Journal of Experimental Botany, Vol. 56, No. 421, pp. 2877–2883, November 2005 doi:10.1093/jxb/eri282 Advance Access publication 12 September, 2005 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) RESEARCH PAPER Diurnal variation of cytokinin, auxin and abscisic acid levels in tobacco leaves Marie Nováková1,2, Václav Motyka1, Petre I. Dobrev1, Jiřı́ Malbeck1, Alena Gaudinová1 and Radomı́ra Vanková1,* 1 Institute of Experimental Botany AS CR, Rozvojova 135, 165 02 Prague 6, Czech Republic 2 Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, 128 43 Prague 2, Czech Republic Received 15 April 2005; Accepted 5 August 2005 As many processes are regulated by both light and plant hormones, evaluation of diurnal variations of their levels may contribute to the elucidation of the complex network of light and hormone signal transduction pathways. Diurnal variation of cytokinin, auxin, and abscisic acid levels was tested in tobacco leaves (Nicotiana tabacum L. cv. Wisconsin 38) grown under a 16/8 h photoperiod. The main peak of physiologically active cytokinins (cytokinin bases and ribosides) was found after 9 h of light, i.e. 1 h after the middle of the light period. This peak coincided with the major auxin peak and was closely followed by a minor peak of abscisic acid. Free abscisic acid started to increase at the light/dark transition and reached its maximum 3 h after dark initiation. The content of total cytokinins (mainly N-glucosides, followed by cis-zeatin derivatives and nucleotides) exhibited the main peak after 9 h of light and the minor peak after the transition to darkness. The main, midday peak of active cytokinins was preceded by a period of minimal metabolic conversion of tritiated trans-zeatin (less than 30%). The major cytokinin-degrading enzyme, cytokinin oxidase/ dehydrogenase (EC 1.5.99.12), exhibited maximal activity after the dark/light transition and during the diminishing of the midday cytokinin peak. The former peak might be connected with the elimination of the long-distance cytokinin signal. These cytokinin oxidase/dehydrogenase peaks were accompanied by increased activity of b-glucosidase (EC 3.2.1.21), which might be involved in the hydrolysis of cytokinin O-glucosides and/or in fine-tuning of active cytokinin levels at their midday peak. The achieved data indicate that cytokinin metabolism is tightly regulated by the circadian clock. Key words: Abscisic acid, auxin, cytokinin, cytokinin oxidase/ dehydrogenase, diurnal rhythm, b-glucosidase, tobacco. Introduction It is assumed that plant growth and development are regulated by the interactions between the environment and endogenous factors, especially hormones. Light is an important environmental signal that activates and modifies various growth and developmental processes in plants, for example, seed germination, flower induction, leaf movements, stem elongation, and de-etiolation. All of these processes are regulated by plant hormones as well. Effects of light and phytohormones can be synergistic (e.g. chloroplast development and cytokinins [CKs]), additive (e.g. inhibition of hypocotyls elongation and CKs), or antagonistic (e.g. germination and abscisic acid [ABA]). There are numerous reports that light itself may influence the levels of different hormones. Light was found to regulate directly the biosynthesis of active gibberellins (Foster and Morgan, 1995) and ethylene (Jasoni et al., 2002) as well as to promote the degradation of ABA (Kraepiel et al., 1994). The effect of light on auxin levels is less transparent and may be realized by influencing auxin * To whom correspondence should be addressed. Fax: +420 220 390 446. E-mail: vankova@ueb.cas.cz Abbreviations: ABA, abscisic acid; CK, cytokinin; CKX, cytokinin oxidase/dehydrogenase; DHZ, dihydrozeatin; DHZR, dihydrozeatin riboside; IAA, b-indolylacetic acid; iP, N 6-(D2-isopentenyl)adenine; iPR, N 6-(D2-isopentenyl)adenosine; iP7G, N6-(D2-isopentenyl)adenine 7-glucoside; iP9G, N6-(D2-isopentenyl) adenine 9-glucoside; Z, trans-zeatin; ZR, trans-zeatin riboside; ZOG, trans-zeatin O-glucoside; ZROG, trans-zeatin riboside O-glucoside; Z7G, trans-zeatin 