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RESEARCH PAPER
Cytokinins in the perianth, carpels, and developing fruit of
Helleborus niger L.
Petr Tarkowski
1,
*, Danus
ˇ
e Tarkowska
´
2
, Ondr
ˇ
ej Nova
´
k
2
, Snjez
ˇ
ana Mihaljevic
´
3
, Volker Magnus
3
,
Miroslav Strnad
2
and Branka Salopek-Sondi
3,†
1
Umea
˚
Plant Science Centre, Department of Forest Genetics and Plant Physiology, Umea
˚
, Sweden
2
Laboratory of Growth Regulators, Palacky University and IEB ASCR, Olomouc, Czech Republic
3
Rudjer Bos
ˇ
kovic
´
Institute, Zagreb, Croatia
Received 12 September 2005; Accepted 9 March 2006
Abstract
Reproductive development in the Christmas rose
(Helleborus niger L.) differs from that in commonly
investigated model plants in two important aspects: (i)
the perianth develops a photosynthetic system, after
fertilization, and persists until seed ripening; and (ii) the
ripe seed contains an immature embryo which contin-
ues to mature off the mother plant. The possible roles
of cytokinins in these processes are investigated here
by analysing extracts of the perianth and the carpels/
maturing fruit prepared during anthesis and four stages
of post-floral development. trans-Zeatin, dihydrozeatin,
N
6
-(D
2
-isopentenyl)adenine, and their ribosides were
identified by tandem mass spectrometry. Single ion
monitoring in the presence of deuterated internal stand-
ards demonstrated the additional presence of the corres-
ponding riboside-59-monophosphates, O-glucosides,
and 9-glucosides, and afforded quantitative data on
the whole set of endogenous cytokinins. Fruit cytoki-
nins were mostly localized in the seeds. Their overall
concentrations increased dramatically during early
seed development and remained high for 6–8 weeks,
until shortly before seed ripening (the last time point
covered in this work). Overall cytokinin levels in the
perianth did not change markedly in the period covered,
but the level of N
6
-(D
2
-isopentenyl)adenine-type cyto-
kinins appeared to increase slightly and transiently
during the greening phase. The perianths of unpolli-
nated or depistillated flowers, which survived, but did
not pass through the complete greening process, con-
tained significantly less cytokinins than observed in
fruit-bearing flowers. This suggests that perianth green-
ing requires defined cytokinin levels and supports the
role of the developing fruit in their maintenance.
Key words: Christmas rose, cytokinin identification and
quantification, fruit and seed development, Helleborus
niger L., perianth greening.
Introduction
It is believed that the photosynthetic system adapts, both
anatomically and functionally, to the rate of assimilate con-
sumption by metabolic ‘sinks’, such as fruits and storage
tubers (Brenner, 1987), but the complexity of most whole-
plant systems imposes practical limits on detailed mecha-
nistic studies. A simple model system can be found in the
flowers of the Christmas rose (Helleborus niger L.,
Ranunculaceae), a herbaceous, winter-green, perennial
native to south-eastern Europe, which is also widely grown
as an ornamental. In mild winters, the flowers may indeed
appear at Christmas time, resembling wild roses with
respect to size and colour (white to pink). After pollination,
the showy elements of the Christmas rose perianth (usually
* Present address: Department of Biochemistry, Faculty of Science, Palacky University, S
ˇ
lechtitelu
˚
11, CZ-783 71 Olomouc, Czech Republic.
y
To whom correspondence should be addressed. E-mail: salopek@irb.hr
Abbreviations: DZ, dihydrozeatin; DZ9G, dihydrozeatin 9-glucoside; DZOG, dihydrozeatin O-glucoside; DZR, dihydrozeatin riboside; DZRMP, dihydrozeatin
riboside-59-monophosphate; DZROG, dihydrozeatin riboside O-glucoside; f. wt., fresh weight; HPLC, high-performance liquid chromatography; iP, N
6
-(D
2
-
isopentenyl)adenine; iP9G, N
6
-(D
2
-isopentenyl)adenine 9-glucoside; iPR, N
6
-(D
2
-isopentenyl)adenine riboside alias N
6
-(D
2
-isopentenyl)adenosine; iPRMP,
N
6
-(D
2
-isopentenyl)adenine riboside-59-monophosphate alias N
6
-(D
2
-isopentenyl)adenosine-59-monophosphate; LC–MS, liquid chromatography–mass
spectrometry; LC-MS/MS, liquid chromatography-tandem mass spectrometry; Z, trans-zeatin; Z9G, trans-zeatin 9-glucoside; ZOG, trans-zeatin O-glucoside;
ZR, trans-zeatin riboside; ZRMP, trans-zeatin riboside-59-monoph osphate; ZROG, trans-zeatin riboside O-glucoside.
Journal of Experimental Botany, Vol. 57, No. 10, pp. 2237–2247, 2006
doi:10.1093/jxb/erj190 Advance Access publication 9 June, 2006
ª The Author [2006]. Published by Oxford Universit y Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: journals.permissions@oxfordjournals.org
characterized as sepals) develop a functional photosynth-
etic system and persist until fruit ripening (Salopek-Sondi
et al., 2000; Aschan and Pfanz, 2003). Comparable pro-
cesses occur in fertilized flowers of a few other plants (Sitte,
1974; van Doorn, 1997), and in the spathes of some Araceans
(Palandri, 1967; Pais, 1972; Gro¨negress, 1974; Chaves das
Neves and Pais, 1980a, b) but, in these species, leaves are
present at the same time to carry out the bulk of photo-
synthesis. In the Christmas rose, the overwintering leaves
are often pressed to the ground by snow and covered with
debris. They also senesce during fruit ripening, and the
appearance of the new generation of leaves can be delayed
by drought and low temperatures. Apart from the stores in
the roots, the green perianth thus represents the most
reliable, if not the only, source of assimilates for the
developing seeds. Notably, flowers ignored by pollinators
survive about as long as their fertilized neighbours, but only
assume a faint greenish tinge (unpublished observations).
