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Endogenous Auxin Profile in the Christmas Rose (Helleborus niger L.) Flower and Fruit: Free and Amide Conjugated IAA

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Abstract

The reproductive development of the Christmas rose (Helleborus niger L.) is characterized by an uncommon feature in the world of flowering plants: after fertilization the white perianth becomes green and photosynthetically active and persists during fruit development. In the flowers in which fertilization was prevented by emasculation (unfertilized) or entire reproductive organs were removed (depistillated), the elongation of the peduncle was reduced by 20–30%, and vascular development, particularly lignin deposition in sclerenchyma, was arrested. Chlorophyll accumulation in sepals and their photosynthetic efficacy were up to 80% lower in comparison to fertilized flowers. Endogenous auxins were investigated in floral and fruit tissues and their potential roles in these processes are discussed. Analytical data of free indole-3-acetic acid, indole-3-ethanol (IEt), and seven amino acid conjugates were afforded by LC-MS/MS in floral tissues of fertilized as well as unfertilized and depistillated flowers. Among amino acid conjugates, novel ones with Val, Gly, and Phe were identified and quantified in the anthers, and in the fruit during development. Reproductive organs before fertilization followed by developing fruit at post-anthesis were the main source of auxin. Tissues of unfertilized and depistillated flowers accumulated significantly lower levels of auxin. Upon depistillation, auxin content in the peduncle and sepal was decreased to 4 and 45%, respectively, in comparison to fruit-bearing flowers. This study suggests that auxin arising in developing fruit may participate, in part, in the coordination of the Christmas rose peduncle elongation and its vascular development. KeywordsAuxin–Indole-3-acetic acid–Amide conjugates–Christmas rose– Helleborus niger L.–Flower and fruit development–Perianth greening–Peduncle elongation–Vascular system
1 23
Journal of Plant Growth Regulation
ISSN 0721-7595
Volume 31
Number 1
J Plant Growth Regul (2012) 31:63-78
DOI 10.1007/s00344-011-9220-1
Endogenous Auxin Profile in the Christmas
Rose (Helleborus niger L.) Flower and
Fruit: Free and Amide Conjugated IAA
Ana Brcko, Aleš Pěnčík, Volker Magnus,
Tatjana Prebeg, Selma Mlinarić, Jasenka
Antunović, Hrvoje Lepeduš, Vera Cesar,
Miroslav Strnad, et al.
1 23
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Endogenous Auxin Profile in the Christmas Rose (Helleborus niger
L.) Flower and Fruit: Free and Amide Conjugated IAA
Ana Brcko Ales
ˇPe
ˇnc
ˇı
´kVolker Magnus Tatjana Prebeg Selma Mlinaric
´
Jasenka Antunovic
´Hrvoje Lepedus
ˇVera Cesar Miroslav Strnad
Jakub Rolc
ˇı
´kBranka Salopek-Sondi
Received: 28 February 2011 / Accepted: 24 May 2011 / Published online: 28 June 2011
ÓSpringer Science+Business Media, LLC 2011
Abstract The reproductive development of the Christmas
rose (Helleborus niger L.) is characterized by an uncommon
feature in the world of flowering plants: after fertilization the
white perianth becomes green and photosynthetically active
and persists during fruit development. In the flowers in which
fertilization was prevented by emasculation (unfertilized) or
entire reproductive organs were removed (depistillated), the
elongation of the peduncle was reduced by 20–30%, and
vascular development, particularly lignin deposition in
sclerenchyma, was arrested. Chlorophyll accumulation in
sepals and their photosynthetic efficacy were up to 80%
lower in comparison to fertilized flowers. Endogenous auxins
were investigated in floral and fruit tissues and their potential
roles in these processes are discussed. Analytical data of free
indole-3-acetic acid, indole-3-ethanol (IEt), and seven amino
acid conjugates were afforded by LC-MS/MS in floral tissues
of fertilized as well as unfertilized and depistillated flowers.
Among amino acid conjugates, novel ones with Val, Gly, and
Phe were identified and quantified in the anthers, and in the
Ana Brcko and Ales
ˇPe
ˇnc
ˇı
´k contributed equally to this work.
Volker Magnus deceased in 2009.
A. Brcko V. Magnus T. Prebeg B. Salopek-Sondi
Ruder Bos
ˇkovic
´Institute, Bijenic
ˇka cesta 54, HR-10002 Zagreb,
Croatia
e-mail: abrcko@irb.hr
T. Prebeg
e-mail: tprebeg@agr.hr
A. Pe
ˇnc
ˇı
´kM. Strnad J. Rolc
ˇı
´k
Laboratory of Growth Regulators, Faculty of Science, Palacky
´
University and Institute of Experimental Botany AS CR,
S
ˇlechtitelu
˚11, CZ-78371 Olomouc, Czech Republic
e-mail: alespencik@seznam.cz
M. Strnad
e-mail: miroslav.strnad@upol.cz
J. Rolc
ˇı
´k
e-mail: jakub.rolcik@seznam.cz
A. Pe
ˇnc
ˇı
´k
Department of Growth Regulators, Faculty of Science,
Centre of the Region Hana
´for Biotechnological and Agricultural
Research, Palacky
´University, S
ˇlechtitelu
˚11,
CZ-78371 Olomouc, Czech Republic
S. Mlinaric
´J. Antunovic
´V. Cesar
Department of Biology, University of J.J. Strossmayer, Trg
Ljudevita Gaja 6, HR-31000 Osijek, Croatia
e-mail: smlinaric@biologija.unios.hr
J. Antunovic
´
e-mail: jantunovic@biologija.unios.hr
V. Cesar
e-mail: vcesarus@yahoo.com
H. Lepedus
ˇ
Agricultural Institute Osijek, Juz
ˇno predgrad
¯e 17,
HR-31000 Osijek, Croatia
e-mail: hlepedus@yahoo.com
T. Prebeg
Department of Ornamental Plants, Landscape Architecture and
History of Garden Art, Faculty of Agriculture, University
of Zagreb, Svetos
ˇimunska cesta 25, Zagreb, Croatia
B. Salopek-Sondi (&)
Department of Molecular Biology, Rud
¯er Bos
ˇkovic
´Institute,
Bijenic
ˇka cesta 54, HR-10002 Zagreb, Croatia
e-mail: salopek@irb.hr
123
J Plant Growth Regul (2012) 31:63–78
DOI 10.1007/s00344-011-9220-1
Author's personal copy
fruit during development. Reproductive organs before fer-
tilization followed by developing fruit at post-anthesis were
the main source of auxin. Tissues of unfertilized and depis-
tillated flowers accumulated significantly lower levels of
auxin. Upon depistillation, auxin content in the peduncle and
sepal was decreased to 4 and 45%, respectively, in compar-
ison to fruit-bearing flowers. This study suggests that auxin
arising in developing fruit may participate, in part, in the
coordination of the Christmas rose peduncle elongation and
its vascular development.