7-glucoside; Z9G, trans-zeatin 9-glucoside. ª The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. The online version of this article has been published under an Open Access model. Users are entitled to use, reproduce, disseminate, or display the Open Access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and the Society for Experimental Biology are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact: journals.permissions@oxfordjournals.org Downloaded from http://jxb.oxfordjournals.org/ by guest on May 29, 2013 Abstract 2878 Nováková et al. Materials and methods Plant material Tobacco plants (Nicotiana tabacum L. cv. Wisconsin 38) were grown in soil in a growth chamber for 6 weeks at a 16 h photoperiod and 130 lmol mÿ2 sÿ1, a day/night temperature of 25/23 8C, and a relative humidity of c. 80%. Mixed samples of leaves (3rd–5th of 7, i.e. with the exception of the youngest and oldest ones) from three plants were collected every hour during a 24 h period. After removal of the main vein the samples were immediately frozen in liquid nitrogen. Incubation with tritiated trans-zeatin Tip parts of the 3rd or 4th leaf (c. 1 g FW) were cut at 3 h intervals and their cut surface was submerged into aqueous tritiated trans-zeatin ([3H]Z, 33104 Bq, specific activity 0.6 TBq mmolÿ1, prepared by Dr Jan Hanuš, Isotope Laboratory, Institute of Experimental Botany AS CR, Prague, Czech Republic). After 1 h incubation (under the same light regime as the intact plants) leaf tips were frozen in liquid nitrogen. The uptake of [3H]Z by the vascular tissue was efficient (35–50% of the applied radioactivity). Plant hormone extraction and purification Phytohormones were extracted and purified according to Dobrev and Kamı́nek (2002). Frozen leaf samples (c. 1 g FW) were ground in liquid nitrogen and extracted overnight with 10 cm3 methanol/water/ formic acid (15/4/1, by vol., pH;2.5, ÿ20 8C). For analyses of endogenous CKs, 50 pmol of each of the following 12 deuteriumlabelled standards were added: [2H5]Z, [2H5]ZR, [2H5]Z7G, [2H5]Z9G, [2H5]ZOG, [2H5]ZROG, [2H6]iP, [2H6]iPR, [2H6]iP7G, [2H6]iP9G, [2H5]DHZ, [2H5]DHZR (Apex Organics, Honington, UK). Tritiated internal standards were used for the determination of auxin (3[5(n)-3H] indolyl acetic acid, Amersham, UK, specific activity 74 GBq mmolÿ1, 53103 Bq) and ABA (Amersham, UK, specific activity 1.74 TBq mmolÿ1, 53103 Bq). The extracts were purified using Si-C18 columns (SepPak Plus, Waters, Milford, MC, USA) and Oasis MCX mixed mode (cation exchange and reversephase) columns (150 mg, Waters, USA) and evaporated. HPLC of radiolabelled trans-zeatin and its metabolic products Evaporated samples were resuspended in 200 mm3 20% (v/v) acetonitrile. Radiolabelled metabolites of [3H]Z were analysed according to Blagoeva et al. (2004) using a Series 200 Quaternary HPLC Pump (Perkin Elmer, Wellesley, MA, USA) coupled to a 235C Diode Array Detector (Perkin Elmer, USA) and a RAMONA 2000 flow-through radioactivity detector (Raytest, Straubenhardt, Germany), column: Luna C18(2), 150 mm/4.6 mm/3 lm (Phenomenex, Torrance, CA, USA). The radioactive metabolites were identified on the basis of coincidence of retention times with authentic standards. HPLC of auxin and abscisic acid Indolyl acetic acid and ABA were determined using two-dimensional HPLC according to Dobrev et al. (2005). The instrumental set-up consisted of a series 200 autosampler (Perkin Elmer, Norwalk, CT, USA), two HPLC gradient pump systems (first pump: ConstaMetric 3500 and 3200 with 500 ll mixer, TSP, Riviera Beach, FL, USA; second pump: Series 200 Quaternary Pump, Perkin Elmer), two columns (first column: ACE-3CN, 15034.6 mm, 3 mm, ACT, Aberdeen, Scotland, UK; second column: Luna C18(2), 15034.6 mm, 3 mm, Phenomenex, Torrance, CA, USA), one 2-position, fluid processor SelectPRO with 1 ml loop (Alltech, Deerfield, IL, USA). The segment containing IAA and ABA obtained in the first dimension was collected in the loop of the fluid processor and redirected to the second HPLC dimension. IAA was quantified using fluorescence detector LC 240 (Perkin Elmer). Quantification of ABA was performed on the basis of UV detection using diode