The greening process is also arrested when the developing
fruit are removed (Salopek-Sondi et al., 2000). It is resumed
when the sepals are treated with cytokinins (Salopek-Sondi
et al., 2002). Endogenous cytokinins could thus play a
role in the greening process, and indeed preliminary anal-
yses by high-performance liquid chromatography (HPLC)
suggested dramatic quantitative changes during fruit
development (Salopek-Sondi et al., 2002). This prompted
the analysis of the endogenous cytokinins in Christmas
rose fruit and sepals using a liquid chromatography–mass
spectrometry (LC–MS) approach which provides more re-
liable identifications and more complete quantitative
data. In fruits, cytokinins tend to be concentrated in the
seeds, changing in kind and quantity as endosperm
and embryo development proceed (Morris, 1997;
Emery et al., 1998, 2000). In the species studied so far,
these processes proceed to completion before seed rip-
ening. In contrast, in Helleborus, embryo maturation is
still in progress when the seeds are released by the mother
plant, and this also appears to be the case in many
perennial ornamentals. Cytokinin housekeeping during the
development of this type of seeds is addressed here for
the first time. This also appears to be the first detailed report
on cytokinins in the perianth during its entire life cycle.
Materials and methods
Plant material
Flowers of the Christmas rose (H. niger L. ssp. niger sensu Damboldt
and Zimmermann, 1965) were collected at their natural habitat in the
mountain forests of Gorski kotar, Croatia (altitude, 800 m; dominant
tree species, Abies alba L., Fagus silvatica L., and Picea abies L.).
The six stages of the normal developmental cycle sampled for
cytokinin analysis are shown in Table 1. Two stages of anthesis were
recognized. The (‘proterogynous’) flowers first passed through their
female phase during which the stigmata were receptive and the
immature anthers were arranged in a ring at the base of the cluster
of carpels. The male phase began with the elongation of the fila-
ments, and ended with anther abortion. Fruit development and the
accompanying metamorphosis of the perianth were monitored at
four subsequent stages: ‘initial perianth greening’ (sepals with first
greenish tinge, barely noticeable fruit growth); ‘advanced perianth
greening’ (light green sepals, fruit weight twice that in late anthesis);
‘perianth greening complete’ (chlorophyll content in the sepals
entering the stationary phase, fruit weight three times that in late
anthesis); and ‘2–3 weeks before seed ripening’ (sepals green, fruit
fully grown, weight ;12 times that in late anthesis).
Non-pollinated flowers (same carpel size as in late anthesis) and
flowers depistillated at bud opening were collected when the seeds of
fertilized flowers at the same location were 2–3 weeks before
ripening.
For cytokinin analysis, the perianths and the clusters of carpels or
developing fruit were separated, immediately frozen, and kept in
Table 1. Stages of the life cycle of Christmas rose flowers sampled for cytokinin analysis
Description Perianth Fruit cluster
Colour
a
Fresh weight (g)
c
Length (mm)
b
Fresh weight (g)
c
Anthesis, female phase
d
White 1.12 Ovaries: 9 0.18
Styles: 6
Anthesis, male phase
e
White 1.31 Ovaries: 10 0.26
Styles: 9
Initial perianth greening Greenish-white 1.00 Bodies: 10 0.26
Beaks: 10
Advanced perianth greening Light green 1.17 Bodies: 15 0.53
Beaks: 10
Perianth greening complete Green 1.25 Bodies: 16 0.79
Beaks: 11
2–3 weeks before seed ripening Green 1.41 Bodies: 26 3.10
Beaks: 13
a
Exposure to intense light induces red pigmentation causing ‘white’ flowers to apear rose and ‘green’ sepals to be tinged with purple in a range
of shades.
b
Arithmetic means (SE 65–10%) for random samples of 10 fruit clusters. The bodies of the follicles (derived from the ovaries) and the beaks (derived
from the styles) were measured separately.
c
Arithmetic means (SE 610–15%) for the samples (n=30–65) from which aliquots for cytokinin analysis were taken.
d
First days after bud opening: stigmata receptive, stamina immature, anthers short, nectaries mostly erect.
e
Stigmata dry, anthers elongated, pollen mature, nectaries spreading.
2238 Tarkowski et al.
sealed plastic bags, at ÿ80 8C, until work-up. Stamens and nectaries
were discarded.
Cytokinin identification
Cytokinins were extracted and separated, essentially as outlined by
A
˚
stot et al. (1998). Frozen plant material (;10 g fresh weight) was
ground with a mortar and pestle in liquid nitrogen and extracted
overnight in methanol–chloroform–formic acid–water (12:5:1:2, by
vol.) (Bieleski, 1964). Passing the extract, in sequence, through
a cation (SCX-cartridge) and an anion [DEAE-Sephadex combined
with an SPE(C18)-cartridge] exchanger afforded fraction 1 contain-
ing the cytokinin free bases, ribosides, and glucosides, and fraction
2 containing the riboside-59-monophosphates. Both fractions were
purified further by immunoaffinity chromatography based on generic
monoclonal anticytokinin antibodies, but fraction 2 was first treated
with alkaline phosphatase. In fraction 1, the O-glucosides did not
bind to the immunoaffinity column. The effluent was thus treated
with b-glucosidase and the hydrolysate was rechromatographed on
the immunoaffinity column to yield the O-glucoside fraction (which
actually contained just the aglycones of the original O-glucosides).
