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Plant Physiol. (1999) 119: 111-122
Dynamics of Cytokinins in Apical Shoot Meristems of a
Day-Neutral Tobacco during Floral Transition and
Flower
Formation1
Walter Dewitte,
Adriana Chiappetta,
Abdelkrim Azmi,
Erwin Witters,
Miroslav Strnad,
Jacques Rembur,
Michelle Noin,
Dominique Chriqui, and
Henri Van Onckelen*
Laboratory for Plant Biochemistry and Physiology, Department of
Biology, University of Antwerp, B-2610 Antwerp, Belgium (W.D., E.W.,
H.V.O.); Laboratory Cytologie Expérimentale et Morphogenèse
Végétale, Pierre and Marie Curie University, F-75252 Paris cedex
05, France (A.C., A.A., J.R., M.N., D.C.); and Laboratory of Growth
Regulators, Department of Botany, Palacky University and Institute
of Experimental Botany, Academy of Sciences of the Czech Republic,
78371 Olomouc, Czech Republic (M.S.)
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ABSTRACT |
This study considered cytokinin
distribution in tobacco (Nicotiana tabacum L.) shoot
apices in distinct phases of development using immunocytochemistry and
quantitative tandem mass spectrometry. In contrast to vegetative apices
and flower buds, we detected no free cytokinin bases (zeatin,
dihydrozeatin, or isopentenyladenine) in prefloral transition apices.
We also observed a 3-fold decrease in the content of cytokinin
ribosides (zeatin riboside, dihydrozeatin riboside, and
isopentenyladenosine) during this transition phase. The group concluded
that organ formation (e.g. leaves and flowers) is characterized by
enhanced cytokinin content, in contrast to the very low endogenous
cytokinin levels found in prefloral transition apices, which showed no
organogenesis. The immunocytochemical analyses revealed a differing
intracellular localization of the cytokinin bases. Dihydrozeatin and
isopentenyladenine were mainly cytoplasmic and perinuclear, whereas
zeatin showed a clear-cut nuclear labeling. To our knowledge, this is
the first time that this phenomenon has been reported. Cytokinins do
not seem to act as positive effectors in the prefloral transition phase
in tobacco shoot apices. Furthermore, the differences in distribution
at the cellular level may be indicative of a specific physiological role of zeatin in nuclear processes.
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INTRODUCTION |
Cytokinins have been arbitrarily defined as factors capable of
promoting growth of cultured plant cells (Skoog and Miller, 1957 ).
Chemically, known natural cytokinins are the
N6-substituted adenines and their riboside,
ribotide, and glycoside conjugates. The diversity of the
N6 substituents is the origin of the different
cytokinin types. Aside from the well-documented stimulatory effect of
added cytokinins on growth and differentiation of cultured plant cells,
flowering is among the many other developmental processes that
cytokinins have been reported to mediate in plants (Mok, 1994). Altered
cytokinin concentrations before and after flower induction have been
reported for some species (Lejeune et al., 1988, 1994; de Bouillé
et al., 1989). Intervention in the signal-transduction cascade caused by decreasing the cytokinin sensitivity in
Arabidopsis resulted in a pleiotropic effect that included the
formation of a single, infertile flower (Deikman and Ulrich, 1995).
This effect was more complex than a dose response; it was demonstrated
in Arabidopsis that the effect of an aromatic cytokinin on
the flowering program was dependent on the developmental stage of the
apical shoot meristem (Besnard-Wibaut, 1981; Venglat and Sawhney,
1996).
Research on the involvement of cytokinins in flowering and other
physiological phenomena requires accurate techniques to study the
distribution and concentration on a cellular and tissue level. To meet
these demands, research groups have adopted two strategies. One
consists of improving the detection limit and specificity of the
analytical chemistry techniques used for quantification of endogenous
hormone levels (Prinsen et al., 1995). The other focuses on the
elaboration of techniques for in situ localization of hormones (Zavala
et al., 1983; Eberle et al., 1987; Sotta et al., 1990; Ivanova et al.,
1994).
We report changes in the endogenous cytokinin content in the shoot apex
of tobacco (Nicotiana tabacum L.) during distinct phases of
the transition from a vegetative to a reproductive status. We obtained
the data by combining an accurate procedure for immunolocalization of
three different cytokinin bases, zeatin, DHZ, and IP, and capillary liquid chromatography-tandem MS (E. Witters, K. Vanhoutte, W. Dewitte,
I. Machackova, E. Benkova, W. Van Dongen, E. Esmans, and H.A. Van
Onckelen, unpublished data), a highly sensitive technique for the
quantification of cytokinins. The immunocytochemical study focused on
cytokinin bases that were postulated to be the main active forms
(Laloue and Pethe, 1982). At the molecular level the potential of
naturally occurring N9-substituted cytokinins to
inhibit starfish p34cdc2/cyclin B kinase activity
by competing with ATP in vitro was less than the inhibiting potential
of the free bases (Veselý et al., 1994); however, it remains
difficult to explain cytokinin action solely by means of this
competition model, because stimulation of a tobacco
p34cdc2-like kinase in vivo by cytokinins was
also reported (Zhang et al., 1996).
Based on the observed dynamics of endogenous levels and in situ
localization of different cytokinins at the cellular and tissue level,
our study discusses their putative roles in developmental processes
such as leaf initiation, floral induction, and flower formation.
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MATERIALS AND METHODS |
Plant Material
Tobacco (Nicotiana tabacum L. var. Petit Havana SR1)
seeds were germinated in compost (Sterlux, Barbin S.A., Rungis, France) in open, plastic containers in the greenhouse with day and night temperatures of about 25°C and 20°C, respectively. A 16-h
photoperiod was obtained with fluorescent tubes (Truelight, Grolux,
Sylvania, and Mazdafluor, Mazda-Philips, Paris, France) dispensing 90 µmol m  2 s1. After 2 weeks the plantlets were transferred to small, individual pots. At the
three-leaf stage they were transferred to larger pots until harvesting.