Keywords Auxin Indole-3-acetic acid Amide
conjugates Christmas rose Helleborus niger L.
Flower and fruit development Perianth greening
Peduncle elongation Vascular system
Introduction
Reproductive plant development, characterized by flower-
ing and fruit formation, is a complex process that relies on
careful timing and coordination of tissue development. The
processes of flower and fruit development are highly med-
iated by the interplay of different plant hormones: auxins,
cytokinins, gibberellins, abscisic acid (ABA), and ethylene
(Ozga and Reinecke 2003; Finkelstein 2004; Srivastava and
Handa 2005). Among them, auxins seem to trigger both the
flower (Cheng and Zhao 2007) and the fruit developmental
program (Alabadi and others 2009). In addition, auxins,
primarily indole-3-acetic acid, appear to participate in the
coordination of processes within and between floral organs
during flower and fruit development, such as anther dehis-
cence, pollen maturation, gynoecium development, fertil-
ization, fruit initiation, seed development, and fruit ripening
(Sundberg and Østergaard 2009). Most of a plant’s endog-
enous IAA is found not as a free and biologically active
form, but rather as an ester linked to sugars or amide linked
to amino acids and peptides. The proposed roles of these
conjugates include storage, transport, compartmentaliza-
tion, protection against degradation, and detoxification
(Cohen and Bandurski 1982). Different plant species have
distinct IAA conjugate profiles. In general, monocots
accumulate mostly ester conjugates, whereas dicots pref-
erably accumulate amide conjugates (see for review Bajguz
and Piotrowska 2009; Ludwig-Mu
¨ller 2011). Studies of the
metabolism and the physiological role of auxins in repro-
ductive development have been focused on commercially
interesting species such as maize (Jensen and Bandurski
1994), pea (Magnus and others 1997), bean (Bialek and
Cohen 1989), tomato (Epstein and others 2002), and wheat
(Hess and others 2002). The largest body of information on
auxin metabolism and its role in both vegetative and gen-
erative development resulted from research on the model
plant Arabidopsis thaliana, particularly from the point of
view of auxin identification and biochemistry to genetics
and molecular biology (O
¨stin and others 1998; Kowalczyk
and Sandberg 2001; Ljung and others 2002; Aloni and
others 2006; Østergaard 2009; Normanly 2010). In horti-
cultural plants, auxin research was focused mostly on the
growth and elongation of the peduncle, with the aim of
improving flower traits according to market requirements.
Early research dealt with the influence of floral organs on
flower peduncle elongation. It was observed that removal of
the flower or floral organs inhibits the elongation of the
floral stalk or peduncle (Op den Kelder and others 1971;
Hanks and Rees 1977). Treatment of emasculated or
decapitated flowers with auxins (Edelbluth and Kaldewey
1976; Hanks and Rees 1977; De Munk 1979; Kohji and
others 1979; Saniewski and De Munk 1981; Ohno and Kako
1991), as well as treatments of isolated stalk explants
(Gabryszewska and Saniewski 1983), led to the conclusion
that these plant hormones originating in anthers, gynoe-
cium, or developing fruit are largely responsible for
peduncle elongation. Identification of endogenous hor-
mones during flowering in the tulip (Okubo and Uemoto
1985; Xu and others 2008) and barley (Wolbang and others
2004) confirm that diffusible auxins from floral organs, in
the first place, and in coordination with gibberellins are
responsible for stalk elongation. Despite these findings,
systematic analyses of auxins during reproductive devel-
opment are still missing, and the role of auxins in the
coordination of floral organ development remains unclear.
Here we investigated flower and fruit development in the
Christmas rose (Helleborus niger L.) during pre-anthesis,
anthesis, and post-anthesis development. Christmas rose
belongs to the genus Helleborus, which comprises about 20
perennial species with large, attractive flowers in various
shades of purple, green, and white (Mathew 1989). Repro-
ductive development of most of these plants is characterized
by an interesting feature: they retain the perianth (formed by
the sepals) until seed ripening with its color changing to
green. Both the original species and hybrid cultivars are
becoming increasingly popular as ornamentals (Burrell and
Tyler 2006). H. niger is a species with an exceptionally long
flowering period (2–6 months starting around Christmas)
and particularly large flowers (6–13 cm), which change
color from white to intensely green, both mixed with purple
in sun-exposed locations (Salopek-Sondi and Magnus 2007).
The greening process in the perianth and elongation of the
peduncle are linked to the presence of developing fruit,
which makes the fertilized Christmas rose flower an inter-
esting model for the study of developmental aspects of sink-
source interactions. It was previously shown that sepal
greening and peduncle elongation of unfertilized or depis-
tillated flowers were stalled at an early stage of development.
The greening process could be stimulated to completion by
64 J Plant Growth Regul (2012) 31:63–78
123
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treating the sepals with gibberellins and cytokinins
(Salopek-Sondi and others 2002). Indeed, endogenous
cytokinins (Tarkowski and others 2006) and gibberellins
(Ayele and others 2010) were identified in floral and fruit
tissues and found to be important regulators of sepal
greening. Treatment of the depistillated flowers with auxins
IAA and 4-Cl-IAA did not affect the greening process, but
4-Cl-IAA stimulated peduncle elongation (Salopek-Sondi
and others 2002). In this study, we examined reproductive
development of H. niger and identified free IAA and seven
amino acid conjugates. Conjugates IAA-Val, IAA-Gly, and
IAA-Phe were found recently by the same authors (Pe
ˇnc
ˇı
´k
and others 2009)inH. niger for the first time in higher plants.
