JXB Advance Access originally published online on September 25, 2003
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Journal of Experimental Botany, Vol. 54, No. 392, pp. 2511-2517,
November 2003
© 2003 Oxford University Press
Cytokinin levels in leaves, leaf exudate and shoot apical meristem of Arabidopsis thaliana during floral transition
Received 29 April 2003; Accepted 15 July 2003
,1
1 Department of Life Sciences, Laboratory of Plant Physiology, University of Liège, B22 Sart Tilman, B-4000 Liège, Belgium
2 Department of Biology, Laboratory for Plant Biochemistry and Physiology, University of Antwerpen, B-2610 Antwerpen, Belgium
* Present address and to whom correspondence should be sent: Max Planck Institute for Plant Breeding Research, Carl von Linne Weg, 10, D-50829 Cologne, Germany. Fax: +49 221 5062 207. E-mail: corbesie{at}mpiz-koeln.mpg.de Present address: Cropdesign, Technologiepark 3, B-9052 Zwijnaarde, Belgium.
Abbreviations: BA, benzyladenine; (diH)Z, dihydrozeatin; (diH)[9R]Z, 9-ß-D-ribofuranosyldihydrozeatin; (diH)[9R-5'P]Z, 5'-monophosphate of (diH)[9R]Z; FW, fresh weight; iP, N6-(
2-isopentenyl)adenine; (9G)iP, 9-glucopyranosyl of iP; [9R]iP, 9-ß-D-ribofuranosyl-iP; [9R-5'P]iP, 5'-monophosphate of [9R]iP; LD, long day; SAM, shoot apical meristem; SD, short day; Z, zeatin; (7G)Z, 7-glucopyranosyl of Z; (9G)Z, 9-glucopyranosyl of Z; [9R]Z, 9-ß-D-ribofuranosyl-Z; (OG)[9R]Z, 9-ß-D-ribofuranosyl of [9R]Z; [9R-5'P]Z, 5'-monophosphate of [9R]Z.
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Abstract |
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Understanding the complete picture of floral transition is still impaired by the fact that physiological studies mainly concern plant species whose genetics is poorly known, and vice versa. Arabidopsis thaliana has been successfully used to unravel signalling pathways by genetic and molecular approaches, but analyses are still required to determine the physiological signals involved in the control of floral transition. In this work, the putative role of cytokinins was investigated using vegetative plants of Arabidopsis (Columbia) induced to flower synchronously by a single 22 h long day. Cytokinins were analysed in leaf extracts, leaf phloem exudate and in the shoot apical meristem at different times during floral transition. It was found that, in both the leaf tissues and leaf exudate, isopentenyladenine forms of cytokinins increased from 16 h after the start of the long day. At 30 h, the shoot apical meristem of induced plants contained more isopentenyladenine and zeatin than vegetative controls. These cytokinin increases correlate well with the early events of floral transition.
Key words: Arabidopsis thaliana, cytokinins, flowering, floral stimulus, immunolocalization, phloem exudate, shoot apical meristem.
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Introduction |
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In photoperiodic plants, daylengths favourable for flowering are essentially perceived by expanded leaves in which they cause floral induction. This process then results in the production and export of a floral stimulus which moves in the phloem and eventually reaches the shoot apical meristem (SAM) where it causes floral evocation followed by flower formation (Thomas and Vince-Prue, 1997).
The implication of cytokinins in the control of these processes has been thoroughly investigated, but so far without converging results. The most simple and clear situation has been described in Sinapis alba, a member of the mustard family (Brassicaceae) like Arabidopsis thaliana. In Sinapis plants induced to flower by a single long day (LD), the cytokinin content increases significantly in the leaves and in the leaf phloem sap at the time of movement of the floral stimulus (Bernier et al., 1981; Lejeune et al., 1994). Subsequently, the cytokinin content increases in the SAM, at the time of early mitotic activation (Jacqmard et al., 2002). In addition, an exogenous cytokinin application to vegetative plants grown in short days (SDs) can induce in the SAM various cellular and molecular changes that are normally associated with floral transition (Bernier et al., 2002).