Shoot apices for immunolocalization and extraction were collected
during the vegetative, transitional, and reproductive phases (Fig. 1).
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| Figure 1.
Summary of the main parameters of vegetative and
reproductive development of the tobacco var Petit Havana cv SR1. The
number of visible leaves and the stem height were recorded starting
with an interval of 1 week, and were plotted against the number of
weeks after sowing. Sampling points for both cytokinin quantification
(S1-S4) and for immunolocalization of cytokinin bases (I1-I6) are
located in the graph. The horizontal bars in the graph indicate the
different morphogenetic events at the apical shoot meristem. The
horizontal bars under the graph indicate the different phases of plant
development.
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Chemicals
Unless stated otherwise, Merck (Overijse, Belgium) provided the
chemicals.
Immunological Reagents
Coupling of cytokinin ribosides (ZR, DHZ, and IPA) (Apex
International, Honiton, UK) to BSA was achieved by the periodate oxidation method (Erlanger and Beiser, 1964). We calculated a coupling
ratio of 3 to 9 mol of the appropriate cytokinin per mol BSA from the
UV spectra of the conjugates. The antigens were dissolved in PBS (25 mM Na2HPO4 and
0.15 M NaCl, pH 7.4) at 0.5 mg mL1
and mixed with an equal volume of Freund's complete adjuvant. New
Zealand White rabbits were immunized by multiple-site subcutaneous injections of 200 µg of conjugates given at d 0, 7, 28, 42, 72, and
132. We bled the animals 7 d after the last injection and stored
the sera at  20°C.
Preparation of Cytokinin-Affinity Columns
Cytokinin ribosides (50 µmol) were dissolved in 500 µL of
dimethylformamide, and 100 µmol of solid NaIO4
was added. The mixture was stirred in the dark for 15 min. Adding 90 µmol of ethylene glycol stopped the reaction. After 5 min the
reaction mixture was applied dropwise to 5 mL of poly-L-Lys
agarose (Sigma) slurried in 1 mL of borate buffer (25 mM
Na2B4O7
and 15 mM NaCl, pH 9.5) and stirred for 60 min in the dark.
The coupling reaction was stabilized overnight at 4°C with an excess
of NaBH3CN (250 µmol). The cytokinin affinity
sorbent (5 mL) was packed into a 10-mL polypropylene syringe and washed
consecutively with 5 volumes of PBS, 2 M NaCl in PBS, PBS,
water, methanol, water, 0.1 M glycine-HCl (pH 2.5, saturated with diethyl ether), water, and PBS containing 0.1% (w/v)
NaN3. We determined a theoretical column capacity
of 7 to 15 mg IgG mL1 of poly-L-Lys
agarose from the difference in the UV spectra of the sorbents.
Purification of Cytokinin Antibodies by Affinity Chromatography
Two milliliters of rabbit serum diluted five times in PBS (pH 7.5, 37°C) was allowed to flow through the affinity column for at least
1 h. The columns were subsequently washed with 5 volumes of PBS,
pH 7.5, and 2 M NaCl in PBS. Columns were eluted with Gly-HCl (pH 2.5, 4°C, saturated with diethyl ether) until the A280 was enhanced (SP8440 XR, Spectra
Physics, San Jose, CA). UV-absorbing fractions (1 mL) were collected
into 350 µL of ice-cold 1 M Tris-HCl, pH 8.0. Fractions were combined and the ether was rapidly removed by rotary
evaporation at 25°C. Purified antibodies were precipitated with 100%
(v/v) of saturated ammonium sulfate solution (1 h at 4°C), followed
by centrifugation at 3000g (4°C). We discarded the
supernatant and dissolved the pellet in 1 mL of PBS. The antibody
solution was dialyzed against five 2-L changes of PBS (supplemented
with 0.04% [w/v] NaN3) at 4°C; we determined the protein content by measuring the A280
(UV-2101PC, Shimadzu, Kyoto, Japan). Glycerin was added until a 50%
(v/v) final concentration was reached. This stock solution was divided
into aliquots of 100 µL and stored at 20°C until use. After
affinity purification and dialysis all affinity-purified sera had a
protein content between 0.8 and 1.5 mg mL1.
Determination of the Cross-Reactivity of the Sera
We used ELISA to screen for cross-reactivity of the sera to
different compounds (Strnad et al., 1992) and coated the wells of an
ELISA plate (Maxisorb, Nunc, Roskilde, Denmark) with 150 µL of 5 µg
mL1 antibody solution in PBS, pH 7.2, at 4°C
overnight. After saturating remaining binding sites with 0.5% (w/v)
BSA in PBS for 1 h, we pipetted 50 µL of PBS, 50 µL of
cytokinin riboside linked to AP (Boehringer Mannheim), and 50 µL of
the different diluted compounds into the wells. After 1 h of
incubation and several consecutive washes with 3× PBS, water, and Tris
buffer (pH 9.6, 2 mM MgCl2), we added
4-nitrophenyl disodium phosphate (1 mg mL1 in
Tris-HCl buffer, 0.1 M, pH 9.6, and 2 mM
MgCl2), and incubated the plate for 1 h at
37°C. We measured competition by monitoring the
A405 (EAR 400, SLT, Grödig, Austria).