array-detector 235C (Perkin Elmer). HPLC/mass spectrometry LC-MS analysis was performed as described by Lexa et al. (2003) using a Rheos 2000 HPLC gradient pump (Flux Instruments, Switzerland) and HTS PAL autosampler (CTC Analytics, Switzerland) coupled to an Ion Trap Mass Spectrometer Finnigan MAT LCQ-MSn equipped with an electrospray interface. Detection and quantification were carried out using a Finnigan LCQ operated in the positive ion, full-scan MS/MS mode using a multilevel calibration graph with deuterated cytokinins as internal standards. The levels of 27 different cytokinin derivatives were measured. The detection limit was calculated for each compound as 3.3 r/S, where r is the standard deviation of the response and S the slope of the calibration curve. Three independent experiments were done. Each sample was injected at least twice. Determination of CKX activity The enzyme preparations were extracted and partially purified using the method of Chatfield and Armstrong (1986) as modified by Motyka et al. (2003). The CKX activity was determined by in vitro assays based on the conversion of [2-3H]isopentenyladenine (prepared by Dr Jan Hanuš) to [3H]adenine. The assay mixture (50 mm3 final volume) included 100 mM TAPS-NaOH buffer containing 75 lM 2,6-dichloroindophenol (pH 8.5), 2 lM substrate ([2-3H] isopentenyladenine, 7.4 Bq molÿ1) and the enzyme preparation equivalent to 250 mg tissue FW (corresponding to 0.02–0.26 mg protein gÿ1 FW). After incubation at 37 8C the reaction was terminated by the addition of 10 mm3 Na4EDTA (200 mM) and 120 mm3 95% (v/v) ethanol. Separation of the substrate from the product of the enzyme reaction was achieved by the same HPLC system as described for tritiated trans-zeatin metabolites according to Blagoeva et al. (2004). The CKX activity was determined in two independent experiments. As the data from both experiments showed Downloaded from http://jxb.oxfordjournals.org/ by guest on May 29, 2013 polar transport (Tian and Reed, 2001) or by the modulation of cell sensitivity towards auxin (Jones et al., 1991). The data on the effect of light on the metabolism of cytokinins are rather scarce. The assumption of a positive effect of light on CK accumulation has been based on the decreased level of trans-zeatin in phytochrome mutants (Kraepiel et al., 1995). Diurnal variation of CK levels was followed in barley (Kurapov et al., 2000) and carrot (Paasch et al., 1997), but these studies were limited only to the determination of CK bases and ribosides. The recent development of analytical methods (especially of mass spectrometry) has enabled the study of the diurnal variation of a broad array of CK metabolites. Diurnal changes in the levels of 27 CK metabolites were studied in tobacco leaves. The potential contribution of the cleavage of CK O-glucoconjugates to the fast changes of the level of physiologically active CKs (CK bases and ribosides) was estimated via the activity of b-glucosidase (EC 3.2.1.21). In order to study CK catabolism, the activity of the main CK-degrading enzyme, cytokinin oxidase/ dehydrogenase (CKX, EC 1.5.99.12) was determined. As CK levels are also affected by other hormones, the diurnal variation of auxin and ABA levels was followed. Cytokinin, auxin, and ABA diurnal rhythms 2879 the same tendencies (but different absolute values), the results of one representative experiment (mean value of three parallel assays for each of three replicate protein preparations) are presented. Determination of b-glucosidase activity Tobacco leaves (0.2 g FW) were ground in liquid nitrogen and transferred into 0.4 cm3 ice-cold extraction buffer [25 mM TRIS-Cl, pH 7.5, 5 mM MgCl2, 1 mM EDTA, and protease Complete EDTAfree cocktail tablet (Roche)]. Samples were centrifuged at 13 000 g for 15 min (4 8C) and 0.02 cm3 supernatant were added into 0.28 cm3 citrate-phosphate buffer (0.02 M citric acid; 0.06 M NaH2PO4; pH 5.5). The reaction was initiated by 0.1 cm3 20 mM p-nitrophenyl a-D-glucopyranoside. After 120 min at 30 8C the reaction was stopped by adding 0.6 cm3 2 M Na2CO3. The released p-nitrophenol was measured at 405 nm (UNICAM 5625 UV/VIS spectrometer). Results