The dried samples were propionylated using N,N-dimethylformamide–
N-methylimidazole–propionic anhydride (5:3:1, by vol.) (A
˚
stot
et al., 1998), evaporated in vacuo and stored at ÿ20 8C until further
analysis.
For analysis, the samples were redissolved in 1% aqueous formic
acid and subjected to LC–MS/MS. The chromatographic separation
was performed on a capillary column (150 mm
30.3 mm; LC
Packings, Amsterdam, The Netherlands) packed with Symmetry
C
18
packing material, particle size 3.5 lm (Waters). The eluent,
applied at a flow rate of 4 ll min
ÿ1
, was 1% aqueous formic acid
(solvent A), mixed with 1% formic acid in acetonitrile (solvent B),
as follows: 0–5.5 min, 10% B; 5.5–20 min, linear gradient to 70%
B; followed by 3 min isocratic elution with 70% B. The effluent
was introduced into a Micromass Quattro Ultima triple-stage quad-
rupole mass spectrometer via an electrospray ion source (capillary
voltage, +2.9 kV; cone voltage, +70 V; source temperature, 90 8C;
desolvation temperature, 120 8C; cone gas flow, 120 l h
ÿ1
; desolvation
gas flow, 520 l h
ÿ1
; collision energy, 15 eV; dwell time, 0.35 s;
scanning, 1 s per scan for mass range 0–600 amu).
Cytokinin quantification
For quantitative analysis, the separation method was modified as
follows (Nova´k et al., 2003). Aliquots of plant material (1 g) were
processed by adding the following internal standards at the extraction
stage: trans-[
2
H
5
]zeatin, trans-[
2
H
5
]zeatin riboside, trans-[
2
H
5
]zeatin
9-glucoside, [
2
H
3
]dihydrozeatin, [
2
H
3
]dihydrozeatin riboside,
[
2
H
3
]dihydrozeatin 9-glucoside, [
2
H
6
]isopentenyladenine, [
2
H
6
]iso-
pentenyladenine riboside, [
2
H
6
]isopentenyladenine 9-glucoside,
trans-[
2
H
5
]zeatin O-glucoside, trans-[
2
H
5
]zeatin riboside O-
glucoside, trans-[
2
H
5
]zeatin riboside-59-monophosphate, [
2
H
3
]dihy-
drozeatin riboside-59-monophosphate, and [
2
H
6
]isopentenyladenine
riboside-59-monophosphate (OlChemIm Ltd, Olomouc, Czech
Republic). After fractionation and purification as for cytokinin iden-
tification (but no propionylation), the samples were subjected to
HPLC on a reversed-phase column (150 mm
32.1 mm; particle size,
5 lm) (Symmetry C
18
, Waters) operated at 30 8C. The components
of the eluent were (A) 15 mM ammonium formate, pH 4.0 and (B)
methanol; they were mixed as follows (difference from 100% is A):
0 min, 10% B; 0–25 min, linear gradient to 50% B; 25–30 min, 50%
B; 30–35 min, 100% B, all at a flow rate of 250 ll min
ÿ1
. Using post-
column splitting (1:1), the eluent was simultaneously introduced into
a diode array detector (Waters PDA 996) and the electrospray source
(source temperature, 100 8C; capillary voltage, +3.0 kV; cone
voltage, +20 V; desolvation temperature, 250 8C) of a single-stage
quadrupole mass spectrometer (ZMD 2000, Micromass, Manchester,
UK). Nitrogen was used as both the desolvation gas (400 l h
ÿ1
) and
the cone gas (50 l h
ÿ1
). Quantification was done by single ion moni-
toring of the quasi-molecular ([M+H]
+
) ions of the plant cytokinins
and the corresponding deuterated internal standards.
Results and discussion
Perianth and fruit development
The metamorphosis of the sepals in pollinated Helleborus
flowers has already been documented in detail (Salopek-
Sondi et al., 2000, 2002). In brief, during anthesis, only
the guard cells contain chloroplasts, resulting in bulk
chlorophyll levels below 1 lgg
ÿ1
fresh weight (f. wt.).
After fertilization, chloroplasts form in the entire peri-
anth, eventually affording chlorophyll concentrations of
;350 lgg
ÿ1
f. wt. At the same time, the interior of the
sepals assumes a sponge-like structure, due to the expan-
sion of intercellular spaces. Cell divisions were not noticed;
cell expansion was limited, and so was overall sepal
growth. The small weight gain during the assimilatory
phase shown in Table 1 was supported by further obser-
vations through several growth seasons (Salopek-Sondi
et al., 2000, 2002, and unpublished data). The weight
fluctuations observed in the present samples during anthe-
sis and ‘initial sepal greening’ (Table 1) are not typical
and appear to reflect intraspecific variability in flower size.
Simultaneously with perianth greening, the fruit clusters
started to grow. Characteristic developmental stages of the
follicles (Fig. 1A) and the seeds (Fig. 1B) are illustrated in
Fig. 1. Interestingly, fruit growth was not restricted to the
ovaries, but included the styles which developed into
elongated beaks (Table 1). Most fruit weight was gained
while the photosynthetic capacity of the perianth was fully
established. The ratio between pericarp and seed weight
depended on seed set (more or less successful pollination).
In several samples analysed 3–4 weeks before ripening, up
to two-thirds of the overall fruit weight was in the pericarp.