Fixation Efficiency Testing
To determine the fixation of the cytokinin bases in vitro, we
coated the wells of an ELISA plate overnight with 150 µL of 0.5%
(w/v) BSA solution, rinsed them with distilled water, and treated them
with 20 nmol of zeatin, DHZ, IP, or their ribosides dissolved in a 3%
(w/v) paraformaldehyde and a 0.5% (v/v) glutaraldehyde (LADD Research
Industries, Williston, VT) mixture in PBS. After 3 h at 4°C,
wells were washed with distilled water, and 150 µL of PBS buffered
primary antibody solution in the same dilution as used for
immunolabeling was pipetted into the wells and incubated overnight at 4°C. After rinsing with PBS, we used AP-conjugated sheep
anti-rabbit immunoglobulins (1:100) (Boehringer Mannhein) as
secondary antibodies. 4-Nitrophenyl phosphate disodium salt (1 mg
mL1) was provided as a substrate for the AP,
and the reaction product was monitored at
A405 after 15 min at room temperature.
The binding of cytokinin bases and ribosides to plant tissues was
determined by means of radioactive cytokinins: 1.7 kBq of tritium-labeled cytokinin base or riboside
(trans[2-3H]zeatin, 0.9 TBq
mmol1; [3H]DHZ, 1.27 TBq mmol1; [2-3H]IP,
1.65 TBq mmol1;
[2-3H]ZR, 0.9 TBq
mmol1; [3H]DHZ, 1.27 TBq mmol1; and
[2-3H]IPA, 1.65 TBq
mmol1; Institute of Experimental Botany,
Prague, Czech Republic) was added to 200 µL of fixative (3% [w/v]
paraformaldehyde and 0.5% [v/v] glutaraldehyde in PBS, pH 7.2). The
fixative, supplemented with the radioactive tracers, was allowed to
react with fresh tobacco stem sections approximately 1 mm thick for
3 h at 4°C. The tissue was washed in PBS (5 × 10 min) and
dissolved in 3 mL of Soluene 350 (Packard Instruments, Groningen, The
Netherlands) before liquid-scintillation counting (Tri Carb 1500, Packard Instruments). The results were expressed as a percentage of the
total radioactivity added, and the fresh weight of the stem sections
was used for normalization of the radioactivity measured in the tissue.
Mitotic Index Determination
Mitotic activity was quantified on longitudinal sections of shoot
apices excised at various developmental stages. The apices were fixed
by ethanol:acetic acid (3:1, v/v), and then stained according to the
Feulgen reaction (1 N HCl hydrolysis for 10 min at 60°C,
and Schiff's reagent for 2 h).
Nuclear DNA Imaging Analysis
Excised vegetative (Fig. 1, S1) and prefloral (Fig. 1, S3) shoot
apices were fixed in acetic acid:alcohol (1:3, v/v), rinsed with 70%
(v/v) EtOH, rehydrated, and stained by the Feulgen reaction, as
described above. We excised stained apical meristems under a
microscope, then flattened and mounted them in DePex (Gurr, BDH, Poole,
UK). An image-analysis system fitted with Ploidy 4.04 software (SAMBA
2005, Alcatel, TITN, Grenoble, France) performed nuclear DNA
quantification. The mean 2C reference value ±2 SD was
determined by analysis of half-telophases on both types of meristems
and submitted to the Kolmogorov-Surinov test of normality. For
each condition around 500, interphase nuclei were analyzed and ranged
into histograms. We estimated the relative frequency of
G0-G1 nuclei based on their
position between the limits of the 2C distribution.
Extraction, Purification, and Analysis of Cytokinins
We dissected the apices and froze them in liquid nitrogen, taking
care to discard all young leaves. Twelve to fifteen apices were pooled,
resulting in a sample of approximately 40 mg fresh weight. Samples were
extracted overnight in Bieleski's solvent. Before centrifugation at
24,000g for 15 min at 4°C, we added deuterated standards
for cytokinins (Apex International) and purified the extract using a
combination of solid-phase and immunoaffinity purification, as
described by Redig et al. (1996a). We performed a quantitative analysis
of cytokinins using capillary column switching on a fully automated
workstation (Famos, LC Packings, Amsterdam, The Netherlands)
coupled to a liquid chromatography setup, consisting of an HPLC pump
(model 325S, Kontron Instruments, Milan, Italy), an in-line UV detector
(model 322, Kontron), and a triple-quadrupole MS (Quattro II, Micromass
UK Ltd., Cheshire, UK).
Twenty-five-microliter sample aliquots were introduced into one
dimension of the system using 10 mM
CH3COONH4 as the mobile phase at a flow rate of 40 µL min1. Analytes
were captured on a C18 column (5 µm, 500 µm
i.d. × 5 mm, LC Packings) for 7 min. A mobile phase switch for 5 min using 10 mM
CH3COONH4 and
water:methanol (30:70, v/v) at a flow rate of 7 µL
min1 introduced the analytes onto the
C18 analytical column (5 µm, 300 µm i.d. × 150 mm; Adsorbosphere, Alltech, Laarne, Belgium). Diagnostic transition
ions as described by Prinsen et al. (1995) recorded the cytokinin
chromatograms in multiple-reaction-monitoring mode. Absolute detection
limits ranged from 2 fmol for IP to 15 fmol for zeatin. Due to
electrospray characteristics under the applied conditions, a lower
sensitivity was obtained for zeatin and DHZ than for IP. A more
detailed description of this technology will be presented elsewhere (E. Witters, K. Vanhoutte, W. Dewitte, I. Machackova, E. Benkova, W. Van
Dongen, E. Esmans, and H.A. Van Onckelen, unpublished data). We
calculated the results according to the principle of isotope dilution
and expressed them in picomoles per gram fresh weight. Calculation of
detection limits and errors took fresh weight and recovery into
account.
