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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 continues 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, N6-(Δ2-isopentenyl)adenine, and their ribosides were identified by tandem mass spectrometry. Single ion monitoring in the presence of deuterated internal standards demonstrated the additional presence of the corresponding riboside-5′-monophosphates, O-glucosides, and 9-glucosides, and afforded quantitative data on the whole set of endogenous cytokinins. Fruit cytokinins 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 N6-(Δ2-isopentenyl)adenine-type cytokinins appeared to increase slightly and transiently during the greening phase. The perianths of unpollinated or depistillated flowers, which survived, but did not pass through the complete greening process, contained significantly less cytokinins than observed in fruit-bearing flowers. This suggests that perianth greening requires defined cytokinin levels and supports the role of the developing fruit in their maintenance.
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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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... It is recognized, however, that the mechanism of regreening in these various plant organs might differ. For example, regreening in sepals of H. niger was believed to be induced (at least in part) by fructification (Salopek-Sondi et al. 2000, Tarkowski et al. 2006, Ayele et al. 2010. Regreening in peel of fruits in C. sinensis on the other hand, was reported to be induced by a decrease in the content of sucrose within the fruits (Huff 1984), while regreening in the yellowing leaf of N. rustica can be induced by the application of cytokinins (Zavaleta-Mancera et al. 1999). ...
... Cytokinins and gibberellins have been shown to stimulate regreening in floral organs or leaves in some species (Pais and Neves 1983, Zavaleta-Mancera et al. 1999, Salopek-Sondi et al. 2002. This role of cytokinins and gibberellins in regreening was confirmed by both exogenous application and analysis of endogenous cytokinins/gibberellins in regreening and regreened tissue (Ananieva et al. 2004, Tarkowski et al. 2006, Ayele et al. 2010. Conversely, a recent study in 'Best Gold' showed that application of cytokinin [6-benzylaminopurine (BAP)] and gibberellin (GA 3 ) either exerted no effect or resulted in an initial delay to the color change from yellow to green in the spathe tissue (Chen et al. 2013). ...
... Similar conclusions have been suggested with regard to other plant systems which regreen, e.g. sepals of H. niger (Salopek-Sondi et al. 2002, Tarkowski et al. 2006, wherein regreening was initiated in the depistillated flower, but did not proceed to completion. However, the mechanism for this potential mode of interconnection between fructification and regreening is unclear. ...
Article
The mature pigmented spathe of Zantedeschia is characterized by a developmental process, wherein the spathe regreens after anthesis and prior to senescence of the inflorescence. Previous research has shown that spathe regreening involves redifferentiation of chloroplasts and reaccumulation of chlorophyll, but the detailed physiological changes associated with regreening are still largely unknown. Using Z. aethiopica and the Z. pentlandii variety ‘Best Gold’ as models, the current study explores the physiological mechanism and possible roles of fructification, 6-benzylaminopurine (BAP) and gibberellin (GA3) in induction or progression of spathe regreening. Application of BAP stimulated regreening in spathe tissue of ‘Best Gold’ by enhancing accumulation of carotenoid and chlorophyll, and also increasing stacking of grana. In contrast, GA3 retarded formation of double-membrane lamella during chloroplast redifferentiation, thus delaying the onset of regreening. We suggest that these actions of BAP and GA3 have a synergistic effect in delaying the onset of regreening in ‘Best Gold’ so that when applied together retardation of chlorophyll accumulation, chloroplast redifferentiation and accumulation of carotenoids was enhanced. The elimination of fructification did not prevent the occurrence of regreening in either Zantedeschia model plants, indicating that fructification was not a prerequisite for the induction of regreening. It is still unclear how regreening in Zantedeschia is triggered. We propose that the onset of regreening in Zantedeschia is likely to be a genetically programmed event.