Selected features of the anatomy of developing Helle-
borus seeds are shown in Fig. 2. Following Schiffner
(1891), the principal storage tissue has so far been claimed
to be the endosperm, but no supporting documentation came
to our attention. In accord with a recent comparative study
(Endress and Igersheim, 1999) on the gynoecium of the
basal eudicots (including the Ranunculaceae), it is therefore
proposed here to attribute the most prominent tissue in
Helleborus seeds to the nucellus (or a ‘pseudonucellus’
derived from the epidermal layers of the embryo sac), with
only ;20% of its overall volume occupied by the enclosed
endosperm. The latter was in its liquid (nuclear) phase
during sepal greening and mostly cellular at seed ripen-
ing (Fig. 2). The embryo started developing during sepal
greening to reach an early cotyledonary stage by the time
the seeds were shed. Its development appears to continue
during several months of after-ripening in moist soil, at
summer temperatures (Hartmann and Kester, 1975; Lockhart
Cytokinins in Helleborus 2239
and Albrecht, 1987). A further 3 months at +4 8C were
then required for germination.
Identification of the cytokinin types present in
Helleborus flowers
To gain qualitative insight into the set of cytokinins present
in Helleborus fruit and sepals, plant extracts were subjected
to a verified fractionation and purification procedure (see
Materials and methods) and the isolates obtained were O-
propionylated and analysed by LC–MS/MS. The results
unequivocally demonstrated the presence of the free bases
and ribosides of zeatin (Z), dehydrozeatin (DZ), and
N
6
-(D
2
-isopentenyl)adenine (iP). The mass spectra of
mono-O-propionyl zeatin, mono-O-propionyl dihydrozea-
tin, tetra-O-propionyl zeatin riboside, and tri-O-propionyl
N
6
-(D
2
-isopentenyl)adenine riboside are shown in Fig. 3;
those of tetra-O-propionyl dihydrozeatin riboside and iP,
which are not presented, were also free of contaminantions,
and showed analogous fragmentation patterns. Generally,
all cytokinins form [M+H]
+
quasi-molecular ions. These
were at m/z 578 for tetra-O-propionyl dihydrozeatin ribo-
side and at m/z 204 for iP; the values for the other
cytokinins identified are shown in Fig. 3. The important
fragments of cytokinin bases correspond to fragmentation
of the side chain, with ions at m/z 136 (adenine) and 148.
O-propionyl-zeatin also shows ions at m/z 220 and 202,
corresponding to protonated Z and loss of water from the
latter. In addition, loss of ammonia from the fragment at m/z
202 results in an ion at m/z 185. O-propionyl-dihydrozeatin
shows a similar fragmentation pattern with ions at m/z
222 and 204. In the mass spectra of cytokinin ribo-
sides, the presence of O-propionylated ribose is indi-
cated by a fragment at m/z 301 (charged tri-O-propionyl
ribose). Further elimination of two equivalents of pro-
pionic acid yields m/z 153, and additional loss of a methyl-
ketene (56) results in a fragment at m/z 97. The mass
spectra were recorded at retention times corresponding to
the retention times of appropriate synthetic standards.
In addition to the above major cytokinins, ions which
may belong to cis-zeatin riboside appeared in some
samples, at the appropriate retention times, but complete
daughter ion spectra with acceptable signal-to-noise ratios
could not be obtained, possibly due to low levels of the
respective cytokinin. The extraction and sample purifica-
tion procedure used is also suitable for the isolation and
identification of aromatic cytokinins. Those were, how-
ever, not detected in Christmas rose fruit and sepals.
Quantitative cytokinin relationships—general features
Mass spectrometric analysis, in the presence of deuterium-
labelled internal standards, combined with a slightly modified
fractionation procedure for the plant extracts, permitted
quantification of the cytokinins present in Helleborus
flowers. The data for six stages of their life cycle are
summarized in Tables 2 (pistils and developing fruit) and
3 (sepals). The analyses were repeated for three series of
samples (2–4 replicates per sample) and mostly agreed
within the error limits indicated in the tables. Only the
results for iP in the sepals varied too much to state definite
numerical values (arithmetic means), but were clustered
around a concentration of 0.2 pmol g
ÿ1
f. wt., throughout
the life cycle of the perianth. The method used was previ-
ously tested with authentic standards and in plant samples,
including extensive validation by enzyme immunoassay
using specific anti-cytokinin antibodies (Nova´ket al., 2003).
In both the perianth and the developing fruit, the 9-
glucosides played a negligible role: only in one sample
(fruit 2–3 weeks before ripening) did they contribute
slightly more than 1% to the overall cytokinin concentra-
tion; elsewhere they were less abundant or undetectable.
Fig. 1. Fruit and seed growth in Helleborus niger L. (glutaraldehyde-
fixed material). (A) Follicles split lengthwise at the following
developmental stages (from left to right): white flower, male phase
(i.e. shortly after fertilization), initial perianth greening, perianth
greening complete, and 2–3 weeks before ripening. The upper half
of the follicle wall was removed. Some seeds had aborted (probably due
to incomplete pollination), as seen most clearly in the fully grown fol-
licles. (B) Seeds sampled at the same developmental stages plus
a ripe seed after rehydration. Helleborus seeds bear an elaiosome
(arill) which was removed from the ripe seed to prevent fungus in-
festation during the rehydration process. The dimensions of the fields of
the grid used as a background in (A) and (B) are 1 mm
31 mm.
2240 Tarkowski et al.
Fig. 2. Longitudinal sections through seeds of Helleborus niger L. at two developmental stages. Left: toluidine blue-stained section prepared when the
sepals started to turn green. Presented is the micropylar end of the nucellus (surrounded by integumental tissue which is not completely shown) enclosing
the liquid endosperm. The intensely stained, large cell visible at the micropylar end of the endosperm is part of the embryo at a very early developmental
stage. The length of the scale bar corresponds to 100 lm. Middle: toluidine blue-stained section through the same region taken at seed ripening. The
now cellular endosperm encloses an embryo at its early cotyledonary stage with its radicle up (i.e. pointing toward the micropyle). The length of
the scale bar corresponds to 200 lm. Right: ripe unstained seed in about the same orientation with the tiny embryo at the upper tip. The total length of the
seed shown was 4 mm.