Preembedding Immunolocalization of Zeatin, IP, and DHZ
Shoot apices were fixed under a 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). The samples were then stored in the fixative for 2.5 h at
4°C. Thick sections (30-50 µm) were cut with a vibratome (model
1000, Technical Products International, St. Louis, MO). When we
used enzyme-linked or colloidal gold-linked secondary antibodies
for detection, we adapted 25% gelatin (w/v) in PBS as a support for
cutting small samples such as apices. After mounting the samples in
gelatin on a microscope slide, they were fixed for 30 min. Sections
were collected in ice-cold PBS in the wells of a tissue-culture plate.
The floating sections were transferred consecutively in blocking buffer
(0.5% [w/v] BSA, and 0.1% [v/v] fish gelatin, 1% [v/v]
normal sheep serum, 20 mM glycine, and PBS, 3 × 10 min) and in a 0.025% (v/v) Tween 20 solution in PBS for 10 min.
Sections were incubated with primary antibody in diluted 1:100 blocking
buffer, supplemented with 0.025% (v/v) of Tween 20 at 4°C overnight,
followed by 1 h at room temperature.
After three 10-min washes with PBS, we administered the secondary
antibody diluted in blocking buffer. Sheep anti-rabbit IgG (1/100, in
blocking buffer) conjugated with either AP or FITC (Molecular Probes,
Eugene, OR) was monitoried by either light microscopy (Axioscop, Zeiss)
or confocal laser microscopy (MRC-600, Bio-Rad). Sections were
incubated with secondary antibodies for 1 h at room temperature,
followed by three 10-min washes with blocking buffer and two 10-min
washes with PBS. Sections treated with AP conjugates were rinsed with
Tris-HCl buffer (0.1 M and 2 mM
MgCl2, pH 9.6) and allowed to react in the
presence of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl
phosphate (Bio-Rad) for 3 min. We added 2 mM EDTA in PBS to
stop the enzymatic reaction. Samples were mounted in PBS:glycerin
(50:50, v/v) containing 2 mM EDTA and observed immediately.
After the rinsing steps (in darkness), we mounted the samples, treated
with FITC-labeled goat anti-rabbit antibodies, in Mowiol (Harlow and
Lane, 1988) to reduce fading during observation by confocal
laser microscopy.
For preembedding ultrastructural immunolocalization, colloidal gold
(<1 nm)-labeled secondary antibodies (1:40, Aurion, Wageningen, The
Netherlands) were applied to the sections, revealing binding of the
primary antibody. After five 10-min washes in PBS, a supplementary fixation with 1.6% (v/v) glutaraldehyde in PBS (15 min), followed by
two 10-min rinses with deionized water (2 × 10 min), was
performed prior to silver enhancement (SM kit, Amersham) of the
colloidal gold (10 min). After washing with water, samples were
dehydrated through an ethanol series and embedded in araldite (Fluka)
via propylene oxide. Sections were embedded flat in the resin between the bottom of a silicon rubber embedding mold and a cover slip. After
polymerization (48 h at 70°C), we selected areas of interest by light
microscopy and placed a gelatin capsule filled with araldite upside
down on the preparation, permitting polymerization. Coverslips were
easily removed after cooling of the specimens with liquid nitrogen, and
blocks were sectioned with a diamond knife on an ultramicrotome (LKB,
Bromma, Sweden). Sections were collected on copper grids and
stained with 6% uranyl acetate (w/v) and 0.3% (w/v) lead citrate in
water for 10 min. We then observed the preparations with a transmission
electron microscope (model 301S, Philips, Eindhoven, The Netherlands,
or model 1200, Jeol) operated at 80 kV.
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RESULTS |
Growth Parameters and Meristematic Activity of Tobacco during
Vegetative and Reproductive Development
The tobacco var Petit Havana cv SR1 is day neutral (the time of
flowering is not affected by day length; McDaniel, 1992). To
define the different sampling points, we determined some of the
developmental characteristics of the vegetative and reproductive phases
of the SR1 tobacco plants grown under our experimental conditions (Fig.
1). The shoot apical meristem entered a
flowering stage after 5 to 6 weeks of vegetative growth, during which
time 15 ± 1 leaves, nodes, and internodes were initiated. The
switch to the prefloral transition phase was identified by the arrest of leaf initiation, accompanied by loss of the apical organization into
lateral and central zones, and doming of the meristem (Fig. 5a).
Inflorescence was initiated next, with the first floral meristems with
sepals and petals observed on 7- to 8-week-old plants. Because the
inflorescence in the Nicotianaea is a raceme of cymes,
several stages of flower development were present on a single plant.
Sampling during the reproductive phase concerned exclusively the
terminal flowers.
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| Figure 5.
Immunolocalization of zeatin and IP in prefloral
and floral shoot apices. Longitudinal sections of a prefloral
transition apex (a), early floral apices (b and c), and floral apices
(d and e) subjected to immunohistochemistry for zeatin (a, c, d, and e)
and IP (b). Localization was with AP-conjugated secondary antibodies
and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as the
chromogenic substrate. a, Prefloral transition apex immunostained for
zeatin, disclosing no reaction of the AP in the meristem. b and c,
Early floral phase with sepal formation showing the absence of label
for IP (b), but the presence of reaction with the anti-zeatin antibody
was reflected in weak purple staining (c). d and e, Floral phase with
sepal, petal, and stamen already initiated; a strong purple staining
occurred in different cells throughout the developing flower after
immunostaining of zeatin. e, Longitudinal section of a developing
stamen immunolabeled with anti-zeatin, showing a strong signal in the
sporogenic tissue. p, Petal; s, sepal; st, stamen. Bars = 90 µm
(a) and 45 µm (b, c, d, and e).