... Similar to auxin, cytokinin is another important plant hormone regulating many aspects of plant growth (Tarkowski et al., 2006;Werner et al., 2008). In plants, the regulation of cytokinin is facilitated by the two-component system (TCS) which consists of four groups of proteins: histidine kinases (AHKs; AHK2, AHK3, and AHK4/WOL1/CRE1), histidine-containing phosphotransfer proteins (AHPs; AHP1-AHP5), type-B response regulators (type-B ARRs; ARR1, ARR2, ARR10-ARR14, and ARR18-ARR21), and type-A ARRs (ARR3-ARR9 and ARR15-ARR17). ...
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Ricebean ( Vigna umbellata ) is a lesser known pulse with well-recognized potential. Recently, it has emerged as a legume with endowed nutritional potential because of high concentration of quality protein and other vital nutrients in its seeds. However, the genes and pathways involved in regulating seed development and size are not understood in this crop. In our study, we analyzed the transcriptome of two genotypes with contrasting grain size (IC426787: large seeded and IC552985: small seeded) at two different time points, namely, 5 and 10 days post-anthesis (DPA). The bold seeded genotype across the time points (B5_B10) revealed 6,928 differentially expressed genes (DEGs), whereas the small seeded genotype across the time point (S5_S10) contributed to 14,544 DEGs. We have also identified several candidate genes for seed development–related traits like seed size and 100-seed weight. On the basis of similarity search and domain analysis, some candidate genes ( PHO1 , cytokinin dehydrogenase , A-type cytokinin, and ARR response negative regulator) related to 100-seed weight and seed size showed downregulation in the small seeded genotype. The MapMan and KEGG analysis confirmed that auxin and cytokinin pathways varied in both the contrasting genotypes and can therefore be the regulators of the seed size and other seed development–related traits in ricebeans. A total of 51 genes encoding SCF TIR1/AFB , Aux/IAA , ARFs , E3 ubiquitin transferase enzyme, and 26S proteasome showing distinct expression dynamics in bold and small genotypes were also identified. We have also validated randomly selected SSR markers in eight accessions of the Vigna species ( V. umbellata : 6; Vigna radiata : 1; and Vigna mungo : 1). Cross-species transferability pattern of ricebean–derived SSR markers was higher in V. radiata (73.08%) than V. mungo (50%). To the best of our knowledge, this is the first transcriptomic study conducted in this crop to understand the molecular basis of any trait. It would provide us a comprehensive understanding of the complex transcriptome dynamics during the seed development and gene regulatory mechanism of the seed size determination in ricebeans.
... Similar to auxin, cytokinin (CK) is another important plant hormone regulating many aspects of plant growth 21,22 . CK plays positive roles in the regulation of shoot apical meristem (SAM) activity 23 . ...
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Auxin plays critical roles in many developmental processes of plants. The auxin signaling pathway is a series of plant responses to auxin stimuli. However, the functions of many genes in this pathway are still obscure. As auxin receptors, TIR/AFB family genes encode F-Box proteins that directly bind auxin and then transduce the stimulus through the signaling pathway. In this paper, we generated an overexpression line of Auxin-signaling F-Box 6 (OsAFB6) in rice, which largely delayed heading, greatly increased spikelets per panicle and primary branch number and ultimately enhanced grain yield by 50%. OsAFB6 is preferentially expressed in young tissues with stronger meristem activities and suppresses flowering by upregulating OsRR1 and downregulating Ehd1 expression levels. Overexpression of OsAFB6 delayed heading, increased cytokinin (CK) by suppressing the expression level of Gn1a and simultaneously decreased the IAA concentration in the young panicle, which promoted inflorescence meristem development and resulted in large panicles with more spikelets per panicle, primary branches and increased grain yield. It would be a beneficial strategy to generate lines with varied expression levels of OsAFB6 to breed high-yielding cultivars for specific regions that can fully utilize the local sunlight and temperature resources.