Fig. 3. Daughter ion mass spectra (electron spray ionization, positive ions) of (A) O-propionyl trans-zeatin, (B) O-propionyl-dihydrozeatin, (C) tetra-
O-propionyl-trans-zeatin riboside, and (D) tri-O-propionyl-N
6
-(D
2
-isopentenyl)adenosine. The fragmentation pattern is indicated in the structural
formulae.
Cytokinins in Helleborus 2241
Table 2. Cytokinin levels in pistils and developing fruit of Helleborus niger
Cytokinin Quantity
Anthesis: female
phase
Anthesis: male
phase
Initial perianth
greening
Advanced perianth
greening
Perianth greening
complete
2–3 weeks before
seed ripening
pmol
g
ÿ1
f. wt.
pmol
f. c.
ÿ1a
pmol
g
ÿ1
f. wt.
pmol
f. c.
ÿ1a
pmol
g
ÿ1
f. wt.
pmol
f. c.
ÿ1a
pmol
g
ÿ1
f. wt.
pmol
f. c.
ÿ1a
pmol
g
ÿ1
f. wt.
pmol
f. c.
ÿ1a
pmol
g
ÿ1
f. wt.
pmol
f. c.
ÿ1a
Z 0.4660.05 0.08 0.4460.08 0.11 0.4360.16 0.11 9.9760.43 5.30 35.8365.42 28.43 63.6266.58 197.01
ZOG 0.0060.00 0.00 0.2060.03 0.05 0.0860.00 0.02 1.4260.29 0.76 7.1861.28 5.70 37.1762.50 115.11
ZR 1.2160.23 0.21 1.0960.26 0.28 0.67960.15 0.21 30.7962.08 16.35 127.06617.14 100.82 72.3569.44 224.07
ZROG 0.3060.05 0.05 0.3860.06 0.10 0.5960.00 0.15 2.3860.66 1.26 10.8161.53 8.57 40.7362.91 126.13
ZRMP 3.0660.45 0.54 2.1660.54 0.55 1.8560.39 0.48 47.2861.44 25.10 51.7062.49 41.02 15.6060.26 48.30
Z9G 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.2160.01 0.11 0.7660.22 0.60 3.3060.92 10.21
All trans-zeatin forms 5.0360.51 0.88 4.2760.61 1.09 3.7560.45 0.97 92.0562.67 48.89 233.33618.26 185.15 232.76612.17 720.83
DZ 0.1360.01 0.02 0.0560.01 0.01 0.0660.00 0.01 0.3460.03 0.18 1.1060.30 0.87 2.0260.16 6.26
DZOG 0.0060.00 0.00 0.0060.00 0.00 0.5060.09 0.13 0.7360.16 0.39 1.0860.27 0.86 1.9760.28 6.09
DZR 4.8960.32 0.86 3.2260.40 0.82 1.5960.21 0.41 6.2160.39 3.30 16.2962.67 12.93 13.2160.38 40.92
DZROG 1.6360.14 0.29 1.3760.12 0.35 0.8960.17 0.23 1.7560.25 0.93 3.8460.34 3.05 11.3761.12 35.22
DZRMP 5.2160.46 0.92 3.4260.33 0.87 1.3160.13 0.34 12.5960.35 6.69 11.4561.15 9.09 6.7060.44 20.76
DZ9G 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0360.01 0.03 0.2960.04 0.92
All dihydrozeatin forms 11.8660.57 2.09 8.0660.53 2.06 4.3460.31 1.12 21.6160.6 11.47 33.8062.96 26.82 35.5761.30 110.17
iP 0.5060.05 0.09 0.2760.01 0.07 5.2061.24 1.34 3.2560.66 1.73 2.6560.35 2.10 1.9060.19 5.89
iPR 1.4660.23 0.26 1.3560.52 0.34 0.7560.11 0.19 1.1860.20 0.63 0.7760.04 0.61 0.3560.03 1.08
iPRMP 9.0660.90 1.59 3.4460.43 0.88 5.7060.68 1.47 32.7966.26 17.42 24.4062.58 19.36 4.1960.49 12.98
iP9G 0.0060.00 0.00 0.0060.00 0.00 0.0260.00 0.00 0.0360.00 0.01 0.0660.02 0.05 0.0460.01 0.11
All isopentenyladenine
forms
11.0160.93 1.94 5.0660.68 1.29 11.6761.42 3.01 37.2666.29 19.79 27.8862.60 22.12 6.4860.52 20.06
Free cytokinins 1.0960.07 0.19 0.7660.08 0.19 5.6961.25 1.47 13.5660.81 7.20 39.5865.44 31.40 67.5466.59 209.1625
Ribosides+ribotides 24.8861.19 4.37 14.6861.04 3.75 11.9960.84 3.10 130.8466.77 69.49 231.67617.75 183.83 112.4169.47 348.11
O-Glucosides 1.9360.15 0.34 1.9660.13 0.50 2.0660.20 0.53 6.2760.78 3.33 22.9162.05 18.18 91.2464.01 282.55
9-Glucosides 0.0060.00 0.00 0.0060.00 0.00 0.0260.00 0.004 0.2460.01 0.13 0.8560.22 0.68 3.6360.92 11.24
Total cytokinins 27.9161.21 4.91 17.3961.05 4.44 19.7661.52 5.10 150.9266.86 80.15 295.01618.68 234.07 274.81612.25 851.06
a
f. c., fruit cluster; the cluster of carpels or developing fruit of a single flower.