The levels of IAA and its amino acid conjugates were
quantified in intact floral and fruit tissues at different stages
of development and in unfertilized and depistillated flowers.
With respect to these data the role of auxin in Christmas rose
development is discussed.
Materials and Methods
Plant Material
Flowers of the Christmas rose (Helleborus niger L. ssp.
niger) were collected at a woodlot in the Pharmaceutical
Botanical Garden ‘‘Fran Kus
ˇan’’ of the Faculty of Phar-
macy and Biochemistry, University of Zagreb, in two dif-
ferent growth seasons. The material was collected to gain
insight into auxin dynamics during the whole life cycle of a
Christmas rose flower, comprising a series of six flower
bud developmental stages (pre-anthesis), two stages of
anthesis (Female and Male phase), and four stages of post-
anthesis development (Light green, Advanced green,
Green, and Almost ripe) (Fig. 1).
Unfertilized flowers were obtained by enclosing emas-
culated flower buds in sacks of gauze to prevent cross-
pollination. In depistillated flowers, pistils and anthers
were surgically removed at the bud stage as described
earlier (Salopek-Sondi and others 2000).
In the flowers collected for auxin analysis, flower parts
were separated (sepals, pistils or developing fruit, anthers,
and peduncles). Some almost-ripe fruits were separated
into seeds and pericarps. The plant material was stored at
-80°C until further use.
Endogenous Auxins Analysis
To assess the dynamics of auxins during flower and fruit
development of the Christmas rose, endogenous auxin
levels (precursor IEt, free IAA, and amide conjugates)
were determined in the different tissues, in two growth
seasons, by LC-MS/MS [consisting of a triple-quadrupole
mass spectrometer (Quatro micro API tandem; Waters) and
an Acquity UPLC System (Waters) equipped with a
Symmetry C18 column (Waters)] using
15
N- and/or
2
H
5
-
labeled internal standards, as described previously (Pe
ˇnc
ˇı
´k
and others 2009). The analytical protocol that we used is
suitable for isolation and quantification of IAA and a broad
range of its amino acid conjugates and does not allow
isolation (quantification) of protein conjugates of IAA.
Regarding ester conjugates of IAA, the capability of the
protocol to retain them has not been tested because corre-
sponding standards were not available to us. IAA and
Fig. 1 Development of the Christmas rose (Helleborus niger L.)
through six bud stages at pre-anthesis (a), Female (b), and Male
(c) stages at anthesis, and Light green (d), Advanced green (e), Green
(f), and Almost ripe (g) stages at post-anthesis
J Plant Growth Regul (2012) 31:63–78 65
123
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related indole compounds were obtained from OlChemIm
(Olomouc, Czech Republic).
15
N- and/or
2
H
5
-labeled
internal standards were prepared according to the method
of Ilic
´and others (1997). All reagents and solvents were
purchased from Sigma-Aldrich (St. Louis, MO, USA).
Briefly, the frozen plant tissue [30 mg FW (fresh weight)]
was ground in liquid nitrogen and extracted with 1 ml of
cold phosphate buffer (50 mM, pH 7.0) supplemented with
0.02% Na-diethyldithiocarbamate, and a mixture of stan-
dards ([
2
H
5
]IAA, [
2
H
5
]IEt, [
15
N,
2
H
5
]IAA-Ala, [
15
N,
2
H
5
]
IAA-Asp, [
15
N,
2
H
5
]IAA-Glu, [
15
N,
2
H
5
]IAA-Gly, [
15
N,
2
H
5
]
IAA-Leu, [
15
N,
2
H
5
]IAA-Phe, and [
15
N,
2
H
5
]IAA-Val) was
added to each extract. Following centrifugation (36,000g,
10 min, 4°C), samples were acidified with 1 M HCl to pH 2.7
and applied on a C8 column (Bond Elut, 500 mg, Varian).
After column washing with methanol (10%) and formic acid
(1%), the analyte was eluted with methanol (70%) acidified
with formic acid (1%), and evaporated to dryness. The
extract residue was dissolved in 100 ll of methanol acidified
with concentrated HCl (0.1%), and methylated with 300 ll
of ethereal solution of diazomethane. Following 10 min of
reaction, the mixture was evaporated under a nitrogen stream
at 40°C, the residue was dissolved in 50 ll of ethanol (70%)
and 450 ll of phosphate buffer (20 mM NaH
2
PO
4
,15mM
NaCl, pH 7.2), and the sample was passed through the pre-
column containing immobilized BSA and was applied on an
immunoaffinity column with polyspecific polyclonal anti-
bodies against IAA (that is, antibodies interacting with all the
studied indole compounds). Polyspecific polyclonal anti-
bodies against IAA and its conjugates were obtained by
immunizing rabbits with an IAA-protein conjugate as
described recently by Pe
ˇnc
ˇı
´k and others (2009). As the
authors reported, the capacity and recovery of the columns,
estimated by application of various amounts of indole-3-
acetamide, was about 3 nmol and 95–100%, respectively.
The column was then washed with water (three times
3 ml) and the analyte was eluted with 3 ml of cold meth-
anol (-20°C) and evaporated to dryness. Separation was
performed on an Acquity UPLC System (Waters) equipped
with a Symmetry C18 column (5 lm, 2.1 9150 mm,
Waters) at 30°C and a flow rate of 250 ll/min using the
following linear gradient of 10 mM aqueous formic acid
(solvent A) and methanol containing 10 mM formic acid
(solvent B): 25% solvent B for 1 min, gradient of solvent B
to 38% (7 min), 40% (12 min), 58% (15 min), and 60%
(26 min). The effluent was introduced into the ion source
of the Quatro micro API tandem quadrupole mass spec-
trometer (Waters) under the following conditions: the
capillary voltage, ?500 V; desolvation gas flow, 500 l/h;
desolvation temperature, 350°C; and source block tem-
perature, 100°C. Instrument settings and multiple reaction
monitoring (MRM) transitions of individual compounds
were applied as reported earlier (Pe
ˇnc
ˇı
´k and others 2009).