... The high level of tZ+iP accumulation in the female flower at stage 3 and exogenous CK treatment inducing bisexual flowers suggest that CK is crucial for carpel development. 62 LOG proteins are produced at specific locations and time points, especially at active growth regions during plant development. 63 In C. henryi, four LOG genes (LOG1, LOG3, LOG5, and LOG7) were predicted, and the fluorescence quantitative data and correlation analysis indicated that LOG3 and LOG5 were dominantly involved in bioactive CK synthesis in flower of C. henryi during sex expression; however, the action mechanism of these two LOG members in C. henryi was not clear at the present. ...
Article
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Chinese chinquapin (Castanea henryi) nut provides a rich source of starch and nutrients as food and feed, but its yield is restricted by a low ratio of female to male flowers. Little is known about the developmental programs underlying sex differentiation of the flowers. To investigate the involvement of phytohormones during sex differentiation, we described the morphology of male and female floral organs and the cytology of flower sex differentiation, analyzed endogenous levels of indole-3-acetic acid (IAA), gibberellins (GAs), cytokinins (CKs) and abscisic acid (ABA) in the flowers, investigated the effects of exogenous hormones on flower development, and evaluated the expression profiles of genes related to biosyntheses and signaling pathways of these four hormones using RNA-Seq combined with qPCR. Morphological results showed that the flowers consisted of unisexual and bisexual catkins, and could be divided into four developmental stages. HPLC results showed that CK accumulated much more in the female flowers than that in the male flowers; GA and ABA showed the opposite results; while IAA did not show a tendency. The effects of exogenous hormones on sex differentiation were consistent with those of endogenous hormones. RNA-Seq combined with qPCR anlyses suggest that several genes may play key roles in hormone biosynthesis and sex differentiation. This study presents the first comprehensive report of phytohormone biosynthesis and signaling during sex differentiation of C. henryi, which should provide a foundation for further mechanistic studies of sex differentiation in Castanea Miller species and other non-model plants.
... The targeted disturbance of this balance, leading to increased activity of inflorescence and floral meristems and higher seed yield in rice (Oryza sativa L.) [14] and Arabidopsis (Arabidopsis thaliana L.) [15], has recently provided evidence for the importance of cytokinins in reproductive development and hence crop productivity. In support of this, high cytokinin activities or concentrations have been reported in immature seeds and fruit from a large number of species, including pea (Pisum sativum L.) [16], white lupine (Lupinus albus L.) [17], Christmas rose (Helleborus niger L.) [18], tomato (Solanum lycopersicum Mill.) [19], strawberry (Fragaria ananassa Duch.) [20], kiwifruit (Actinidia deliciosa (A. Chev.) ...
Article
Full-text available
Background: Cytokinins are known to play an important role in fruit set and early fruit growth, but their involvement in later stages of fruit development is less well understood. Recent reports of greatly increased cytokinin concentrations in the flesh of ripening kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang & A.R. Ferguson) and grapes (Vitis vinifera L.) have suggested that these hormones are implicated in the control of ripening-related processes. Results: A similar pattern of isopentenyladenine (iP) accumulation was observed in the ripening fruit of several grapevine cultivars, strawberry (Fragaria ananassa Duch.) and tomato (Solanum lycopersicum Mill.), suggesting a common, ripening-related role for this cytokinin. Significant differences in maximal iP concentrations between grapevine cultivars and between fruit species might reflect varying degrees of relevance or functional adaptations of this hormone in the ripening process. Grapevine orthologues of five Arabidopsis (Arabidopsis thaliana L.) gene families involved in cytokinin metabolism and signalling were identified and analysed for their expression in developing grape berries and a range of other grapevine tissues. Members of each gene family were characterised by distinct expression profiles during berry development and in different grapevine organs, suggesting a complex regulation of cellular cytokinin activities throughout the plant. The post-veraison-specific expression of a set of biosynthesis, activation, perception and signalling genes together with a lack of expression of degradation-related genes during the ripening phase were indicative of a local control of berry iP concentrations leading to the observed accumulation of iP in ripening grapes. Conclusions: The transcriptional analysis of grapevine genes involved in cytokinin production, degradation and response has provided a possible explanation for the ripening-associated accumulation of iP in grapes and other fruit. The pre- and post-veraison-specific expression of different members from each of five gene families suggests a highly complex and finely-tuned regulation of cytokinin concentrations and response to different cytokinin species at particular stages of fruit development. The same complexity and specialisation is also reflected in the distinct expression profiles of cytokinin-related genes in other grapevine organs.