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During vegetative growth the mitotic activity in the apical meristems
was highest in the lateral zones (mitotic index = 6.9% ± 1.3),
followed by the rib meristem (mitotic index = 4.9% ± 1.3), and
the axial zone (mitotic index = 3.3% ± 0.3). In prefloral transition meristems, the mitotic activity in the axial zone increased (mitotic index = 4.9% ± 0.5), whereas the lateral zones (mitotic index = 7.1% ± 1.5) and the rib meristem (mitotic index = 6.4% ± 0.6) maintained their previous activity.
In addition, image analysis of DNA contents indicated that the nuclei
of both vegetative and prefloral transition shoot apices were
predominantly in G0-G1
(Fig. 2). The fraction of proliferating cells, revealed by the relative frequency of the
S+G2 nuclei, increased during the transition
phase. Considering mitotic indices and DNA levels, we concluded that
the prefloral condition is characterized by increased cell-cycling
activity.
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| Figure 2.
DNA content distribution in apices during the
vegetative and prefloral transition phase. Histogram of the nDNA
content during the vegetative phase (a) and the prefloral transition
(b). The percentage of cells in G0,G1 and
S,G2 is also presented.
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Specificity of the Antibodies and Immobilization of Cytokinin Bases
in Situ
Due to the ubiquity of adenine derivatives in living cells, the
occurrence of cytokinins as modified nucleosides in the tRNA of some
species (Vreman et al., 1974; Edwards and Armstrong, 1981; Sprinzl and
Gauss, 1983), and the presence of isoprenylated proteins (Biermann et
al., 1994), the immunolocalization of cytokinins requires highly
specific antibodies. The three anti-cytokinin antibodies displayed
significant cross-reactivity only with their respective free bases,
ribosides, and 9-glucosides (Table I). The anti-ZR antibodies did not react with
zeatin-O-glucoside. No cross-reactivity was found
against yeast tRNA,
N-acetyl-S-farnesyl-L-cystein, N-acetyl-S-geranyl-L-cystein,
or
N-acetyl-S-geranyl-geranyl-L-cystein, excluding possible artifacts caused by odd tRNA bases or by
farnesylation, geranylation, or geranylgeranylation of proteins.
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Table I.
Cross-reactivity of three different
affinity-purified anti-cytokinin antibodies for different classes of
cytokinins, yeast tRNA, and isoprenylated cysteins
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Immunostaining detected binding of cytokinin bases and ribosides by
aldehyde fixation to BSA. The absorbance of the AP reaction product of
wells in which the fixation solution included the cytokinin bases IP,
DHZ, and zeatin was higher (A405 = 3) than
that measured in wells containing cytokinin ribosides or BSA
(A405 = 0.5) (Table II). Thus we can deduce that a short and
weak aldehyde fixation resulted in the linkage of only cytokinin bases
to BSA, and that the epitopes on the cytokinin bases were kept intact
by this fixation.
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Table II.
Efficiency of the aldehyde mixture (3% [w/v]
paraformaldehyde and 0.5% [v/v] glutaraldehyde in PBS) to link
cytokinin bases (IP, DHZ, and zeatin) and cytokinin ribosides (IPA,
DHZR, and ZR) to BSA and plant material
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We observed the same preferential binding of IP, DHZ, and zeatin in
plant material upon aldehyde fixation of radioactive cytokinins (Table
II). The slightly higher values observed for the isopentenyl-type cytokinins in the in planta test were probably caused by the more hydrophobic nature of these compounds. These results are in accordance with previous reports (Sossountsov et al., 1988). In contrast to
N-glycosylated cytokinins that do not contain a reactive NH group, O-glucosides are retained by the fixative but are not
recognized by the antibodies (Table I).
Bearing in mind that free bases and O-glucosides are
preferentially linked in planta, and considering the absence of
cross-reactivity of the anti-cytokinin antibodies for
O-glucosides, we can conclude that the cytokinin bases
zeatin, DHZ, and IP are selectively immunolabeled following the
immunocytochemical process.
In Situ Distribution of Cytokinin Bases in Vegetative and
Reproductive Shoot Apices
During vegetative growth (Fig. 1, I1), the three cytokinin bases
were present in apical shoot meristems and in leaf primordia (Fig.
3, a-c). At the end of the vegetative
phase (Fig. 1, I2), we observed an overall decrease in immunolabeling.
Whereas zeatin and IP were still detectable (Fig. 3, d and f), the
signal of DHZ disappeared completely (Fig. 3e). During the experiments
it became apparent that some sections displayed a stronger
immunolabeling in the lateral zone before the emergence of the leaf
buttress (Fig. 3, a, b, d, and f), which could eventually be associated with the initiation of leaf primordia. When, as a control, we saturated
the primary antibodies with their antigens, we observed a decrease in
signal close to the limit of visual detection (Fig. 3, g and h).
Omitting the primary antibody resulted in an absence of labeling (data
not shown). Without counterstaining, all of the control sections
displayed a yellow background conferred by phenolics in the first
tunica layer of the meristem and in the various parenchyma. Cell
outlines in differentiated tissues were visible due to light refraction
at the level of the cell walls.
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| Figure 3.
Immunolocalization of cytokinin bases within
vegetative shoot apices. The location of cytokinin bases in
longitudinal sections was determined using immunohistochemistry.
Nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate was used as
chromogenic substrate for the AP, resulting in a purple reaction
product. Immunolocalization of zeatin (a and d), DHZ (b and e), and IP
(c and f) in vegetative shoot apices at developmental stages I1 (a, b,
and c) and I2 (d, e, and f) (Fig. 1). Purple staining is present for
zeatin (a and d) in the nucleus and cytoplasm, whereas for DHZ (b and
e) and IP (c and f) a more perinuclear purple staining is visible.
