were probably destroyed before the process of senescence could begin.     

Regulators of cell division in plant tissues. XXX. Cytokinin metabolism in relation to radish cotyledon expansion and senescence

Kinetic studies of formation of glucosides of 6-benzylaminopurine (BAP) in excised radish cotyledons indicated that the 3-, 7-, and 9-glucosides (N-glucosides) were each formed directly from BAP. The 7- and 9-glucosides of BAP and the 7-glucoside of zeatin exhibited great stability in the cotyledons, but the 3-glucoside was converted to free BAP and to the 7- and 9-glucosides of BAP. When³H-labeled zeatin was supplied to developed cotyledons, at high concentrations (100 M), 7-glucosylzeatin was the principal metabolite, but an appreciable proportion of the extracted³H was due to O-glucosylzeatin. In immature cotyledons, as used in the radish cotyledon cytokinin bioassay, this O-glucoside was shown to be converted into zeatin 7-glucoside probably via free zeatin.Metabolism of BAP and zeatin in radish cotyledons was studied in relation to cytokinin-induced cotyledon expansion. Cytokinin N-glucosides were not metabolites responsible for the observed cytokinin-induced expansion, and were not detoxification products, or deactivation products formation of which was coupled with cytokinin action. However, the free base, its riboside, and nucleotide were possible active forms of BAP associated with cotyledon expansion. The possible significance of cytokinin N-glucosides is discussed.Senescent and nonsenescent cotyledons differed in their metabolism of BAP, zeatin, and zeatin riboside. Senescence was associated principally with a reduction in ability to form 7-glucosylzeatin, enhanced metabolism to adenine derivatives, and an inability to form appreciable amounts of 3-glucosyl-BAP.A two-dimensional thin layer chromatography (TLC) system, based on adjoining layers of cellulose and silica gel, for separating zeatin metabolites is described. This does not completely separate zeatin and zeatin riboside from the corresponding dihydro-compounds. A reversed phase TLC method for achieving these separations is also reported.     

Changes in Concentrations of Cytokinins (CKs) in Root and Axillary Bud Tissue of Miniature Rose Suggest that Local CK Biosynthesis and Zeatin-Type CKs Play Important Roles in Axillary Bud Growth

Involvement of cytokinins (CKs) in axillary bud growth of miniature rose was studied. Variation in root formation and axillary bud growth was induced by two indole 3-butyric acid (IBA) pretreatments in two cutting sizes. At six physiological developmental stages around the onset of axillary bud growth, concentrations of CKs were determined in both root and axillary bud tissue by liquid chromatography combined with electrospray tandem mass spectrometry (LC-ESP-MS/MS). Chronological early onset of axillary bud growth occurred in long cuttings pretreated at low IBA concentration, whereas physiological early root formation was associated with long cuttings and high IBA concentration. The CKs zeatin (Z), isopentenyl adenine (iP), zeatin riboside (ZR), dihydrozeatin riboside (DHZR), isopentenyl adenosine (iPA), zeatin O-glucoside (ZOG), zeatin riboside O-glucoside (ZROG), zeatin riboside 5′-monophosphate (ZRMP), and isopentenyl adenosine 5′-monophosphate (iPAMP) were detected. Concentrations of CKs in axillary bud tissue far exceeded those in root tissue. Indole 3-butyric acid pretreatment influenced the concentration of CKs in axillary bud tissue more than did cutting size, whereas pretreatments only slightly affected CKs in root tissue. The dominant CKs found were iPAMP and ZR. An early and large increase in iPAMP indicated rapid CK biosynthesis in rootless cuttings, suggesting that green parts, including the axillary bud, can synthesize CKs. At the onset of axillary bud growth an increase in concentration of Z, ZR, ZRMP, ZOG, and ZROG was largely coincident with a decrease in iPAMP, iPA, iP, and DHZR. After the onset of axillary bud growth, CK content largely decreased. These results strongly indicate a positive role for CKs in axillary bud growth, and presumably ZRMP, ZR, and Z are active in miniature rose.     

Phytohormones,Rhizobium mutants,and nodulation in legumes. VII. Identification and quantification of cytokinins in effective and ineffective pea root nodules using radioimmunoassay