... In contrast, the sepals of unpollinated or depistillated H. niger flowers have the same life-span as pollinated ones, but tend not to regreen significantly. Active fruit development is required in H. niger for regreening of sepals to occur and removal of developing fruit arrests the regreening process (Salopek-Sondi et al. 2002;Tarkowski et al. 2006). Leucadendron differ in that pollination or subsequent seed development does not seem to be a prerequisite for regreening of involucral leaves. ...
Article
Involucral leaves of Leucadendron have the remarkable ability to turn yellow upon flowering and regreen naturally as the florets of the inflorescence wilt. This colour change results from degradation of chlorophyll and to a lesser degree carotenoids, resulting in the unmasking of yellow colour. Chlorophyll levels were restored upon regreening. Degreening coincided with the complete dismantling of the thylakoid system, while keeping the outer plastid envelope intact. Regreening resulted from the complete redifferentiation of these gerontoplast-like plastids into functional chloroplasts. The colour change was directly linked to the development of the inflorescence. Complete removal of the inflorescence before flowering prevented the colour change while removal at full bloom, when involucral leaves were yellow, resulted in significantly faster regreening. This designates the inflorescence or florets as the possible origin of the colour change trigger and suggests that the colour change is involved with attraction of pollinators. Degreening and regreening also took place in a growth chamber under continuous high light intensity. Therefore neither pollination nor the presence of roots is required for regreening. It appears that colour change in Leucadendron results from a well-regulated degradation and subsequent synthesis of photosynthetic pigments.
... However, there has been no report about the MS behaviors of auxins when using electrospray ionization in the positive ion mode (ESI+). The MS behavior of CKs has been studied mostly in the positive ion mode, where ions m/z 148 and 136 have both been observed in the spectra of KT [7] and tZ [8], and the ion at m/z 136 was found as the product ion for the protonated molecule of iP [9]. However, few MS behavior studies of the CKs in negative ion mode have been reported. ...
Article
Auxins, cytokinins (CKs), gibberellins (GAs), and abscisic acid (ABA) are four classes of plant hormones, which play important roles in phytophysiology. However, few mass spectrometric fragmentation pathway studies of these compounds have been performed using a high-resolution mass spectrometer. Therefore, there is an urgent need to research the fragmentation pathways of classic plant hormones. In this study, the fragmentation pathways of four types of plant hormones were studied by employing an electrospray ionization-quadrupole/time-of-flight (ESI-Q/TOF) mass spectrometer. The accurate masses of the ions in the mass spectrometer (MS) and the product ions in the MS/MS were combined and used to obtain the MS fragmentation pathways of these compounds. The exact mass-to-charge ratio for each product ion was determined to deduce the elemental compositions for each compound. Most of the ions were assigned according to the collected high-resolution accurate mass data, and typical fragmentation pathways of the four classes of plant hormones were proposed. Furthermore, a comprehensive MS/MS spectra library of the plant hormones was established for the first time using ESI-Q/TOF. For instance, a characteristic mass signal of m/z 130 was identified for the product ions of the indole 3-acetic acid homologue auxins in the positive ion mode and for the characteristic neutral loss of CO2CO2H2O for gibberellin in the negative ion mode. These findings are valuable for the identification of a variety of plant hormones and could also provide a basis for developing MS-based methods of detecting or screening a target class of plant hormones in plant extracts.