Analyses of endogenous cytokinins in other species have revealed that most of them exhibit similar increases in, and/or supply to, the shoot apex at floral transition (Bernier et al., 1981; Kinet et al., 1993,1994; Machácková et al., 1993). A contrary observation was, however, reported for a day-neutral tobacco where a sharp transient decrease of cytokinins occurred at the end of vegetative growth (Dewitte et al., 1999). On the other hand, exogenous cytokinins have been reported to stimulate flowering in various species, but most often in environmental conditions that are marginally favourable for the floral transition (Bernier et al., 1981; Kinet et al., 1993).
New approaches using mutants or transgenic plants have not led to more conclusive results. On account of a positive effect of cytokinins on flowering, the late-flowering uniflora mutant of tomato shows a marked acceleration of flowering when supplied with a cytokinin (Dielen et al., 2001), and tobacco plants constitutively overexpressing the Arabidopsis cytokinin oxidase gene, thus deficient in cytokinins, exhibit a 3-month delay in flowering (Werner et al., 2001). On the other hand, flowering is delayed in lettuce or pea plants that are enriched in cytokinins by ectopic expression of the Agrobacterium IPT gene (Wang et al., 1997; McCabe et al., 2001), although lettuce plants enriched in cytokinins by overexpression of the KNAT1 gene are early flowering (Frugis et al., 2001). This discrepancy could be due to the fact that cytokinin effects are strongly dose-dependent, with supraoptimal levels being invariably inhibitory to flowering (Bernier et al., 1981; Kinet et al., 1993). One can not dismiss either that differences in the localization of cytokinin enrichment due to different promoters associated with the transgenes may account for distinct phenotypes. Indeed, the increases in cytokinin levels at floral transition are mostly found in apical buds, as mentioned above.
Previous work on the possible participation of cytokinins in the control of flowering time in Arabidopsis thaliana is scarce. Exogenous applications have been reported to accelerate flowering in various ecotypes (Michniewicz and Kamienska, 1965; Besnard-Wibaut, 1981; He and Loh, 2002). In the Columbia ecotype, such a stimulation is observed only when light irradiance is low (Dennis et al., 1996). On the other hand, high endogenous levels of cytokinins are associated with early flowering in Columbia plants treated with triacontanol, cerium and lanthanum (He and Loh, 2002), as well as in the amp1 mutant (Chaudhury et al., 1993; Nogué et al., 2000). Early flowering in this last case might however be pleiotropic since the AMP1 gene was found to encode a putative glutamate carboxypeptidase (Helliwell et al., 2001).
In this paper, the aim was to assess whether quantitative and/or qualitative changes in endogenous cytokinins occur during the transition to flowering in Arabidopsis thaliana ecotype Columbia. Changes were analysed in leaf tissues, leaf phloem exudate, and the SAM of plants induced to flower by exposure to a single LD. A major advantage of this experimental system is that floral transition is induced synchronously, and proceeds with reproducible timing. When plants are induced to flower by one 22 h LD, floral stimulus export by the leaves begins 24 h after the start of the LD and is followed, 20 h later, by the first visible signs of floral transition: enlargement of the SAM and initiation of the first floral meristems (Corbesier et al., 1996; Jacqmard et al., 2003).
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Materials and methods |
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Plant material and growth conditions
The growing conditions were as described elsewhere (Corbesier et al., 1996). Briefly, seeds of Arabidopsis (Columbia) were first vernalized in the dark at 2 °C on wet filter paper for 6 weeks, then sown and grown on a mixture of leaf mould, clay and sand. Six plants were grown in a tray containing 1.0 l of substrate and were watered daily with tap water. All plants were grown in controlled cabinets at 20 °C and at a relative humidity of about 80%. Light (40 µE m–2 s–1, PAR) was provided exclusively by Very High Output Sylvania fluorescent tubes (Sylvania, Zaventem, Belgium).