Arrows in a, b, d, and f point to staining in the lateral zones of the
meristems. g and h, Sections (from stage I1, see Fig. 1) incubated with
anti-zeatin (g) and anti-DHZ (h) antibodies saturated with zeatin and
DHZ as a control, respectively. Bars = 90 µm (a, b, d, e, g, and
h); 45 µm (c); and 22 µm (f).
|
|
At higher magnification the compartmentation of the cytokinin bases DHZ
and IP appeared mainly cytoplasmic and perinuclear. A very specific and
pronounced nuclear labeling was observed on sections processed for
zeatin localization (Figs. 3, a and d, and
4). We used ultra-small colloidal gold
particles (Fig. 4, A and C) and a fluorescence probe (Fig. 4B) linked
to the secondary antibody to check for artifacts caused by a
nonspecific deposition of the AP reaction product. Both light
microscopy and confocal laser microscopy revealed strong signals in the
nuclei of some cells upon labeling for zeatin (Fig. 4, A and B). The
nuclear appearance of this labeling was confirmed by electron
microscopy. Silver particles resulting from the enhancement of
colloidal gold particles were clearly present in the nucleus and
cytoplasm of some cells (Fig. 4C).
View larger version (153K):
[in this window]
[in a new window]
| Figure 4.
Immunolocalization of zeatin in vegetative shoot
apices. Zeatin was localized in the nucleus of vegetative meristem
cells upon silver-enhanced gold-labeling by light and electron
microscopy (A and C). A, Yellow color, originating from light
diffraction by the silver particles, was distributed throughout the
apex. C, A large number of silver particles (arrows) were detected in
the nucleus (N) of some cells by electron microscopy. Confocal laser
microscopy after immunolocalization with FITC as a probe revealed a
strong fluorescence signal in the nucleus of some cells (B). Bars = 90 µm (A), 100 µm (B), and 1 µm (C).
|
|
The prefloral transition apex (Fig. 1, I3) was characterized by the
total disappearance of detectable cytokinin bases in the apical
meristem (Fig. 5a). Additionally, we
observed a reduced immunostaining in the differentiated leaf and stem
tissues and in the axillary buds (data not shown). The onset of
flowering (Fig. 1, I4), which is marked by sepal and petal initiation,
was accompanied by a progressive reappearance of the immunolabeling in the meristem and in the new perianth sections (Fig. 5, b and c),
with zeatin being detectable (Fig. 5c) earlier than DHZ and IP (Fig.
5b). At the later stages of flower development (Fig. 1, I5 and I6),
when carpels and stamens were formed, we observed a further increase in
detectable cytokinin bases (Fig. 5d). Because all three anti-cytokinin
antibodies gave similar results (except for the pronounced nuclear
labeling of zeatin in some cells), only labeling of zeatin was
presented for stages I5 and I6 (Figs. 5, d and e, and
6). The staining was particularly strong
in sporogenous tissue and young pollen grains in developing anthers
(Fig. 5e). Developing ovules (Fig. 6, a and b) were also labeled.
View larger version (69K):
[in this window]
[in a new window]
| Figure 6.
Immunolocalization of zeatin at two stages of
ovule formation and at the end of seed formation. Cross-sections of
ovule primordia arising from the placenta (a, arrowheads) and
developing ovule (b, arrow indicates the archesporial cell in a
developing ovule). Purple staining resulting from labeling of zeatin
was detected in ovule primordia (a, arrowheads), the ovary wall (a and
b), and the developing ovules (b). Archesporial cells are also
significantly labeled (b). c, Cross-section of a seed immunolabeled
with anti-zeatin antibody. The cytoplasm of embryo and endosperm are
heavily stained. e, Embryo; en, endosperm; f, funiculus; o, ovule; ow,
ovary wall; p, placenta. Bars = 180 µm (a) and 90 µm (b and
c).
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|
Among the ovular cells, the archesporial cell (Fig. 6b, arrow) and
later the embryo sac were significantly labeled. At the end of stamen
differentiation (Fig. 1, I6), no label was found in mature pollen
grains, whereas the vascular bundle of the connective and the cells of
the longitudinal slits provided with secondary thickenings were highly
labeled (data not shown). The vascular strands of the placenta were
heavily stained as well (data not shown). After fertilization we
observed strong cytoplasmic labeling both in the endosperm and in the
embryo (Fig. 6c).
These observations during the development of tobacco shoot apices were
highly reproducible; we obtained similar results for three different
populations of plants arising from sowing at different dates.
Quantification of Endogenous Cytokinins in the Shoot Apex
By means of capillary column switch technology, in combination
with tandem MS, endogenous cytokinins could be measured with high
sensitivity in minute plant samples. This methodology permitted quantification of 11 cytokinins in a single pool of 12 to 15 apices, representing approximately 40 mg fresh weight. Results of these analyses are presented in Table III. No
N-glucoside cytokinin conjugates were detected in any of the
samples analyzed (detection limit, 0.5 pmol g1
fresh weight). About 10 pmol g1 fresh weight of
cytokinin bases were recorded in samples S1 (Fig. 1), corresponding to
vegetative apices still initiating leaves, nodes, and internodes. No
cytokinin bases could be detected during the lapse between the arrest
of leaf formation and the initiation of flower buds (Fig. 1, S2 and
S3). Cytokinin ribosides were present in all samples analyzed, and a
correlation with the kinetics of the free bases was observed. Indeed,
significantly lower riboside concentrations coincided with
nondetectable levels of cytokinin bases in samples S2 and S3 (Fig. 1).
Compared with ZR and IPA, consistently lower concentrations of DHZR
were found. Initiation of flower parts (Fig. 1, S4) coincided with
increased levels of both free bases and ribosides.
View this table:
[in this window]
[in a new window]
|
Table III.