Article
Peace lily (Spathiphyllum wallisii Regel) is a herbaceous, commercially important ornamental plant characterized by greening of its originally white spathe during a post-anthesis period and fruit development. It is a useful model plant for gaining insight into source–sink relationship. To clarify what is the triggering signal for spathe greening and its potential physiological role, the greening process has been examined in fertilized vs unfertilized and decapitated (with spadix removed) plants. Results showed that spathe of fertilized plants is viable for a longer period of time and greening process is more intensive in comparison with decapitated or unfertilized plants. Greening process comprised chloroplast differentiation as well as chlorophyll accumulation, and resulted in the photosynthetically active organs. Photosynthesis performance index (PIABS) of spathe in fertilized flowers was significantly higher than that in unfertilized and decapitated plants, indicating its better overall photosynthetic performance. Spathe removal during fruit development revealed approximately 30% smaller fruits in comparison with intact ones, implicating that green spathe contribute significantly as assimilate source for developing fruits. Cytokinin analysis showed that t-Z-type cytokinins were the most abundant in the spadix (t-Z and t-ZR) and the greening spathe (t-ZR), while overall cytokinin pool was significantly lower in the spathe of the decapitated plants. Exogenous treatment of spathe in decapitated plants with t-Z enhanced the intensity of greening. We may conclude that spathe greening in peace lily is genetically programmed process that may be mediated by fructification and, in part, by cytokinins originated in the developing fruit which was confirmed herein for the first time.
Article
Cytokinin dehydrogenase is responsible for regulating the endogenous cytokinin content by oxidative removal of the side chain. Herein, we have applied fluorescence method to study the interaction between cytokinin dehydrogenase and cytokinin in vitro, and obtain some parameters of their interaction. We found that addition of isopentenyl adenine can quench the fluorescence of cytokinin dehydrogenase and the quenching mechanism was to be a static quenching procedure. We have measured the number of binding sites n and apparent binding constant K, and have calculated the thermodynamics parameter ΔH, ΔG and ΔS by fluorescence quenching method. Based on thermodynamics parameter's results we concluded that their binding reaction was both entropy-driven and the enthalpy-driven, and the Van der Waals force and hydrogen bond force played major role in the interaction. Based on the synchronous fluorescence spectrometry results we demonstrated that the binding site between isopentenyl adenine and cytokinin dehydrogenase is in the microenvironment of both tryptophan and tyrosine. The fluorescence signal of coenzyme, flavin adenine dinucleotide, decreases gradually with the addition of isopentenyl adenine. And this method can be used for isopentenyl adenine routine assay. Under optimized experimental parameters, the linear segment increases from 0.6 µM to 100 µM with a regression equation ofΔF = 0.04 + 0.15cip(r = 0.999, cip in µM) with the detection limit of 0.15 µM iP.
Article
Although, the ripening of climacteric fruits such as bananas is known to be mainly triggered by ethylene, cytokinins may also intervene in the control and regulation of this process. Bananas were treated by dipping for 24 h in solutions of cytokinins at concentrations ranging from 10-6 to 10-3 M to determine effects of these phytohormones on some parameters during the fruit ripening. At all concentrations used, benzylaminopurine induced significant retentions of water in the peel of banana fruits. Only 10-3 M kinetin solution was significantly effective in inhibiting the decrease of water content in the peel. There was an inhibition of the accumulation of water in the pulp of bananas after treatments with kinetin or benzylaminopurine. Qualitative analysis of pigments in peel extracts revealed an inhibition of chlorophyll degradation after application of cytokinins. Thus, banana fruits treated with 10-3 M kinetin still contained only chlorophyll a whereas, fruits treated with 10-3 M benzylaminopurine contained both chlorophyll a and chlorophyll b at ripening stage 7. At this ripening stage, no chlorophyll could be detected in extracts from the peel of control fruits. There were increased accumulations of monoacylglycerol, triacylglycerol and free fatty acids in the peel of banana fruits treated with 10-3 M benzylaminopurine. Contrarily to apolar lipids, no remarkable effects of treatments of bananas with 10-3 M benzylaminopurine on polar lipids could be observed. These results indicated that cytokinins modulated the metabolism of photosynthetic pigments and lipids and changes in the water content and that chlorophyll b was converted into chlorophyll a before its degradation in the peel of banana fruits during the ripening process.