Photoperiodic treatments
After 8 weeks of culture in 8 h SDs, plants were induced to flower by a single 22 h LD, then returned to the SD regime (Fig. 1). The photoperiodic extension was given with the same light source and at the same irradiance as during standard SDs. Dissection of shoot apices 2 weeks after the experiment showed that all induced plants had initiated flowers while all plants continuously kept in SDs (controls) remained vegetative.
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Collection of leaf tissues
For cytokinin analysis by LC-MS/MS, each experimental batch was of 40 plants. The seven youngest mature leaves per plant were collected 16 h and 20 h (Experiment 1) or 16, 20 and 24 h (Experiment 2) after the start of the experiment, simultaneously on LD-induced and SD-control plants in two independent experiments (Fig. 1). Collected leaves were directly frozen in liquid nitrogen, ground into powder, and stored at –70 °C until analysis.
Collection of leaf phloem sap
Leaf exudates were collected using the EDTA-method as previously described (Corbesier et al., 1998). Briefly, the seven youngest mature leaves per plant were collected and placed in a 500 µl microcentrifuge tube containing 400 µl of 10 mM EDTA (pH 8.5) for 16 h. In two independent experiments 30–40 plants were used at each sampling time. The vessels containing the leaves were enclosed in airtight humid clear chambers. During exudation, leaves were subjected to the same light–dark regime as intact plants (Fig. 1). After collection, exudates were stored at –20 °C until analysis.
Cytokinin analysis by LC-MS/MS
Leaf samples were extracted overnight in 40 ml Bieleski solution (chloroform/methanol/formic acid/water, 25/60/5/10, by vol.) at –20 °C. The extracts were centrifuged (20 000 g, 15 min, 4 °C), re-extracted in 10 ml 80% methanol for 1 h at 4 °C in the dark, and recentrifuged. The supernatants were pooled and loaded on a C18 cartridge (1 g of solid phase) to remove pigments after the addition of the following deuterated internal tracers: 2H5-Z, 2H5-[9R]Z, 2H5-(9G)Z, 2H5-(7G)Z, 2H5-(OG)Z, 2H5-(OG)[9R]Z, 2H6-iP, 2H6-[9R]iP, and 2H6-(9G)iP (Apex Organics, Oxford, UK). Methanol was evaporated under vacuum, and the dry residue dissolved in 20 ml ultrapure water. Plant extracts and leaf exudates were adjusted to pH 7.0 and then purified on an immunoaffinity-column as described by Redig et al. (1996). The O- and N7-glucoside derivatives were not retained on the immunoaffinity-column and were therefore separated from other cytokinins before analysis by LC-MS/MS, in a separate run. Samples, the volume of which was reduced to 20 µl after the purification step, were analysed by HPLC linked to a Quatro II mass spectrometer equipped with an electrospray interface ((+)ES LC-MS/MS) under Multiple Reaction Monitoring (VG Micromass, Manchester, UK). The column was a LiChrosphere C8 reversed phase (125x4 mm, 5 µm particles, Merck-Eurolab, Overijse, Belgium) and cytokinins were eluted with methanol/0.01 M ammonium acetate (70/30, v/v; 25/75, v/v for O- and N7-glucoside fractions) at 800 µl min–1. Using a post-column split of 1/20, the effluent was introduced into the electrospray source (source temperature 80 °C, capillary voltage +3.5 kV, cone voltage 20 V, collision energy of 20 eV and Argon pressure of 4x10–5 Pa). Quantification was done by Multiple Reaction Monitoring of [MH]+ (dwell time 0.1 s) and the appropriate product ion (Prinsen et al., 1995). All data were processed by Masslynx software (VG Micromass, Manchester, UK). Results were expressed in fmol of cytokinin per unit of leaf fresh weight (FW) for the leaf tissue and in fmol of cytokinin per unit of leaf dry weight (DW) for the exudate. Note that in our growth conditions, the FW/DW ratio is approximately equal to 10.