Fig. 2. Endogenous levels of CK N-glucosides, CK phosphates, and ciszeatin derivates in tobacco leaves during a 24 h period. Other details as for Fig. 1. Cytokinin O-glucosides and activity of b-glucosidase The level of CK storage forms (O-glucosides) was found to increase around the dark/light transition. Another peak of O-glucosides was detected after 7 h of light, preceding the peak of active CKs as well as that of CK deactivation metabolites. However, this CK O-glucoside peak was significantly lower than those of the deactivation products. Minor peaks of CK O-glucosides were detected after 10 h of light, when the level of active CKs was decreasing, and 2 h after the light/dark transition. The activity of b-glucosidase, the enzyme that catalyses the cleavage of glucosyl moieties from O-glucosides releasing, in the case of CK O-glucosides, active CKs, exhibited the first maximum after 1 h of light, which correlated with the decrease of the peak of CK O-glucosides found around the dark/light transition (Fig. 3). The major peak of b-glucosidase activity was found 7 h after the light was switched on, preceding the midday peak of active CKs. Downloaded from http://jxb.oxfordjournals.org/ by guest on May 29, 2013 Cytokinins Physiologically active cytokinins, i.e. trans-zeatin, isopentenyladenine, dihydrozeatin, and their corresponding ribosides, exhibited in tobacco leaves a sharp maximum after 9 h of light, i.e. 1 h after the middle of the light period (Fig. 1). Another, considerably lower, peak was detected after the transition to darkness and the lowest one was found at the beginning of the light phase. The content of cis-zeatin and its riboside exhibited a minor peak coinciding with the maximum of active CKs. Their maximum followed the second peak of active CKs after the light/ dark transition (with 2 h delay). The content of total CKs (the sum of physiologically active CKs together with CK phosphates, cis-zeatin derivatives, N- and O-glucosides) have an increasing trend during the light period, up to a maximum 9 h after the light was switched on (i.e. 1 h after midday). The second half of the light period was characterized by a sharp decrease in CKs. After the light/dark transition another transient CK peak occurred. During the course of the dark period, the total CK level gradually declined. (The levels of individual CKs are given in a table as supplementary material that can be found at JXB online.) The concentration of the CK deactivation metabolites (N-glucosides) increased at the end of the dark period and remained at an elevated level up to 6 h of light (Fig. 2). The main maximum of CK N-glucosides coincided with (or closely followed) the peak of active CKs after the middle of the light period. Then, a sharp decrease took place. The second main peak, nearly as large as the first one, was found after the light was switched off, coinciding with a minor peak of physiologically active cytokinins. The level of cis-zeatin derivatives followed a trend similar to the N-glucosides, with the exception of the second maximum after the light/dark transition, which was, in the case of cis-zeatin derivatives, delayed and much less profound. The levels of CK phosphates exhibited a transient peak around 4 h of light. Their maximum was found after the middle of the light period. Fig. 1. Endogenous levels of total cytokinins, physiologically active cytokinins (trans-zeatin, isopentenyladenine, dihydrozeatin, and the corresponding ribosides) and cis-zeatin (riboside) in tobacco leaves during a 24 h period. The photoperiod is represented by the black and white box on the abscissa. Means 6SE of three independent experiments are shown. 