Endogenous cytokinin bases and riboside
concentration in apical shoot meristems at different developmental
stages ±SE (S1-S4 as described in Fig. 1)
|
|
| |
DISCUSSION |
By using aldehyde fixation (Sossountsov et al., 1988) in
combination with affinity-purified antibodies for immunocytochemical analysis of nonembedded sections, we obtained new and accurate information on the distribution of the cytokinin bases zeatin, DHZ, and
IP. The absence of embedding medium resulted in a high antigenicity and
a low background. No artifactual tRNA labeling was observed, which was
confirmed experimentally by the absence of signal in the prefloral
transition stage. Although some reports indicate the presence of
cytokinins as modified bases next to the anticodon in tobacco (Murai et
al., 1975), the sterical organization of the tRNA probably obstructed
recognition by the antibodies. This preembedding technology coupled
with the use of specific antibodies proved to be very accurate for a
highly specific localization of the three cytokinin bases. A good
correlation was also obtained between quantification of cytokinin bases
by HPLC-tandem MS and results obtained by immunolocalization. The
absence of immunostaining in the prefloral transition meristem
coincided with an endogenous cytokinin base content below the detection
limit. In flowering buds, cytokinin bases became detectable by both
techniques. Data on the endogenous cytokinin content in dissected
apices (±1.5 mm3) could only be obtained with a
capillary HPLC column switch system linked to an electrospray-tandem
MS.
The presence of detectable free cytokinin bases, as measured by both
immunocytochemistry and tandem MS, was positively correlated with
increased concentrations of the cytokinin ribosides. This suggests that
threshold amounts of ribosides could be required to produce detectable
amounts (>1-2 pmol g1 fresh weight) of free
cytokinin bases by the action of adenosine nucleosidases (Letham and
Palni, 1983).
Our results show, for the first time to our knowledge, that
immunocytochemistry can provide reliable qualitative and quantitative information on the cellular distribution of specific cytokinins.
Using these techniques we studied the kinetics of the endogenous levels
and cellular localization of specific cytokinins in shoot apices
developing from vegetative to reproductive functioning. In extracts of
vegetative apices that initiate leaves, we detected nodes and
internodes, significant amounts of zeatin, DHZ, IP bases, and ribosides
by HPLC-tandem MS. The concentrations found for the free bases (varying
around 10 pmol g1 fresh weight) were apparently
sufficient to produce a clear signal in most of the cells when the
anti-IPA antibody was used. We observed a more patchy signal when using
both the anti-ZR and anti-DHZR antibodies. Because the overall
concentration of the three bases was quite similar, this observation
might be a reflection of a less homogenous distribution of zeatin and
DHZ. Toward the end of the vegetative phase, the signal observed for
all three cytokinin bases decreased significantly, reflecting the
transition of the vegetative apex toward a prefloral status.
This prefloral phase, during which the shape of the meristem changed
from flat to dome, was characterized by the arrest of leaf initiation
and the loss of the apical organization into lateral and central zones.
In contrast to what was described for white mustard
(Gonthier et al., 1987), the nuclear DNA quantification in
tobacco prefloral apices disclosed no blocking of cells in G2. Moreover, an overall mitotic activity of
about 6% indicated that during the prefloral phase meristematic cell
division proceeded normally, with an increase in the
S+G2 fraction of the nuclei population. The
absence of organogenesis during this phase coincided with significantly
decreased endogenous cytokinin levels in the meristem. The riboside
concentrations dropped to less than 50% of the values observed in
vegetative meristems, whereas the concentration of free bases dropped
even below detection limits.
These quantitative HPLC-tandem MS data matched exactly the
immunocytochemical observations, with no labeling used for any of the
anti-cytokinin antibodies. Once cytokinin concentrations increased
again, as detected both by HPLC-tandem MS and immunolabeling, organogenesis resumed, with the initiation of petals and sepals. During
further development of the flowers, all vascular strands and developing
reproductive tissues were strongly labeled. This observation might be
indicative for a sink effect of areas with high division activity, both
mitotic and meiotic.
Previously reported results on increased cytokinin levels during flower
development (Bernier et al., 1988; Lejeune et al., 1988, 1994; de
Bouillé et al., 1989) in other species pointed to a
putative role for cytokinins in floral evocation. However, the
decreasing cytokinin levels in tobacco apical meristems at the end of
the vegetative phase and the complete disappearance of free cytokinin
bases during the reorganization of the meristem prior to flowering
(being reported here for the first time to our knowledge), may incite a
re-evaluation of the question as to whether cytokinins positively
regulate the switch from a vegetative toward a floral meristem.
Earlier work in Chenopodium showed that after the
photoperiodic induction of flowering, cytokinin content decreased
(Machackova et al., 1993). Exogenous cytokinin application after the
inductive photoperiod also inhibited flowering in this species (I. Machackova, personal communication). Recently, by measuring the
cytokinin content in xylem sap, Beveridge et al. (1996, 1997)
demonstrated that root-derived cytokinins do not control flowering in
pea. Apparently, changes in the levels of endogenous cytokinins in the
shoot apex itself (not just in leaves or other parts of the plant)
seem to be involved in the flowering transition.
Although it is certainly premature to propose a general conceptual
framework for the role of cytokinins in flowering, the results
presented in this report may introduce some new insights on the
involvement of cytokinins in the functioning of the apical meristem of
tobacco plants. It can be concluded from our results that relatively
high endogenous cytokinin levels are correlated with organogenesis
(either vegetative or reproductive). Estruch et al. (1991, 1993) showed
that upon expression of the Agrobacterium tumefaciens ipt
gene, the enhanced endogenous cytokinin content resulted in
adventitious vegetative buds on leaves of vegetative plants and flower
buds on leaves of flowering plants. Our observations support the idea
that enhanced cytokinin levels are essential for cell differentiation
and organogenesis, but not for floral evocation in the strictest
sense. This seems to be substantiated by the observation
that during the prefloral phase, when floral evocation apparently
occurs, no organogenesis takes place, a phenomenon that eventually may
prove to be provoked by the significantly reduced cytokinin levels.