Photosynthesis Measurement
Sepal greening was monitored in vivo using a Chlorophyll
Content Meter (CCM-200, Opti-Sciences, Inc., Hudson,
NH, USA) which afforded chlorophyll levels in arbitrary
units based on the difference of the absorbances at
600-700 nm (attributed to chlorophyll a?b) and at
900-1000 nm (attributed to light scattering by cell struc-
tures and nonspecific absorbance). Measurements were
done for each sepal separately in its subterminal part.
To gain information about photosynthetic efficacy of
sepals, we performed chlorophyll afluorescence measure-
ments in vivo on the growing flowers. Flowers (intact
controls, unfertilized, and depistillated) were examined in
April when fertilized flowers were at the Green develop-
mental stage. Chlorophyll fluorescence was measured with
a pulse-amplitude-modulated photosynthesis yield analyzer
(Mini Pam, Waltz). Sepals were dark-adapted for 30 min
before measurements. The maximum quantum yield (F
v
/
F
m
) in dark-adapted samples and effective quantum yields
(DF/F
m
0) at 50, 250, and 500 lmol photons m
-2
s
-2
were
determined according to Schreiber and others (1994).
Measurements were done in five repetitions.
Enzyme Assays and H
2
O
2
Measurement
Activities of ascorbate peroxidase (APX; EC 1.11.1.11),
guaiacol peroxidase (GPOD; EC 1.11.1.7), and superoxide
dismutase (SOD; EC 1.15.1.1), as well as hydrogen per-
oxide levels were assayed in sepal extracts at the Green
stage of development. Three replicates were taken from
each flower treatment. The tissue was ground into fine
powder using liquid nitrogen.
The extractions for APX activity were done in ice-cold
100 mM potassium phosphate buffer (pH 7.0) containing
5 mM sodium ascorbate and 1 mM ethylenediamine tetra-
acetic acid (EDTA) with the addition of polyvinylpyrroli-
done (PVP). The reaction mixture contained 50 mM
potassium phosphate buffer (pH 7.0) with 0.1 mM EDTA,
50 mM ascorbic acid, and enzyme extract. The reaction was
started with the addition of 12 mM H
2
O
2
. The APX activity
was measured spectrophotometrically by observing the
decrease in absorbance at 290 nm (Nakano and Asada 1981).
The extractions for GPOD activity were done in ice-cold
100 mM Tris-HCl buffer (pH 8.0). The reaction mixture
consisted of 5 mM guaiacol and 5 mM hydrogen peroxide in
200 mM phosphate buffer (pH 5.8). GPOD activity was
measured at 470 nm as described by Siegel and Galtson
(1967).
The extractions for SOD activity were made in ice-cold
100 mM potassium phosphate buffer (pH 7.5) with the
addition of PVP. SOD activity was assayed by measuring its
ability to inhibit the photochemical reduction of nitroblue
66 J Plant Growth Regul (2012) 31:63–78
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tetrazolium (NBT) as described by Giannopolitis and Ries
(1977). The reaction mixture consisted of 50 mM potas-
sium phosphate buffer (pH 7.5), 13 mM methionine, 75 lM
NBT, 0.1 mM EDTA, 2 lM riboflavin, and enzyme extract.
The reaction mixture, that was not exposed to light, did not
develop color and served as a control. The absorbance was
measured at 560 nm. One unit of SOD activity was defined
as the amount of enzyme required to cause 50% inhibition
of the rate of NBT reduction at 560 nm.
Concentrations of hydrogen peroxide (H
2
O
2
) were
determined according to Mukherjee and Choudhuri (1983).
Sepal tissue was homogenized and extracted with ice-cold
acetone. Following centrifugation, titanium sulfate and
NH
4
OH were added into extracts. Precipitate was dissolved
with 2 M H
2
SO
4
. For the determination of H
2
O
2
levels, the
absorbance was measured at 415 nm and the concentration
was calculated using an extinction coefficient of 1.878/nM/
cm.
Lignin Measurement
Lignins and lignin-like polymers were semiquantitatively
estimated in the plant material as previously described
(Sancho and others 1996). Christmas rose peduncles (1 g
FW) were pealed (to avoid the contribution of anthocy-
anidin-containing peduncle envelope to the spectrophoto-
metric measurement of lignin content), homogenized in
liquid nitrogen, and rinsed in boiling water. The insoluble
material was pelleted by centrifugation and resuspended in
100% ethanol. The dry residue was dissolved in 2.5 ml of
18% (v/v) HCl in ethanol for 2.5 h. After this period,
200 ll of 2.5% phloroglucinol-HCl (Sigma-Aldrich) was
added to the mixture and the absorbance of the final
supernatant was measured after 4 h at 540 nm. The results
were calculated as A
540nm
/g FW and expressed per total
weight of the peduncle.
Histological Study of Flower Peduncle
For histological studies of vascular tissue, small pieces of
peduncles were fixed in 2% glutaraldehyde in 0.05 M
cacodylate buffer (pH 7.2) for 45 min at 4°C, and postfixed
in 1% osmium tetroxide in the same buffer for 1 h at 4°C.
After dehydration in a graded series of ethanol, the tissue
was embedded in Spurr’s resin. Semithin sections (1 lm
thick) of the tissue were stained with a mixture of 2%
toluidine blue and 2% borax (1:1). For lignin examination,
fresh, hand-made sections of peduncles were stained with
1% phloroglucinol in 70% ethanol for 2 min, followed by
treatment with the concentrated HCl (Yuan and others
2007). Samples were examined using an Olympus BX51
light microscope.