2880 Nováková et al. Cytokinin oxidase/dehydrogenase A comparison of the diurnal variations in CKX activity and in the levels of CKs, which are the substrates of this enzyme, i.e. isopentenyladenine, trans-zeatin, and their corresponding ribosides (but not dihydrozeatin or its derivatives), is shown in Fig. 4. The main CKX maximum was found 3 h after the transition from dark to light. The second maximum followed the midday peak of active CKs. The third, least profound peak of CKX activity accompanied the minor peak of CKs after the light/dark transition (with a 1 h delay). Metabolic conversion of radiolabelled trans-zeatin Diurnal changes in the levels of endogenous CKs and the activity of CK metabolic enzymes were also evaluated following the metabolic conversion of radiolabelled trans-zeatin (Fig. 5). A high peak of labelled adenine and adenosine, products of CK breakdown, was found close to the dark/light transition and a minor one around 9 h of light, i.e. at the periods preceding the detected maxima of CKX activity. The peak of non-metabolized trans-zeatin, corresponding to the period of the diminished trans-zeatin breakdown, preceded the midday peak of active CKs. The maximum of the non-metabolized labelled trans-zeatin correlated with the minimum level of the ribosylated and phosphoribosylated forms. Fig. 3. Endogenous levels of CK O-glucosides and activity of b-glucosidase in tobacco leaves during a 24 h period. Other details as for Fig. 1. Fig. 5. Metabolism of radiolabelled trans-zeatin in tobacco leaves during a 24 h period (incubation time 1 h). Other details as for Fig. 1. Fig. 4. The activity of cytokinin oxidase/dehydrogenase (CKX) and endogenous levels of cytokinins, which are the substrates of this enzyme (trans-zeatin, isopentenyladenine, and the corresponding ribosides), in tobacco leaves during a 24 h period. Other details as for Fig. 1. Fig. 6. Endogenous levels of indole-3-acetic acid (IAA), abscisic acid (ABA), and active cytokinins in tobacco leaves during a 24 h period. Other details as for Fig. 1. Downloaded from http://jxb.oxfordjournals.org/ by guest on May 29, 2013 Indolyl acetic acid and abscisic acid The midday CK peak coincided with the maximum level of indolyl acetic acid and was accompanied (or closely followed) by a minor peak of ABA (Fig. 6). No other profound peak of indolyl acetic acid was detected during the diurnal cycle. At the end of the light period, a gradual increase in ABA level was found. This enhancement was accelerated after the light was switched off, the maximum Cytokinin, auxin, and ABA diurnal rhythms being reached after 3 h of dark. The high level of ABA was only transient. A low, sharp peak of ABA was also detected immediately after the light was switched on. Discussion who reported a stimulation of ABA biosynthesis at dark, in order to maintain the stomata in the closed position during the night to ensure maximal tissue rehydration. The sharp decrease in ABA concentration, which was found shortly after the beginning of the light period, seems to be connected with the depletion of endogenous ABA and the activation of its catabolism. According to Tallman (2004), after irradiation ABA levels drop due to the activation of the xanthophyll cycle (conversion of ABA precursors to zeaxanthin), isomerization into physiologically inactive trans-forms, as well as to ABA degradation by cytochrome P450 mono-oxygenase. Phytochrome-mediated activation of ABA degradation was also reported by Kraepiel et al. (1994). The decrease in ABA concentration in the guard cells and surrounding apoplast in the morning enables stomatal opening, which positively affects plant photosynthesis. Later, at the midday phase, elevated apoplastic ABA around guard cells, delivered by transpiration, is necessary for the regulation of the stomata aperture in order to balance the rate of photosynthesis and plant turgor maintenance (Tallman, 2004). The minor peak of ABA, which was found 1 h after the middle of the light phase, might be related to this ‘after-midday’ regulation or, more probably, may serve to counterbalance the midday peak of CKs, as under temperate conditions stomatal aperture is regulated by the CK/ABA ratio (Fusseder et al., 1992). Fusseder et al. (1992) reported that the increase in xylem-delivered CKs preceded the increase in leaf conductance. They found the peak of xylem CKs in the morning and their rapid decrease in the afternoon in desert-grown almond trees. Beck and Wagner (1994) suggested that the high concentration of xylem CKs in the morning originated from the steady rate of CK biosynthesis in the roots and of their exudation