Cytokinin immunolocalization within the SAM
The in situ immunolocalization procedure used in this paper, allowing the detection of Z and iP free bases, is similar to that described by Dewitte et al. (1999) and adapted for small apical buds by Jacqmard et al. (2002). Briefly, apical buds were collected 30 h after the start of the experiment simultaneously in SD and LD (Fig. 1). Three independent experiments were performed, each comprising two buds of each type of plants. Buds were fixed under vacuum for 30 min in a 0.5% (v/v) glutaraldehyde and 3% (w/v) paraformaldehyde mixture in PBS (135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8 mM K2HPO4, pH 7.2). They were frozen at –70 °C and sectioned longitudinally (15 µm) at –20 °C. Sections were incubated with rabbit primary antibody raised against [9R]iP or [9R]Z. The following controls were realized: (i) omission of the primary antibody and (ii) replacement of primary antibody with blocked antibody prepared by incubation with [9R]iP or [9R]Z at saturating concentrations. Sections were then incubated with secondary antibody. Sheep anti-rabbit IgG (Roche Diagnostics, Brussels, Belgium) conjugated with alkaline phosphatase was visualized by light microscopy. Sections were incubated for 2 h at room temperature, allowed to react in the presence of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (Bio-Rad, Brussels, Belgium) and immediately photographed.
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Results |
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Leaf cytokinins
Figure 2 shows the cytokinin profile observed for leaf tissues in two independent experiments. In the first experiment, plants were collected 16 h and 20 h after the start of the LD (Fig. 2A, B). In the second experiment, the sampling time was extended to 24 h (Fig. 2C–E). In both experiments, it was observed that in the SD-controls, the Z-type cytokinins were mainly represented by (9G)Z and [9R-5'P]Z while the most abundant iP-types were [9R]iP and [9R-5'P]iP.
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The first experiment showed that exposure to the LD resulted in a doubling of the leaf contents in [9R-5'P]Z and [9R-5'P]iP at 16 and 20 h, and in [9R]iP at 16 h. In the second experiment (Fig. 2C–E), it was observed that the leaf content in [9R-5'P]iP increased from 16 h after the start of the LD, while other cytokinins remained almost unchanged or somewhat decreased. The [9R-5'P]iP content still rose afterwards and was about 6-fold higher in LD than in SD at 24 h. Although less abundant, the ribotide [9R-5'P]Z was also detected in increasing amounts at 20 h and 24 h of the LD.
These results confirmed preliminary data obtained by analysing cytokinins in leaf extracts by high performance liquid chromatography and radio-immunoassays, as described by Lejeune et al. (1994). Three independent experiments indeed showed an increase in leaf cytokinins from 16 h after the start of the LD (results not shown). LC-MS/MS was preferred for final analyses because of the inherent limitation of immunological techniques, mainly due to variable specific affinity of the antibodies for the different cytokinins.
Leaf exudate cytokinins
The LC-MS/MS analysis of leaf exudate showed that the iP-types were by far the most abundant, especially [9R-5'P]iP and [9R]iP: their contents were at least 10–20-fold higher than those of Z-types (mostly [9R-5'P]Z) (data not shown). In LD-induced plants, after a slight decrease observed during the first exudation period (8–24 h), an increased content of the major cytokinin [9R-5'P]iP started to be observed during the second exudation period (16–32 h) (Fig. 3). A 3-fold enrichment of this cytokinin was detected during the last exudation period (24–40 h) which actually occurred just after the LD (Fig. 1).