Another interesting observation involved the preembedding
immunolocalization technique. In both vegetative and floral apices, we
found a patchy nuclear labeling, but only when using anti-zeatin antibodies. These results indicate that among the free bases only zeatin is detectable in nuclei. Both confocal laser microscopy and
electron microscopy confirmed the presence of zeatin in some nuclei, as
was observed by using AP-conjugated secondary antibodies. This is the
first report, to our knowledge, of such differential behavior of the
different cytokinin types, which might be related to a specific
function of the zeatin cytokinin types at the nuclear level in tobacco.
This nuclear labeling observed with anti-zeatin antibody is intriguing
in light of the observations made by Redig et al. (1996b). By using a
synchronized cytokinin autotrophic cell culture (BY2) (Nagata et al.,
1992), they found that only zeatin was present during the
whole-cell cycle, with a short, transient accumulation of this
cytokinin type at the end of the S phase and during the M phase. One
might be tempted to correlate the patchy appearance of the nuclear
label with these specific phases in the cell cycle. However, the
absence of label from prefloral apices, in which mitosis proceeds
throughout the meristem at the same rate as in the lateral zones of
vegetative apices, seems to contradict this assumption. It is possible
that this is a matter of concentration and detection limits.
Nevertheless, the nuclear compartmentation observed exclusively for the
zeatin-type cytokinins provides good experimental evidence that, at
least in tobacco, a specific mode of action within the nucleus might be
attributed to zeatin. Colocalization experiments on zeatin with
nuclear-marker proteins (Hemerly et al., 1992) for specific phases of
the cell cycle are planned.
The practical approach for immunolocalization of cytokinins described
in this paper opens new perspectives to address the cellular and
molecular functions of cytokinins in plant growth and development. It
has already permitted the introduction of some novel aspects of the
involvement of cytokinins in the transition from a vegetative to a
reproductive apex. We were also able to show the possibility of a
unique mode of action at the nuclear level for cytokinins of the zeatin
type. Because a good correlation was found between immunostaining and
endogenous cytokinin levels, as measured by HPLC-tandem MS, this
immunocytochemical technique will prove to be helpful in identifying
cytokinin-driven events, such as the regulation of gene expression at
the cell and tissue levels.
| |
FOOTNOTES |
1
W.D. was a recipient of an Individual Human
Capital and Mobility Fellowship (no. ERBCHBICT941211). H.V.O. is a
Research Director at the Flemish Fund for Scientific Research, Belgium.
This research was also supported by a grant from the Belgian Program on
Interuniversity Poles of Attraction (Prime Minister's Office, Science
Programming, no. 15) and by the granting agency of the Czech Republic
(no. 206 196/K 188).
*
Corresponding author; e-mail hvo{at}uia.ua.ac.be; fax
1-32-3-820-22-71.
Received July 6, 1998;
accepted September 9, 1998.
| |
ABBREVIATIONS |
Abbreviations:
AP, alkaline phosphatase.
DHZ, dihydrozeatin.
DHZR, dihydrozeatin riboside.
FITC, fluorescein-5-isothiocyanate.
IP, isopentenyladenine.
IPA, isopentenyladenosine.
ZR, zeatin riboside.
| |
ACKNOWLEDGMENTS |
The authors wish to thank Dr. M.S. Tsuji for advice on
preembedding methodology, Dr. D. Stickens and Dr. T. Weiha for
operation of the confocal microscope, W. van Dongen for help on tandem
mass spectrometry, and I. Bernaert and M. Prouteau for skillful
sectioning. Dr. W. Jacob and Dr. J.P. Verbelen are gratefully
acknowledged for the use of the electron and confocal microscopes. The
authors are also thankful to Dr. P.B. Gahan and Dr. D. Inzé for
critical reading of the manuscript.
| |
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T. Werner, V. Motyka, M. Strnad, and T. Schmulling
Regulation of plant growth by cytokinin
PNAS,
August 10, 2001;
(2001)
171304098.
[Abstract]
[Full Text]
[PDF]
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G. Frugis, D. Giannino, G. Mele, C. Nicolodi, A. Chiappetta, M. B. Bitonti, A. M. Innocenti, W. Dewitte, H. Van Onckelen, and D. Mariotti
Overexpression of KNAT1 in Lettuce Shifts Leaf Determinate Growth to a Shoot-Like Indeterminate Growth Associated with an Accumulation of Isopentenyl-Type Cytokinins
Plant Physiology,
August 1, 2001;
126(4):
1370 - 1380.
[Abstract]
[Full Text]
[PDF]
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I. B. D'Agostino, J. Deruère, and J. J. Kieber
Characterization of the Response of the Arabidopsis Response Regulator Gene Family to Cytokinin
Plant Physiology,
December 1, 2000;
124(4):
1706 - 1717.
[Abstract]
[Full Text]
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G. Frugis, D. Giannino, G. Mele, C. Nicolodi, A. M. Innocenti, A. Chiappetta, M. Beatrice Bitonti, W. Dewitte, H. Van Onckelen, and D. Mariotti
Are Homeobox Knotted-Like Genes and Cytokinins the Leaf Architects?
Plant Physiology,
February 1, 1999;
119(2):
371 - 374.
[Full Text]
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T. Werner, V. Motyka, M. Strnad, and T. Schmulling
Regulation of plant growth by cytokinin
PNAS,
August 28, 2001;
98(18):
10487 - 10492.
[Abstract]
[Full Text]
[PDF]
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