into the xylem sap during the night when the transpiration rate is low. In the morning, the flow of xylem sap sharply increases, delivering accumulated CKs throughout the plant. Due to the increased volume of the transpiration stream during the day and the steady rate of CK biosynthesis in roots, CK concentration in the xylem sap gradually decreases. The importance of the velocity of the transpiration stream on the translocation of CKs produced in roots to other parts of plant was recently demonstrated by Aloni et al. (2005). In our experiments xylem transport of CKs was not measured. Indirect evidence of the existence of such translocation may be given by the position of the main maximum of CKX activity that was detected early in the light period. The other peaks of CKX activity (after midday and following the light/dark transition) correlated well with the decrease of endogenous CKs in leaf blades, indicating that CK breakdown by CKX is involved in the down-regulation of these endogenous CK peaks. However, the main maximum of CKX activity, which appeared early in the light phase, did not coincide with substantial changes of endogenous CK levels in leaf Downloaded from http://jxb.oxfordjournals.org/ by guest on May 29, 2013 Timing of the main CK peak in tobacco leaves shortly (1 h) after the middle of the light period is in accordance with the diurnal profile in barley leaves (main peaks of CK bases and ribosides around 14.00 h; Kurapov et al., 2000) and slightly differs from that in carrot plants kept under continuous light (maximum of CK bases and ribosides around 16.00 h; Paasch et al., 1997). As both CKs and auxin are necessary for the progression of cell division, this finding of the coincidence of the main CK peak with the maximum of IAA indicates that cell division may be initiated at this time. The close connection between CK increase and growth stimulation was reported in barley (at the stage of the third leaf), when the CK peak at 14.00 h was shown to precede the period of maximal growth from 15.00 h to 18.00 h (Kurapov et al., 2000). The minimal growth was determined before dawn (at 04.00–05.00 h). A correlation between growth and the hormone level was also found in the case of auxin, the link between the oscillation in the level of IAA in the rosette leaves and the stem growth rate in Arabidopsis was reported by Jouve et al. (1998, 1999). The results on the midday peak of IAA are in accordance with the data of Pavlova and Krekule (1984), who reported a peak of IAA content after the middle of the light phase in Chenopodium rubrum plants grown under very long day (20 h). In shorter periods (16 h, 12 h, 8 h), connected with flower induction, this peak was shifted towards the dark phase. It should be kept in mind that these results reflect the situation in still developing but not the youngest tobacco leaves, in other tissues and/or species hormones may exhibit a different diurnal pattern. The effect of developmental stage was observed in barley. Leaves at the stage of the third leaf had the main CK peak at 14.00 h, while at the stage of tillering the peak was shifted to 17.00 h (Kurapov et al., 2000). Another point is that the experiments were aimed at the determination of the diurnal profile of isoprenoid CKs. Due to the differences in physiological function, diurnal rhythms of isoprenoid CKs are likely to differ from those of their aromatic counterparts. The peaks of aromatic CKs m-methoxytopolin and its riboside were detected at 09.30 h in poplar leaves (Tarkowska et al., 2003), which corresponded approximately to 3 h after the light period initiation, i.e. 6 h earlier than for isoprenoid CKs in tobacco. The finding of the ABA maximum at the beginning of the dark phase is in accordance with the survey of diurnal changes in ABA metabolism summarized by Tallman (2004), 2881 2882 Nováková et al. Supplementary data Supplementary data for this paper can be found at JXB online. Acknowledgements The authors wish to acknowledge Eva Kobzová and Marie Korecká for skilful technical assistance. This work was supported by grants from the Grant Agency of the Czech Republic, No. 206/03/0369, and