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SAM cytokinins
In situ immunolocalization of cytokinins was performed on sections of apices collected from SD-controls and LD-induced plants both harvested 30 h after the start of the inductive LD. The technique used only allows the detection of Z or iP free bases since the other forms are washed out during the fixation procedure (Dewitte et al., 1999). The vegetative SAM was found to be poorly immunoreactive (Fig. 4A, C): the signal was slightly higher than in the immune control sections incubated (i) without the primary antibody (Fig. 4E), or (ii) with either [9R]iP- or [9R]Z-saturated primary antibody (Fig 4F, G). Thirty hours after the start of the LD, both the iP- and Z-signals were increased in the SAM, leaf primordia and procambium (Fig. 4B, D), but were not detected in the subapical pith. Thus the increased iP- and Z-immunoreactions seemed to be confined to tissues where cell division is known to occur. Staining was not uniform, suggesting that adjacent cells may have uneven contents of cytokinins. In all experiments performed, the immunolocalization patterns within apices were reproducible for the iP-signal. For the Z-signal, two-thirds of the samples showed similar increases, while changes were weaker in the remaining third.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this work with Arabidopsis plants induced to flower by exposure to a single 22 h LD, increases in iP-forms of cytokinins were found in both leaves and leaf phloem exudate from 16 h after the start of the inductive photoperiod. More iP and Z were detected later in the SAM, 30 h after the start of the LD. These increases correlate with the successive steps of the floral transition: (i) the increases in leaves and leaf exudate are concomitant with transport in the phloem of the floral stimulus from leaves to SAM, which is known to occur in this system 24 to 36 h after the start of the LD (Corbesier et al., 1996), and (ii) elevation of the cytokinin content in the SAM of induced plants is coincident with the observed increase in the rate of cell division in this tissue (Jacqmard et al., 2003). Thus these results suggest that endogenous cytokinins might play a role in the control of floral transition in Arabidopsis and act as a component of the floral stimulus of leaf origin.
These observations are in close agreement with previous data showing that early flowering in Arabidopsis correlates with increased levels of endogenous cytokinins, and also with the situation described in the related species, Sinapis alba (see Introduction). Minor differences are observed in the nature of the cytokinins detected in Arabidopsis and Sinapis. For example, in the leaf exudates of Arabidopsis [9R-5'P]iP and [9R]iP were mainly detected whereas, in Sinapis, it was essentially [9R]iP and iP (Lejeune et al., 1994). The significance of this difference is unclear since it is still unknown whether different cytokinins have specific biological activities (Mok and Mok, 2001), and whether their interconversions are specifically regulated or merge the general metabolism of purines (Chen, 1997). A difference is also observed in SAM tissues in which an increase in both iP and Z occurs in Arabidopsis at floral induction whereas only iP increases in Sinapis (Jacqmard et al., 2002). Both compounds may arise from the elevation in iP-forms of cytokinins observed in leaf exudates of the two species. However, since apical buds are known to be able to synthesize their own cytokinins (Letham, 1994), another possibility, although not mutually exclusive, would be that both iP and Z result from an increased local biosynthesis.
Both iP and Z are restricted to apical tissues of Arabidopsis containing dividing cells, i.e. the whole SAM, leaf primordia, and provascular tissues. These compounds are conspicuously absent from the subapical pith tissues. A similar situation was reported in tobacco and Sinapis apices (Dewitte et al., 1999; Jacqmard et al., 2002). These observations are in line with the essential role played by this class of hormones in the control of cell proliferation (Francis and Sorrell, 2001; Stals and Inzé, 2001).
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Acknowledgements |
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We thank Dr S Melzer for critical reading of the manuscript and N Detry for technical help in the immunolocalization of cytokinins, S Oden for LC-MS/MS analyses, and A Havelange for taking care of the plants. LC and PL are grateful to the FNRS for the award of a Postdoctoral Research and a Scientific Research Worker fellowship, respectively. This research was supported by grants from the Interuniversity Poles of Attraction Programme (Belgian State, Prime Minister’s Office, Federal Office for Scientific, Technical and Cultural Affairs; P4/15) and the University of Liège (Fonds Spéciaux).
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