Results and Discussion
Flower Development: Pre-anthesis, Anthesis,
and Post-anthesis
The Christmas rose has extraordinarily long-lasting flow-
ers, with the life cycle spanning about 2–6 months,
depending on the weather conditions. Thus, flower devel-
opment may start at any time from late November to mid-
April. The seeds generally mature by May or early June
(Salopek-Sondi and Magnus 2007). Because flower
development is highly dependent on ambient temperature
and could not be defined on an absolute time scale, we
defined the developmental stages herein by morphological
characteristics (Fig. 1). Six pre-anthesis bud stages were
collected in February and categorized according to total
peduncle length (stage I: 1–2.5 cm, stage II: 2.5–4 cm,
stage III: 4–6 cm, stage IV: 6–8 cm, stage V: 8–10 cm, and
stage VI: 10–12 cm). When the bud appeared above
ground it was already 1–1.5 cm long (measured from the
base of the bud to the tip of unopened sepals) and its length
increased slightly while its peduncle elongated up to ten-
fold during investigated pre-anthesis stages (Fig. 1a). Bud
length then increased about twice (up to 3 cm) before
unfolding at anthesis. Flower opening was followed by a
period during which only the pistils were receptive, while
the immature anthers were arranged in a ring at the base of
the carpels (Female phase, Fig. 1b). Depending on ambient
temperatures, the Male phase (Fig. 1c) began, usually
1–2 weeks later, with the elongation of the filaments, and
ended with anther abscission. Following fertilization, the
post-anthesis period of flower development began and was
categorized by the degree of chlorophyll accumulation in
the sepals (which persisted until fruit maturity) as Light
green (the first greenish tinge, Fig. 1d), Advanced green
(greening well advanced, Fig. 1e), Green (sepal greening
reached stationary phase; fruit in the phase of significant
growth and seed filling, Fig. 1f), and Almost ripe (seeds
almost ripe; sepals at the phase when chlorophyll levels
start to decline, Fig. 1g). Anthesis and post-anthesis
developmental stages were described and characterized
also by the weights of the sepals and the pistils or devel-
oping fruit, and by fruit lengths, as published earlier
(Salopek-Sondi and others 2002; Salopek-Sondi and
Magnus 2007). Fruit increased gradually, and its overall
growth proceeded in concert with sepal greening. Fruit
length increased about threefold, while fresh weight
accumulation of the fruit increased six- to sevenfold until
the Almost ripe stage. The peduncle grew significantly in
the pre-anthesis and anthesis periods, after which it reached
its full length (approximately 20 cm) and eventually
increased only slightly during the post-anthesis period.
J Plant Growth Regul (2012) 31:63–78 67
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How Does Emasculation or Depistillation Influence
Flower Development?
The fate of the attractive, white Christmas rose flowers is
strongly influenced by fertilization and developing fruits
(Salopek-Sondi and others 2000,2002; Salopek-Sondi and
Magnus 2007). Development of intact flowers is charac-
terized by processes such as intensive peduncle elongation
at pre-anthesis and anthesis, flower opening and sepal
growth at anthesis, and sepal greening at post-anthesis
during which fruit is developing. In contrast, flowers in
which only anthers were removed and fertilization pre-
vented (unfertilized) or entire reproductive organs were
excised (depistillated) in the bud stage (size of bud was
2–2.5 cm) did not pass through the entire developmental
process. The impact of emasculation or depistillation on
perianth and peduncle development was investigated and is
discussed below.
Impact on Perianth Greening
The greening process of Christmas rose sepals was inves-
tigated previously at the ultrastructural and biochemical
levels through several stages of development (Salopek-
Sondi and others 2000). In our study, chlorophyll accu-
mulation in sepals was monitored in detail through the
entire post-anthesis period of flowers by use of a chloro-
phyll meter (Fig. 2). As can be seen in the sepals of fer-
tilized flowers, the chlorophyll level increased to a value of
approximately 3.5 at the stationary phase (Green stage,
which is also the period of intensive fruit growth). As
development proceeded towards the Almost ripe stage, the
chlorophyll level started to decline. The greening process
of sepals was significantly inhibited upon depistillation or
emasculation. Chlorophyll accumulation in the sepals of
unfertilized and depistillated flowers never reached the
level of fertilized ones, being more impaired by depistil-
lation than by emasculation. Thus, the chlorophyll level in
depistillated flower sepals reached about 70% of the level
identified in fertilized flower sepals, after which chloro-
phyll rapidly declined. The chlorophyll accumulation curve
of unfertilized flowers was similar to that of fertilized ones,
with a significantly lower maximum. To gain data on the
photosynthetic efficacy of Green stage sepals, chlorophyll
fluorescence measurement in vivo was performed. An
effective quantum yield (DF/F0
m
) of photosystem II (PSII)
was calculated for Green stage sepals of fertilized and their
unfertilized and depistillated neighbors (Fig. 3). The
maximum quantum yield (measured in dark adopted plants,
F
v
/F
m
) of unfertilized and depistillated flowers was 76 and
67%, respectively, of the value measured for a fertilized
control. Effective quantum yields of PSII (DF/F0
m
) were
decreased significantly in treated flowers, reaching only
20–30% of the value measured in fertilized flowers.
Because DF/F0
m
corresponds to the proportion of the light
absorbed by chlorophyll associated with PSII (Maxwell
and Johnson 2000), the decrease in DF/F0
m
(Fig. 3) was in
accordance with the values of chlorophyll meter readings
in the sepals of the examined Christmas rose flowers
(Fig. 2).