from the Ministry of Education, Youth and Sports, LN LN00A081. References Aloni R, Langhans M, Aloni E, Dreieicher E, Ullrich CI. 2005. Root-synthesized cytokinin in Arabidopsis is distributed in the shoot by the transpiration stream. Journal of Experimental Botany 56, 1535–1544. Beck E, Wagner BM. 1994. Quantification of the daily cytokinin transport from the root to the shoot of Urtica dioica L. Botanica Acta 107, 342–348. Blagoeva E, Dobrev PI, Malbeck J, Motyka V, Strnad M, Hanuš J, Vanková R. 2004. Cytokinin N-glucosylation inhibitors suppress deactivation of exogenous cytokinins in radish, but their effect on active endogenous cytokinins is counteracted by other regulatory mechanisms. Physiologia Plantarum 121, 215–222. Chatfield JM, Armstrong DJ. 1986. Regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv. Great Northern. Plant Physiology 80, 493–499. Dobrev PI, Havlı́ček L, Vágner M, Malbeck J, Kamı́nek M. 2005. Purification and determination of plant hormones auxin and abscisic acid using solid phase extraction and two-dimensional high performance liquid chromatography. Journal of Chromatography A 1075, 159–166. Dobrev P, Kamı́nek M. 2002. Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. Journal of Chromatography A 950, 21–29. Foster KR, Morgan PW. 1995. Genetic-regulation of development in Sorghum bicolor. 9. The Ma(3)(R) allele disrupts diurnal control of gibberellin biosynthesis. Plant Physiology 108, 337–343. Fusseder A, Wartinger A, Hartung W, Schulze ED, Heilmeier H. 1992. Cytokinins in the xylem sap of desert-grown almond (Prunus dulcis) trees: daily courses and their possible interactions with abcisic acid and leaf conductance. New Phytologist 122, 45–52. Hare PD, Van Staden J. 1997. The molecular basis of cytokinin action. Plant Growth Regulation 23, 41–78. Jasoni RL, Cothren JT, Morgan PW, Sohan DE. 2002. Circadian ethylene production in cotton. Plant Growth Regulation 36, 127–133. Jones AM, Cochran DS, Lamerson PM, Evans ML, Cohen JD. 1991. Red light-regulated growth. 1. Changes in the abundance of indole acetic-acid and 22-kilodalton auxin-binding protein in the maize mesocotyl. Plant Physiology 97, 352–358. Jouve L, Gaspar T, Kevers C, Greppin H, Agosti RD. 1999. Involvement of indole-3-acetic acid in the circadian growth of the first internode of Arabidopsis. Planta 209, 136–142. Jouve L, Greppin H, Agosti RD. 1998. Arabidopsis thaliana floral stem elongation: evidence for an endogenous circadian rhythm. Plant Physiology and Biochemistry 36, 469–472. Kraepiel Y, Marrec K, Sotta B, Caboche M, Miginiac E. 1995. In vitro morphogenic characteristic of phytochrome mutants in Downloaded from http://jxb.oxfordjournals.org/ by guest on May 29, 2013 blades, suggesting that this CKX peak may serve in the regulation of xylem CKs. To evaluate the role of CK reversible conjugation in the regulation of their levels, the diurnal rhythm of bglucosidase activity was followed. In the first hours of the light period, the increase in b-glucosidase activity created conditions for the cleavage of CK O-glucosides (one of the CK transport forms) necessary for their further degradation with CKX. The b-glucosidase increase could, of course, also result in hydrolysis of other unidentified compounds, for example, ABA-glucosylester. However, at this time-point, when the level of free ABA was decreasing (Tallman, 2004), the function of b-glucosidase activity stimulation did not seem to be ABA-glucoside deconjugation. The occurrence of b-glucosidase peak in the period before and after the main peak of CKs indicates that hydrolysis of CK O-glucosides may be involved in the finetuning of active CK levels at midday, even if this deglucosylation cannot be the only source of the midday peak of physiologically active CKs. As CK translocation from the roots, which is the primary site of CK biosynthesis (Letham, 1963; Aloni et al., 2005), was reported to be high in the morning, declining rapidly during the day (Beck and Wagner 1994), CK biosynthesis seems to be a more probable source of the sharp midday increase of CKs. As only free ABA was determined, hardly any conclusion may be drawn about the role of