2242 Tarkowski et al.
Table 3. Cytokinin levels in the sepals of Helleborus niger during anthesis and fruit ripening
Cytokinin Quantity
Anthesis: female
phase
Anthesis: male
phase
Initial perianth
greening
Advanced perianth
greening
Perianth greening
complete
2–3 weeks before
seed ripening
pmol
g
ÿ1
f. wt.
pmol
perianth
ÿ1
pmol
g
ÿ1
f. wt.
pmol
perianth
ÿ1
pmol
g
ÿ1
f. wt.
pmol
perianth
ÿ1
pmol
g
ÿ1
f. wt.
pmol
perianth
ÿ1
pmol
g
ÿ1
f. wt.
pmol
perianth
ÿ1
pmol
g
ÿ1
f. wt.
pmol
perianth
ÿ1
Z 0.2460.02 0.27 0.2960.02 0.38 0.2260.03 0.22 0.1960.01 0.22 0.6360.17 0.79 1.4960.11 2.09
ZOG 0.1160.01 0.13 0.1760.02 0.23 0.1960.03 0.19 0.1660.01 0.19 0.2460.02 0.30 0.5260.04 0.73
ZR 1.4460.21 1.62 1.1160.08 1.45 0.5560.05 0.55 0.3660.04 0.20 0.5760.08 0.72 1.2560.24 1.76
ZROG 0.2860.05 0.32 0.4160.05 0.53 0.2060.04 0.20 0.2060.03 0.24 0.256 0.01 0.31 0.6460.12 0.91
ZRMP 1.8060.27 2.03 2.5960.35 3.39 1.5560.19 1.54 0.8960.13 1.05 1.3360.06 1.66 0.7460.16 1.04
Z9G 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00
All trans-zeatin forms 3.8860.35 4.36 4.5860.37 5.98 2.7160.20 2.69 1.8060.14 2.11 3.0260.20 3.78 4.6460.34 6.53
DZ 0.1060.02 0.11 0.0560.01 0.07 0.0660.01 0.06 0.0660.01 0.07 0.0460.01 0.05 0.0560.01 0.07
DZOG 0.4660.06 0.51 0.4860.06 0.62 0.5560.10 0.55 0.5760.08 0.67 0.6260.14 0.78 0.7860.15 1.10
DZR 4.7760.24 5.35 2.6760.17 3.48 2.0060.22 1.99 1.3360.07 1.56 1.3060.08 1.62 0.6960.09 0.97
DZROG 1.0560.10 1.18 1.0360.07 1.34 1.2360.10 1.22 1.1860.07 1.39 1.6660.12 2.08 1.3960.16 1.95
DZRMP 2.0660.40 2.32 3.3860.21 4.41 1.9460.34 1.93 2.2260.11 2.61 1.1860.19 1.47 0.3260.05 0.45
D9G 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00
All dihydrozeatin forms 8.4360.48 9.48 7.6060.29 9.93 5.7860.43 5.75 5.3760.17 6.30 4.8060.28 6.00 3.2260.24 4.53
iP 0.1660.02 0.18 0.1860.01 0.23 ***
a
***
a
***
a
***
a
***
a
***
a
0.1860.02 0.26
iPR 0.9460.07 1.05 0.7260.03 0.94 0.5560.07 0.55 0.4360.04 0.51 0.4760.03 0.59 0.2860.04 0.39
iPRMP 4.3160.35 4.85 3.2560.33 4.24 7.0761.50 7.07 9.1861.47 10.77 5.1160.84 6.38 4.1760.95 5.86
iP9 0.0160.00 0.01 0.0260.00 0.02 0.0160.00 0.01 0.0260.00 0.02 0.0260.00 0.02 0.0660.00 0.08
All isopentenyladenine forms 5.4260.36 6.09 4.1660.33 5.44 7.6361.50 7.63 9.6361.47 11.30 5.6060.84 6.99 4.6960.95 6.59
Free cytokinins 0.5060.03 0.56 0.5260.02 0.68 0.2860.03 0.28 0.2560.01 0.29 0.6760.17 0.84 1.7260.11 2.42
Ribosides + ribotides 15.3260.68 17.22 13.7260.56 17.91 10.4460.49 12.63 14.4161.48 16.70 9.9660.87 12.44 7.4561.00 10.47
O-Glucosides 1.9060.13 2.14 2.0960.11 2.72 2.1760.15 3.37 2.1160.11 2.49 2.7760.19 3.47 3.3360.25 4.69
9-Glucosides 0.0160.00 0.01 0.0260.00 0.02 0.0160.00 0.07 0.0260.00 0.02 0.0260.00 0.02 0.0660.00 0.08
Total cytokinins 17.7360.69 19.93 18.4060.57 21.33 12.9060.51 16.35 16.7961.48 19.50 13.4260.91 16.77 12.5661.04 17.66
a
The results of the individual analyses were too divergent to justify calculation of arithmetic means, but suggested isopentenyladenine levels in the same range as in earlier and later
developmental stages. The value 0.0060.00 was used when computing cumulative parameters (i.e. all iP forms, free and total cytokinins).
Cytokinins in Helleborus 2243
Also, while LC–MS (single ion monitoring) data suggested
the presence of picomolar concentrations of cis-zeatin
cytokinins in some samples, no consistent pattern could be
established. Therefore the focus will be on the intercon-
vertible forms of Z, DZ, and isopentenyladenine cyto-
kinins: free bases, ribosides, riboside-59-monophosphates,
and O-glucosides.