Article
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A comprehensive range of cytokinins (CK) was identified and quantified by gas chromatography-mass spectrometry in tissues of and in xylem and phloem serving developing white lupine (Lupinus albus) fruits. Analyses were initiated at anthesis and included stages of podset, embryogenesis, and seed filling up to physiological maturation 77 d post anthesis (DPA). In the first 10 DPA, fertilized ovaries destined to set pods accumulated CK. The proportion of cis-CK:trans-CK isomers was initially 10:1 but declined to less than 1:1. In ovaries destined to abort, the ratio of cis-isomers to trans-isomers remained high. During early podset, accumulation of CK (30–40 pmol ovary 1) was accounted for by xylem and phloem translocation, both containing more than 90% cis-isomers. During embryogenesis and early seed filling (40–46 DPA), translocation accounted for 1% to 14% of the increases of CK in endosperm (20 nmol fruit 1) and seed coat (15 nmol fruit 1), indicating synthesis in situ. High CK concentrations in seeds (0.6 mol g 1 fresh weight) were transient, declining rapidly to less than 1% of maximum levels by physiological maturity. These data pose new questions about the localization and timing of CK synthesis, the significance of translocation, and the role(s) of CK forms in reproductive development.
Article
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Trans-isomers of cytokinins (CK) are thought to predominate and have greater biological activity than corresponding cis-isomers in higher plants. However, this study demonstrates a system within which the predominant CK are cis-isomers. CK were measured at four developmental stages in developing chickpea (Cicer arietinum L. cultivar Kaniva) seeds by gas chromatography-mass spectrometry. Concentrations were highest at an early endospermic fluid stage and fell considerably when the cotyledons expanded. Thecis-isomers of zeatin nucleotide ([9R-MP]Z), zeatin riboside ([9R]Z), and zeatin (Z) were present in greater concentrations than those of correspondingtrans-isomers: (trans)[9R-MP]Z, (trans)[9R]Z, (trans)Z, or dihydrozeatin riboside. Dihydrozeatin, dihydrozeatin nucleotide, and the isopentenyl-type CK concentrations were either low or not detectable. Root xylem exudates also contained predominantlycis-isomers of [9R-MP]Z and [9R]Z. Identities of (cis)[9R]Z and (cis)Z were confirmed by comparison of ion ratios and retention indices, and a full spectrum was obtained for (cis)[9R]Z. Tissues were extracted under conditions that minimized the possibility of RNase hydrolysis of tRNA following tissue disruption, being a significant source of thecis-CK. Since no isomerization of (trans)[²H]CK internal standards occurred, it is unlikely that the cis-CK resulted from enzymic or nonenzymic isomerization during extraction. Although quantities of total CK varied, similar CK profiles were found among three different chickpea cultivars and between adequately watered and water-stressed plants. Developing chickpea seeds will be a useful system for investigating the activity of cis-CK or determining the origin and metabolism of free CK.
Chapter
The movement of photoassimilates from sites of synthesis in leaf tissue (source) to the sites of net accumulation in a different tissue (sink) potentially can be regulated at numerous points. Regulation of the net flow of photoassimilates is an integrated process. It is generally accepted that the concentration gradient of photoassimilates between the source and sink is the primary determinant of the current rate of transport and pattern of partitioning (14, 19, 60). However, close examination of the various components involved in the overall process of partitioning indicates that endogenous plant hormones may serve as modulators of many of the specific rate limiting components. This chapter will focus on the involvement of plant hormones as natural regulators of partitioning of photoassimilates especially to developing seeds.