b-glucosidase in the regulation of free ABA levels at the ‘after-midday’ peak. This will be the subject of further research. Determination of the bulk leaf ABA content represents another methodological limitation, as for regulation of stomata aperture ABA concentration in the guard cells and the surrounding apoplast is crucial, and the bulk ABA concentration includes the ABA stored in mesophyll chloroplasts as well. The physiological significance of CK peaks may be related, apart from the stimulation of cell division in leaves or the regulation of stomatal aperture, to the generation of a potential CK signal to other plant parts (e.g. flower induction, Macháčková et al., 1996) or the induction/ repression of CK responsive genes (Hare and van Staden, 1997). These genes usually display circadian oscillations in their levels of abundance with maximal expression during the light period (Thomas et al., 1997). The circadian clock represents an important adaptation of life to the conditions on the earth and provides the necessary external and internal co-ordination of the metabolic processes. The results here have shown that CKs, auxin, and ABA levels undergo significant variations in leaves during the day and night. These data provide, to our knowledge, the first report of a diurnal rhythm of CK metabolic enzymes and CK metabolites other than CK bases and ribosides. The coincidence of the peak of CKs and auxin indicates that the changes in plant hormone levels have physiological relevance and correlate with the diurnal changes of plant growth and development. Cytokinin, auxin, and ABA diurnal rhythms View publication stats Paasch K, Lein C, Nessiem M, Neumannn HK. 1997. Changes in the concentration of some phytochromes in cultured root explants and in intact carrot plants (Daucus carota L.) during the day. Angewandte Botanik 71, 85–89. Pavlová L, Krekule J. 1984. Fluctuation of free IAA under inductive and non-inductive photoperiods in Chenopodium rubrum. Plant Growth Regulation 2, 91–98. Tallman G. 2004. Are diurnal patterns of stomatal movement the result of alternating metabolism of endogenous guard cell ABA and accumulation of ABA delivered to the apoplast around guard cells by transpiration? Journal of Experimental Botany 55, 1963–1976. Tarkowská D, Doležal K, Tarkowski P, Astot C, Holub J, Fuksová K, Schmülling T, Sandberg G, Strnad M. 2003. Identification of new aromatic cytokinins in Arabidopsis thaliana and Populus3canadensis leaves by LC-(+)ESI-MS and capillary liquid chromatography/frit-fast atom bombardment mass spectrometry. Physiologia Plantarum 117, 579–590. Thomas TH, Hare PD, van Staden J. 1997. Phytochrome and cytokinin responses. Plant Growth Regulation 23, 105–122. Tian Q, Reed JW. 2001. Molecular links between light and auxin signaling pathways. Journal of Plant Growth Regulation 20, 274–280. Downloaded from http://jxb.oxfordjournals.org/ by guest on May 29, 2013 Nicotiana plumbaginifolia are modified and correlated to high indole-3-acetic acid levels. Planta 197, 142–146. Kraepiel Y, Rousselin P, Sotta B, Kerhoas L, Einhorn J, Caboche M, Miginiac E. 1994. Analysis of phytochrome- and ABAdeficient mutants suggests that ABA degradation is controlled by light in Nicotiana plumbaginifolia. The Plant Journal 6, 665–672. Kurapov PB, Skorobogatova IV, Sal’nikova EI, Sorkina GL, Siusheva AG. 2000. Diurnal courses of endogenous phytohormones in barley. Izvestiya Akademii Nauk Seriya Biologicheskaya 1, 108–114. Letham DS. 1963. Zeatin, a factor inducing cell division from Zea mays. Life Science 8, 569–573. Lexa M, Genkov T, Malbeck J, Macháčková I, Brzohohatý B. 2003. Dynamics of endogenous cytokinins pool in tobacco seedlings: a modelling approach. Annals of Botany 91, 585–597. Macháčková I, Eder J, Motyka V, Hanuš J, Krekule J. 1996. Photoperiodic control of cytokinin transport and metabolism in Chenopodium rubrum. Physiologia Plantarum 98, 564–570. Motyka V, Vaňková R, Čapková V, Petrášek J, Kamı́nek M, Schmülling T. 2003. Cytokinin-induced upregulation of cytokinin oxidase activity in tobacco includes changes in enzyme glycosylation and secretion. Physiologia Plantarum 117, 11–21. 2883