Cytokinin dynamics in developing fruit
The overall cytokinin levels in the carpels (Table 2) de-
creased slightly as anthesis proceeded from the female to the
male phase, to rise dramatically during fruit ripening. This
was mainly due to a transient increase in the concentrations
of trans-zeatin riboside (ZR) and dihydrozeatin riboside
(DZR), with maxima at about the time when sepal green-
ing was complete. During later fruit development, the corres-
ponding free bases increased on account of their ribosides,
leaving overall cytokinin levels almost constant until 2–3
weeks before seed ripening, the most advanced developmen-
tal stage investigated. At anthesis, DZ-type cytokinins were
slightly more abundant than their zeatin analogues. During
fruit development, this proportion was inverted, with Z and
derivatives attaining up to seven times higher concentrations.
As shown in Fig. 4, the cytokinins in Helleborus fruit
sampled 3–4 weeks before ripening are mostly localized
in the seeds. This is probably the case during most of
fruit development, as suggested, for instance, by analogy
with the morphologically similar legume fruits for
which detailed data on cytokinin distribution during the
entire developmental cycle are available (Emery et al.,
1998, 2000). In the seed types which have been studied so
far, such as cereal grains (Morris, 1997) and chick peas
(Emeryetal.,1998),cytokininlevelsrisedramaticallyshortly
after pollination, and drop abruptly as seed differentiation
begins, thus correlating with cell division rates in the
endosperm (Morris, 1997), in accordance with the stimu-
latory role of cytokinins in the cell division cycle (Roef
and van Onckelen, 2004). In Helleborus, cytokinin levels
also increase dramatically during early fruit development
(while the sepals are going through the greening pro-
cess). This coincides with rapid growth, not only of the endo-
sperm, but also of the nucellus, both of which ultimately
occupy >90% of the volume of the seed. Cell division rates
inthese tissues were not recorded, but should decrease to
zero before, or when, the seeds reach their maximal
size. This occurs 2–3 weeks before ripening (Fig. 1).
Why is there no corresponding decrease in cytokinin levels?
Is it because the embryo is still developing?
In somatic embryos, which are more easily accessible
than zygotic embryos, the role of cytokinins appears to
depend on the developmental stage, the type and concen-
tration of the cytokinin, and, possibly, the plant species. In
Corydalis yanhusuo W. T. Wang (Fumariaceae), a species
with relatively close phylogenetic ties to Helleborus,Zor
benzyladenine were required for embryo development from
the globular to the cotyledonary stage (Sagare et al., 2000),
with optimal results at a concentration of 500 ng ml
ÿ1
(;2250 pmol ml
ÿ1
). This is close to the overall concentra-
tion of interconvertible cytokinins in Helleborus seeds 3–4
weeks before ripening (1357 pmol g
ÿ1
; Fig. 4). In carrot
(Daucus carota L.) cell suspension cultures, 1000 pmol
ml
ÿ1
Z increased the rate of embryogenesis, affording about
the same range of developmental stages as described above
for Corydalis (Nomura and Komamine, 1985). When the
embryos were induced on hypocotyl sections, exogenous
cytokinins were not required. However, purine riboside
inhibited embryogenesis and its effect could be compen-
sated by simultaneous addition of as little as 100 pmol ml
ÿ1
ZR (Tokuji and Kuriyama, 2003). This is less than the con-
centration of this cytokinin which was found in Helleborus
seeds 3–4 weeks before ripening (340 pmol g
ÿ1
; Fig. 4).
It is therefore suggested that the high cytokinin levels
during late seed development in Helleborus are related to
the fact that the embryo is still developing towards the
cotyledonary stage. In the model systems investigated
so far (Morris, 1997; Emery et al., 1998, 2000), early
embryogenesis and rapid endosperm proliferation are
simultaneous processes, a fact which would rationalize
the observation that there is only a single short cytokinin
maximum during early seed development.
Cytokinin dynamics in the perianth
In the sepals, only the levels of N
6
-(D
2
-isopentenyl)adenine
riboside-59-monophosphate (iPRMP) appeared to be linked
Fig. 4. Pool sizes of zeatin and dihydrozeatin cytokinins in the seeds (S)
and pericarps (P) of an individual fruit cluster (cluster of 4–7 follicles
present in a single flower) collected 3–4 weeks before maturity. The
respective amounts of isopentenyladenine cytokinins were below the
detection limit except for iP, 0.10; iPR, 0.09; and iPRMP, 2.37 pmol in
the seeds, and iP, 0.10 pmol in the pericarps. The values shown are the
arithmetic means of two (seeds) to three (pericarps) independent analyses;
sampling errors were in the same range as for the data shown in Table 2.
Cytokinin concentrations can be calculated considering that the mean
weight (n=43) of a fruit cluster was 1.88 g, of which 1.223 g was in the
pericarps and 0.657 g in the seeds. The figure is based on fruit collected
in a growth season for which material completely identical to that in
Tables 1–3 was not available.
2244 Tarkowski et al.
to perianth greening, increasing by a factor of two to three
as chlorophyll started to appear (Table 3). However, the
changes were small in absolute terms, and further experi-
ments may be required to confirm a causal relationship. The
overall cytokinin concentration in the sepals decreased
slightly after anthesis, showing only minor fluctuations
during further development, and most individual cytokinins
followed this general trend. However, the concentrations of
Z and most of its derivatives increased slightly, but
consistently, towards the end of the life cycle of the
perianth and, taken together, eventually exceeded the
pooled concentration of all DZ forms. Earlier in perianth
development, the latter were up to three times more
abundant than the total zeatin cytokinins. However, the
DZ in the sepals was mostly in two bound forms: the
riboside and its O-glucoside; the concentration of the free
cytokinin barely exceeded the detection limit.