Article
The possible role of phytohormones in light-dependent plastogenesis is reviewed particularly in respect to the influence of cytokinins in this plant-specific differentiation process. The following aspects of cytokinin action in chloroplast formation are considered in detail: Ultrastructure and replication of chloroplasts, chlorophyll accumulation, plastid enzyme synthesis and activity, nucleic acid and protein biosynthesis. Some remarks are made about the importance of the physiological state of the responding tissue. Possible modes of action on the cellular and molecular levels are discussed in relation to plastogenesis.
Chapter
Shortly after pollination, developing cereal grains exhibit significant transient increases in two of the major plant hormones, auxins and cytokinins. A peak in active cytokinins occurs between three to eight days after pollination in all species examined so far. Both zeatin and zeatin riboside concentrations increase by as much as one hundred fold for a period of three days and then decrease equally rapidly. The peak always coincides in time with the peak in endosperm cell division rate, although no causal relationship has been demonstrated between the two events. The enzyme cytokinin oxidase increases shortly after the increase in active cytokinins and is responsible for reducing cytokinins back to basal levels. The cytokinin peak is followed by a peak in free indole-3-acetic acid (approximately 10 to 15 days after pollination) and is associated with the endosperm cell enlargement and endoreduplication phase. The high levels of free IAA are not destroyed but are converted to various conjugates which probably supply the developing seedling with a source of free IAA. Studies with defective kernel mutants suggest that free IAA levels are causally related to endoreduplication in the endosperm.
Article
Gynoecium and ovule structure was comparatively studied in representatives of the basal monocots, including Acorales (Acoraceae), Alismatales (Araceae, Alismataceae, Aponogetonaceae, Butomaceae, Hydrocharitaceae, JuncÍaginaceae, Limnocharitaceae, Potamogetonaceae, Scheuchzeriaceae, Tofieldiaceae), Dioscoreales (Dioscoreaceae, Taccaceae), and Triuridaceae as a family of uncertain position in monocots. In all taxa studied the carpels or gynoecia are closed at anthesis. This closure is attained in different ways: (1) by secretion without postgenital fusion (Araceae, Hydrocharitaceae); (2) by partly postgenitally fused periphery but with a completely unfused canal (Alismataceae, Aponogetonaceae, Butomaceae, Limnocharitaceae, Scheuchzeriaceae, Dioscoreaceae, Taccaceae); (3) by completely postgenitally fused periphery but with an unfused canal in the centre (Acoraceae, Tofieldiaceae); (4) by complete postgenital fusion and without an (unfused) canal (Juncaginaceae, Potamogetonaceae). In many Alismatales (but without Araceae) carpels have two lateral lobes. The stigmatic surface is restricted to the uppermost part of the ventral slit (if the carpel is plicate); it is never distinctly double-crested (Butomaceae?). Stigmas are commonly unicellular-papillate and secretory in most taxa. The locules are filled with a (often) mucilaginous secretion in a number of taxa. Superficial (probably intrusive) ethereal oil cells were found in the carpel wall of Acorus gramineus (as in Piperales!). Idioblasts in carpels are otherwise rare. A number of basal monocots has orthotropous ovules, which is perhaps the plesiomorphic condition in the group. The presence of almost tenuinucellar (pseudocrassinucellar) ovules is relatively common (Acoraceae, many Araceae, some Alismatales s.s.), whereas completely tenuinucellar ovules are rare and do not characterize larger groups. However, crassinucellar ovules occur in the largest number of families among the study group (basal Araceae, many Alismatales s.s.) The outer integument is always annular in orthotropous ovules. The inner integument is often lobed and it mostly forms the micropyle, whereas the outer integument is always unlobed. Gynoecium structure supports the isolated position of Acoraceae as sister to all other monocots. However, in an overall view, if compared with all other families, Acoraceae clearly shows the greatest similarities with Araceae.