Based on the visual observation of flowers through
development and on biochemical measurements, including
chlorophyll accumulation (Fig. 2) and PSII efficiency
Fig. 2 Sepal greening dynamics monitored by a chlorophyll meter in
fertilized control (circles), unfertilized (squares), and depistillated
(triangles) flowers during post-anthesis. Data are arithmetic
mean ±SE (n=20–25)
Fig. 3 Photosynthetic efficacy of sepals of fertilized (circles),
unfertilized (squares), and depistillated (triangles) flowers. Measure-
ments were done in vivo on the flowers at Green stage. The maximum
quantum yield (F
v
/F
m
) was determined in dark-adapted samples (at 0
PPFD), and effective quantum yields (DF/F
m
0) were determined at 50,
250, and 500 lmol photons m
-2
s
-2
. Data are arithmetic mean ±SE
(n=5)
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(Fig. 3), it may be that the unfertilized and, especially,
depistillated flowers, also entered the process of senescence
earlier than fertilized ones. Data on the H
2
O
2
level and
activities of antioxidant enzymes, particularly in the sepals
of depistillated flowers, are in agreement with this state-
ment (Fig. 4). The activity of GPOD peroxidase (Fig. 4a)
was decreased in the unfertilized and depistillated flowers,
whereas that of APX was mostly unchanged (data not
shown). The level of H
2
O
2
was elevated significantly in
depistillated flowers compared with that in control and
unfertilized flowers (Fig. 4c). The activity of SOD
(Fig. 4b), which dismutases O
2-
to H
2
O
2
, correlated with
the level of H
2
O
2
found in the investigated sepals. The
levels of lipid peroxidation and protein carbonyls that were
also measured did not show significant changes (data not
shown). An increased level of endogenous H
2
O
2
is reported
to be connected with senescence of the leaf (Niewiadomska
and others 2009) and flower tissue (Panavas and Rubinstein
1998; Kumar and others 2008). The decrease of protective
enzyme activity is usually in accordance with the increase
in H
2
O
2
levels during plant tissue senescence. In our study,
depistillation treatment seems to cause a stronger tissue
oxidation state and leads the flower into the process of
earlier senescence in comparison to fertilized and unfer-
tilized counterparts.
Impact on Peduncle Elongation and Vascular Anatomy
The growth of the peduncle was monitored in intact flowers
and their emasculated and depistillated counterparts
through pre-anthesis, anthesis, and post-anthesis develop-
ment. The most prominent elongation of the peduncle was
observed in the pre-anthesis period (Fig. 1a) when its length
increased up to tenfold (from 1 to 10 cm). Thereafter, the
peduncle additionally elongated about twice during the
anthesis and early post-anthesis periods, reaching approxi-
mately 18-20 cm at the stationary phase (Fig. 5). In the
unfertilized and depistillated flowers, peduncle growth was
stunted throughout their life span and never reached the
length of those of fruit-bearing flowers. Thus, the peduncles
Fig. 4 H
2
O
2
and enzyme assays: guaiacol peroxidase (GPOD) and
superoxide dismutase (SOD) detected in the sepals of fertilized,
unfertilized, and depistillated flowers at the Green stage of post-
anthesis. Data are arithmetic mean ±SE (n=3)
Fig. 5 Peduncle elongation dynamics of fertilized controls (circles),
unfertilized (squares), and depistillated (triangles) flowers. Measure-
ment started at the pre-anthesis when the length of peduncle was
7.6 ±0.267 cm, and the surgical treatments (emasculation and
depistillation) were done in two selected groups, which were therefore
monitored as unfertilized and depistillated flowers, respectively. Data
are arithmetic mean ±SE (n=20–25)
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of unfertilized and depistillated flowers at the stationary
phase reached 83 and 68% of the length of fertilized flower
peduncle, respectively (Fig. 5). A similar effect of flower
stalk growth inhibition upon bud decapitation or emascu-
lation was observed in the miniature Cymbidium orchid
(Ohno and Kako 1991), the tulip (Hanks and Rees 1977;
Saniewski and De Munk 1981), and the daffodil (Edelbluth
and Kaldewey 1976; Hanks and Rees 1977).
In addition to monitoring the dynamics of peduncle
elongation, we also examined the peduncle cross-section
anatomy of fruit-bearing versus depistillated flowers, with
particular attention placed on the vascular apparatus. The
vascular bundles of Helleborus peduncles appeared widely
spaced, collateral, and typically with the V-shaped xylem
concave on the side toward the phloem (representative
shown in Fig. 6f), as usually reported for the herbaceous
plants of the Ranunculaceae family (Metcalfe and Chalk
1972). Looking through the development of the peduncle,
semithin sections of vascular tissue showed phloem and
xylem being gradually fully developed until the last stage
(Fig. 6b, d, f). As flower development proceeded, an inter-
fascicular cambium layer was clearly distinguished between
phloem and xylem. For more detailed analyses, lignin
detection, in a qualitative manner, was employed at the
anatomical level by using the phloroglucinol-HCl (Wiesner)
reaction (Yuan and others 2007), whereas semiquantitative
estimation of lignin was done spectrophotometrically
(Sancho and others 1996). The major change in the vascular
bundle during peduncle growth and development was in
lignin deposition in the sclerenchyma (Fig. 6a, c, e). Fur-
thermore, the sclerenchyma was not even noticeable at the
bud stage of development (Fig. 6a). As perianth greening
occurred and the peduncle elongated, lignin deposition
began, thus forming a sclerenchyma noticeable as a light red
color mostly adjacent to the tip points of vascular tissue
(Fig. 6c). As flower development proceeded toward the final
Almost ripe stage, a very red sclerenchyma was observed
surrounding the vascular bundle (Fig. 6e). In contrast, lignin
deposition in the depistillated flower peduncle was arrested
somewhere at the stage between Light green and Green: only
a faint phloroglucinol-HCl staining is evident close to vas-
cular tissue (Fig. 6g). Furthermore, vascular bundles found
in the peduncle of the depistillated flowers were generally
smaller in comparison to that found in the peduncle of fruit-
bearing flowers. Semiquantitative estimation of total lignins
during peduncle development correlated with anatomical
examination (Fig. 7). The amount of lignin and lignin-like
polymers increased gradually in the peduncle from bud stage
III (in which peduncle length was 4–6 cm and peduncle FW
was 0.69 g) until the Almost ripe stage of intact flowers (in
which peduncle length was approximately 20 cm and
peduncle FW was 3.56 g). Depistillated peduncles at the
Almost ripe stage (peduncle length was approximately
13 cm and peduncle FW was 1.03 g) accumulated signifi-
cantly lower amounts of total lignin, only 19% of the value
measured in intact controls.