To verify the effect of developing seeds on cytokinin
levels in the perianth, depistillated and unpollinated flowers
were analysed (Fig. 5). The seedless flowers contained no
ribotides, and the levels of most other cytokinins were also
smaller than in the sepals of fruit-bearing flowers of the
same physiological age (i.e. 2–3 weeks before seed ripen-
ing). The seedless flowers also weighed less and did not
pass through a complete greening process. This indicates
that cytokinins play a part in the normal life cycle of the
perianth including the greening response following fertil-
ization. So far, the impact of these hormones on the pho-
tosynthetic apparatus has mostly been investigated in
model systems based on the prevention of chlorophyll
loss as one of the symptoms of senescence (Richmond
and Lang, 1957; Medford et al., 1989; Gan and Amasino,
1995), an analogy which now appears less straightforward
than originally believed (Werner et al., 2001; Ananieva
et al., 2004). The reverse process, cytokinin-mediated
conversion of etioplasts into chloroplasts (Parthier, 1979),
was, for instance, observed in a line of tobacco callus which
grew well without external cytokinin but nevertheless
required it for the formation of chloroplasts when the
normally dark-grown tissue was transferred to the light
(Stetler and Laetsch, 1965). Greening processes of this
kind are akin to what happens in the Christmas rose
perianth after fertilization when the leucoplasts present at
anthesis develop into chloroplasts (Salopek-Sondi et al.,
2000, 2002). Further research will show if the accom-
panying, comparatively small, changes in the relative
abundance of individual cytokinins (in the perianth) are
by themselves sufficient to trigger the formation of a
photosynthetic apparatus, and to keep it functional during
seed ripening. They may also be just one set of com-
ponents in a complex signalling network.
Concluding remarks
Seeds have long been known to be a rich source of
cytokinins, but their origin has been a controversial issue.
They were long thought to be synthesized in the root tips
and transported to their sites of action via the xylem and
the phloem (Letham, 1994), but recent data now demon-
strate that this is an oversimplification (Faiss et al., 1997;
Emery et al., 2000; Nordstro¨m et al., 2004). In particu-
lar, Lee et al. (1989) demonstrated that wheat (Triticum ae-
stivum L.) ears cultured in vitro, in a hormone-free
liquid medium, maintain a normal cytokinin pattern. Also,
Miyawaki et al. (2004) found some of the genes encod-
ing isopentenyltransferases involved in cytokinin bio-
synthesis expressed in immature seeds. It is thus
plausible to assume that most of the cytokinins detected
in Helleborus seeds are synthesized in situ. If so, two
observations suggest that this preferentially occurs via
iPRMP, i.e. (i) the latter is always the most abundant
isopentenyladenine-cytokinin; and (ii) the dramatic increase
of zeatin and DZ cytokinins during perianth greening is
preceded by an (albeit less prominent) increase in the
concentration of iPRMP. Also, as fruit approach maturity,
iPRMP starts to decrease while zeatin and DZ cytokinins
are still maintaining top levels.
The significance of the changing patterns of cytokinin
forms during perianth and fruit development requires
further research. It has been widely assumed that only the
free bases have genuine hormonal activity, the ribosides
play a role in long-range transport, the O-glucosides are
storage forms, and the 7- and 9-glucosides are catabolites
(Mok and Mok, 2001). This picture is changing, however,
as all plant species studied so far contain several cytokinin
receptors with complex expression patterns (Maxwell and
Kieber, 2004) and, apparently, widely different substrate
specificities. In Arabidopsis, for instance, one of the recep-
tors (CRE1/AHK4) is specific for trans-zeatin, but another
one (AHK3) also recognizes cis-zeatin and DZ as well as
cytokinin ribosides and ribotides (Spichal et al., 2004).
Similar differences in the substrate specificities of indi-
vidual cytokinin receptors were also reported for maize
(Yonekura-Sakakibara et al., 2004).
Does the obvious correlation of fruit growth and perianth
greening imply that cytokinins synthesized in the seeds
are exported to the sepals? A complete answer is not yet
possible, but clearly defined cytokinin levels in the sepals
are required to induce the formation of a photosynthetic
system and to keep it functional during seed filling, because
(i) the perianth of seedless flowers, whether depistillated
at the bud stage or just ignored by pollinators at flowering
time, is cytokinin deficient and such flowers do not pass
through a complete greening process; and (ii) exogenous
cytokinins stimulate chlorophyll accumulation in such
flowers (Salopek-Sondi et al., 2002). During advanced
fruit development, cytokinin pool sizes are indeed much
larger in the fruit than in the perianth (Tables 2, 3).
However, when the greening process is initiated (‘initial
perianth greening’), the perianth of an individual flower
contains a larger cytokinin pool than the fruit cluster
Cytokinins in Helleborus 2245
of that same flower. Under these circumstances, the
biosynthetic machinery in the fruit would have to work
preferentially for the perianth. That this is not impossible is
demonstrated by an example from the developmental
physiology of the pea (Pisum sativum L.) fruit. Indirect
evidence shows convincingly that the seeds supply auxin to
the pericarp (Ozga and Reinecke, 2003), even at a stage
when the latter is about finger-long and contains a signifi-
cantly larger auxin pool than the pinhead-sized seeds
(Magnus et al., 1997).
Acknowledgements
The work presented was supported by grant no. MSM 6198959216
from the Ministry of Education, Youth, and Sports of the Czech
Republic and by grants no. 0098080 and 0119111 from the Ministry
of Science, Education, and Sports of the Republic of Croatia. We are
indebted to E Yeung, University of Calgary, who helped us to
understand the anatomy of developing Christmas rose seeds.
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