Radioimmunoassays (RIA), employing antisera raised in rabbits against bovine serum albumin conjugates of zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine, were used to estimate levels of these cytokinins and their corresponding bases in samples of effective (nitrogen-fixing, Fix⁺), ineffective (nonnitrogen-fixing, Fix^–) pea root nodules and uninoculated roots. Assays were done on extracts of nodule tissue, 1–2 g fresh weight, or approximately 10 g fresh weight of root tissue, and high specific activity [³H]zeatin riboside was added during preparation of the extract for use as a recovery marker. Two different purification procedures were employed, each involving several purification steps. High performance liquid chromatography (HPLC) was the final step in both procedures. Fractions from HPLC were analyzed by RIA using the appropriate antiserum. The cytokinins, zeatin, zeatin riboside, dihydrozeatin riboside, isopentenyl adenine, and isopentenyladenosine were detected and quantified in nodule tissue, and similarly, in root tissue (with the exception of zeatin, which we were unable to quantify in root tissue). Cytokinin levels in nodule tissue were higher than those in root tissue. The major cytokinins detected in nodule tissue were zeatin, followed by zeatin riboside and then dihydrozeatin riboside. The levels of zeatin and zeatin riboside estimated in nodules in the present study by RIA were of the same order of magnitude, though tending to be a little higher, than values obtained previously by bioassay. Dihydrozeatin riboside was identified with confidence for the first time in nodule tissue. There was a general decline with age in cytokinin levels in nodules, but no major qualitative change in nodule cytokinins with age. For theRhizobium strains examined, the data did not indicate a clear correlation between nodule cytokinin levels and the effectiveness of nodules in nitrogen fixation.     

Cytokinin biochemistry in relation to leaf senescence. VIII. Translocation, metabolism and biosynthesis of cytokinins in relation to sequential leaf senescence of tobacco

Cytokinin bases (zeatin and dihydrozeatin) and ribosides (zeatin riboside and dihydrozeatin riboside) were identified as major cytokinins in tobacco xylem sap by radioimmunoassay. When ³H-labelled zeatin riboside or dihydrozeatin riboside were supplied to tobacco plants via the xylem, leaves of differing maturity did not differ appreciably in level of radioactivity or in metabolism of the cytokinin. The major metabolites of zeatin riboside in leaves were adenine, adenosine and adenine nucleotides, whereas that of dihydrozeatin riboside was dihydrozeatin 7-glucoside. Incorporation of [¹⁴C]adenine into zeatin was evident in upper green leaves. indicating that young leaves have the capacity to synthesize cytokinins in situ. In contrast, fully expanded green leaves and senescing tobacco leaves exhibited little or no incorporation of [¹⁴C]adenine into cytokinins. This difference in cytokinin biosynthetic capacity may contribute to the differing cytokinin levels in leaves of different matirity, and may participate in control of sequential leaf senescence in tobacco.     

Changes in endogenous levels of three cytokinins during development of excised watermelon cotyledons

We have determined by an immunological method the endogenous levels of three cytokinins: dihydrozeatin riboside (DHZR), transzeatin riboside (tZR) and isope-ntenyladenosine (IPA) in watermelon (Citrullus vulgaris Schrad., cv. Fairfax) cotyledons that were either attached to the seedling or excised from the seed after imbibition and then grown on water. Both seedlings and cotyledons were grown either for 5 days in continuous light or for 3 days in the dark and 2 days in light. Our aim was to verify whether endogenous cytokinin levels are lower in excised than in attached cotyledons as could be expected since excised cotyledons are much more sensitive to exogenous cytokinin application. The levels of the three cytokinins were very low immediately after imbibition, but gradually increased during the following days. They were higher in excised cotyledons after 5 days of culture in the dark than in cotyledons of the same age that had developed on the seedling. Dihydrozeatin riboside was by far the most abundant of the three cytokinins in cotyledons as well as in the hypocotyl and the root.
Irradiation reduced the level of DHZR, negating the concept that light promotes cotyledon development by increasing endogenous cytokinins. Transzeatin riboside when supplied exogenously, stimulated cotyledon development at a lower concentration than the other two cytokinins. Exogenous supply of ben-zyladenine (BA) induced a strong increase in endogenous tZR already after 24 h.     

Endogenous cytokinins in the vascular cambial region of Pinus sylvestris during activity and dormancy

Elucidation of the role of endogenous cytokinins in cambial activity and wood formation requires knowledge of their identity and concentrations in the cambial region. Here, we have used capillary liquid chromatography/frit-FAB mass spectrometry to identify endogenous cytokinins in the vascular cambial region of mature Pinus sylvestris (L.) trees. Full-scan mass spectra were obtained for isopentenyladenine, isopentenyladenosine, zeatin riboside, dihydrozeatin and dihydrozeatin riboside. Of these, isopentenyladenine, dihydrozeatin and dihydrozeatin riboside are demonstrated by rigorous physico chemical methods for the first time in a conifer. In addition, an adenine glycoside was found for the first time in a plant. The identified cytokinins were quantified in active and dormant cambial region tissues by isotope dilution techniques using the appropriate deuterated isotope for each cytokinin species. The concentration of the detected cytokinins ranged between 1.3 and 5.5 pmol g^-1 fresh weight, and did not vary greatly between dormant tissues, and in tissues actively dividing and differentiating. This observation indicates that cessation and reactivation of cell division activity in the vascular cambium is controlled by factors other than cytokinin availability.