The post-anthesis period of the intact flower is marked by
fruit development. It is the period of intensive assimilate
Fig. 6 Vascular cross-section
anatomy of peduncles in the
intact control: bud stage III (a,
b), Light green (c,d), and
Almost ripe (e,f), as well as
depistillated flower at the last
stage of development (Almost
ripe) (g,h). Panels a,c,e, and
gpresent lignin detection (red/
dark) obtained with
phloroglucinol on the fresh,
hand-made, peduncle cross
sections. Panels b,d,f, and
hpresent semithin sections
(1 lm thick) of flower peduncle
stained with a mixture of 2%
toluidine blue and 2% borax
(1:1). Xxylem; Sc
sclerenchyma; Ph phloem; Ic
interfascicular cambium. Scale
bar =100 lm (Color figure
online)
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production in photosynthetically active sepals and leaves
and their transport from ‘‘source’’ to ‘‘sink’’ plant organs.
Due to transport of assimilates and water, there is a strong
demand for a well-developed vascular tissue in the peduncle.
Endogenous Auxin Profile of Intact Flowers During
Development
The auxin profile during development of the Christmas
rose flower, involving pre-anthesis, anthesis, and post-
anthesis, was determined in the different tissues by
LC-MS/MS as described earlier (Pe
ˇnc
ˇı
´k and others 2009).
Free IAA and amide conjugates, as well as a precursor of
IAA biosynthesis, IEt, were identified and quantified.
Indole-3-ethanol (IEt)
IEt or tryptophol has been found as an endogenous
metabolite in many plants as part of IAA metabolism
(Lac
´an and others 1985). It appears to be a reversible side-
branch product in the biosynthesis of indole-3-acetalde-
hyde, the precursor of IAA (Ljung and others 2002;
Quittenden and others 2009). In this study, we identified
IEt mostly in the immature anthers and sporadically in
developing pistils during pre-anthesis development (in the
range of 1-3.5 pmol/g FW) (Fig. 8a), and later in immature
seeds (at the level of 28.0 ±15.3 pmol/g FW, Table 1).
The occurrence of IEt seems to be organ-specific and does
not necessarily follow the level of IAA.
Free IAA Profile
In the pre-anthesis developmental period auxins were
measured in tissues through six bud stages characterized by
intensive peduncle elongation (Fig. 8b). Pistils (gynoe-
cium), followed by anthers, were the major source of free
IAA. There was a slight tendency for the level of free IAA
to decrease toward advanced bud stages. The perianth and
peduncle contained a lower level of free IAA in compari-
son to reproductive organs, with minor fluctuations
(approximately 10 and 15 pmol/g FW, respectively).
Fig. 7 Semiquantitative estimation of lignins and lignin-like poly-
mers in the peduncle of intact control through development from bud
stage III to Almost ripe, as well as in depistillated flower at the last
stage of development (Almost ripe). Data are arithmetic mean ±SE
(n=3)
Fig. 8 Endogenous IAA
analyzed in different floral
tissues and fruits in intact
control flowers during pre-
anthesis (b), anthesis (c), and
post-anthesis (d). IEt has been
identified and quantified only at
pre-anthesis stages (a). Data are
arithmetic mean ±SE (n=2)
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However, the peduncle was intensively elongated at the
observed period (Fig. 1a); peduncle weight was increased
gradually (from 0.3 g at the first bud stage to approxi-
mately 1.5 g at the last bud stage). Thus, the sum of
quantified auxins per peduncle increased approximately
five times during the pre-anthesis period.
At anthesis, anthers, until their abscission at the
advanced male stage, accumulated free IAA up to
400 pmol/g FW (Fig. 8c). Anthers were also reported as a
rich source of auxins in Arabidopsis during flower devel-
opment (Cecchetti and others 2008). It is suggested that
auxin synthesized in the anthers contributes to the regula-
tion of filament elongation and plays a major role in
coordinating anther dehiscence and pollen maturation. In
addition to regulating processes inside male reproductive
tissues, it is also suggested that auxins originating in
anthers may be basipetally transported and may stimulate
peduncle elongation in Cymbidium orchids (Ohno and
Kako 1991).
Post-anthesis was characterized by fruit growth and
development as well as sepal greening. At the post-anthesis
stages, developing fruit became a tremendous source of
auxin, reaching over 50,000 pmol free IAA/g FW at the
last stage (Fig. 8d). Looking into auxin abundance inside
the Almost ripe fruits, approximately 98% of IAA was
present in the seeds and only 2% in pericarps (Table 1). In
accordance with auxin increase in developing fruit, sig-
nificant accumulation of free IAA was noticed in the
peduncle, reaching almost 100 pmol/g FW at the last stage
(Fig. 8d).
Amide Conjugates of IAA
The profile and dynamics of amino acid conjugates of IAA
during reproductive development are shown in Fig. 9. IAA
conjugates with Asp and Glu were the most abundant
amide conjugates in the flower tissues during development,
generally, following the distribution of free IAA. During
pre-anthesis, IAA-Asp and IAA-Glu were found mostly in
the anthers, with peaks at the advanced stages (Fig. 9a).
Pistils at the first two developmental stages contained about
half of the total conjugates found in the anthers, with a
significant decrease toward the advanced stages. Perianth
and peduncle generally contained lower levels of total
amide conjugates in comparison to reproductive organs.
Results showed that abundance of IAA-Asp and IAA-Glu
varied in the various flower parts. Thus, anthers contained
predominantly IAA-Glu over IAA-Asp; pistils and perianth
showed the opposite situation (more IAA-Asp in compar-
ison to IAA-Glu), whereas the peduncle contained only a
low level of IAA-Glu. It was also reported earlier that
distribution of IAA-Asp and IAA-Glu follows the distri-
bution of free IAA during vegetative development of
Table 1 Contents of IAA and amino acid conjugates of IAA identified in seeds and pericarp of ‘Almost ripe’ fruit of the Christmas rose
Fruit tissue Contents of respective auxin compounds (pmol/g FW)
IAA IEt IAA-Asp IAA-Glu IAA-Ala IAA-Gly IAA-Leu IAA-Phe IAA-Val
Seeds 49499 ±2623 28.0 ±15.3 20312 ±66 303 ±15 2.99 ±0.01 2.98 ±0.06 0.383 ±0.097 1.37 ±0.13 0.544 ±0.016
Pericarp 993 ±59 n.d. 205 ±17 5.75 ±0.22 0.244 ±0.077 n.d. n.d. n.d. 0.035 ±0.001
Data are arithmetic mean ±SE (n=2)
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Arabidopsis, although their abundance may vary at the
spatial and temporal base (Kowalczyk and Sandberg 2001).
At anthesis, IAA-Glu and IAA-Asp were found in
anthers predominantly in the Female and early Male stages
until their abscission, which corresponded well with the
IAA level. The conjugates accumulated slightly in the
pistils and perianth, whereas in the peduncle they retained
very low or undetectable levels (Fig. 9b). In addition to
IAA-Glu and IAA-Asp, some novel amide conjugates such
as IAA-Ala and IAA-Val were identified in anthers of the
early Male stage in the amount of 1.489 and 0.217 pmol/g
FW, respectively.
At the post-anthesis stages, developing fruit became a
tremendous source of conjugated auxins, reaching up to
20,800 pmol of amide conjugates per g FW at the last stage
(Fig. 9c, d). The most abundant conjugate in Almost ripe
fruit was IAA-Asp (98% of total conjugates) at the level of
20,500 pmol/g FW, followed by IAA-Glu (about
309 pmol/g FW). In addition to conjugates with Glu and
Asp, amide conjugates with Val, Phe, Leu, Gly, and Ala
were identified in fruits at the last two developmental
stages (Fig. 9d). Alongside IAA amino acid conjugates
described earlier in different plant species (reviewed by
Bajguz and Piotrowska 2009), novel conjugates with Val,
Gly, and Phe were identified (Pe
ˇnc
ˇı
´k and others 2009) and
quantified in the Christmas rose fruits for the first time in
vascular plants. Amide conjugates with Val, Phe, Leu, Gly,
and Ala were accumulated at a lower rate, mostly in the
seeds (from 0.4 to 3 pmol/g FW), and some of them in the
pericarps (Table 1). IAA-Asp was the first detected amide
conjugate of IAA in pea seedlings (Andreae and Good
1955) and was the predominant form in most dicotyle-
donous plants (see for review Ludwig-Mu
¨ller 2011). It has
been postulated that IAA conjugated to Asp (and possibly
Glu) is an irreversible catabolite. So far, IAA-Ala has been
detected in spruce (O
¨stin and others 1992), and IAA-Ala
and IAA-Leu together with IAA-Asp and IAA-Glu have
been found in Arabidopsis (Tam and others 2000;
Kowalczyk and Sandberg 2001). It has been suggested that
other amide conjugates (with Ala, Leu, Gly, Phe, Val),
which are good substrates for auxin amidohydrolases in
vitro (reviewed by Ludwig-Mu
¨ller 2011), participate in
storage and hormone homeostasis as a slow-releasing
source of free IAA (Hangarter and Good 1981). Seeds of
many species are reported as rich in amide conjugates
(Cohen and Bandurski 1982; Bialek and Cohen 1989;
Ljung and others 2002), which are the main source of
active auxins in the period of germination until young
seedlings are capable of setting up their own de novo auxin
biosynthesis (Rampey and others 2004).
In accordance with the increase in auxin in developing
fruit, significant accumulation of free IAA was noticed in
the flower peduncle as post-anthesis development pro-
ceeded (Fig. 8d), although we did not detect a significant
Fig. 9 Endogenous amide
conjugates of IAA in different
floral tissue of fertilized flowers
during reproductive
development. IAA-Asp and
IAA-Glu, the most abundant
conjugates, are shown at pre-
anthesis (a), anthesis (b), and
post-anthesis (c), whereas other
amide conjugates are detected at
post-anthesis (d). Data are
arithmetic mean (n=2). SEs
were in the same range as for
the data shown on Fig. 8
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level of conjugates (about 90 pmol/g FW of free IAA, and
just about 2.4 pmol/g FW of total amide conjugates). It is
interesting that the peduncle contained almost 40 times
more free IAA than amide conjugates, whereas that ratio
was significantly lower in the perianth (4.3) and the fruit
(2.4). It is an intriguing question: What is the purpose of
such a high level of free IAA in the peduncle during post-
anthesis development? Post-anthesis is a period of inten-
sive production of assimilates and fruit filling, considering
water and assimilate transport through the peduncle.
Results of vascular cross-section anatomy during flower
development (Figs. 6,7) in accordance with the data of
auxin analyses indicate that IAA may be involved in the
development and maintenance of the peduncle vascular
system, necessary for successful transport of water and
assimilates, particularly at post-anthesis. Because other
factors such as cytokinins and gibberellins are known to
be involved in peduncle vascularization (Aloni and others
1990; Wolbang and others 2004; Dettmer and others
2009), additional experiments that will shed light on the
auxin role in these processes will be performed in the
future.
Endogenous Auxin Profile in Unfertilized
and Depistillated Flowers
The fate of the Christmas rose flowers, with special
attention paid to the perianth and the flower peduncle, upon
emasculation or depistillation has been described earlier
(Salopek-Sondi and Magnus 2007) and investigated in
more detail in our study. To investigate the impact of
removal of floral parts (anthers or complete reproductive
organs) on the auxin profile, we measured endogenous
auxins in unfertilized and/or depistillated flowers and
compared them with intact controls of the same age.
Firstly, we compared auxins in flowers at the 5th, 7th, and
10th day after depistillation with corresponding controls.
At those time points, control flowers were at the Female to
Male stages of development with a corresponding peduncle
elongation of approximately 3.5 cm (from 11.3 to 14.8 cm)
and a double increase of peduncle FW (from 1.48 to
3.72 g). At the same time, peduncles of depistillated
flowers were slightly elongated (from 7.9 to 8.2 cm with
FW increase from 1.40 to 1.54 g). As can be seen in
Fig. 10, free IAA was significantly accumulated in the
Fig. 10 Free IAA and amide
conjugates measured in tissues
of fertilized and depistillated
flowers at the 5th (a,b), 7th
(c,d) and 10th (e,f) day upon
depistillation. Data are
arithmetic mean ±SE (n=2).
SEs on the right panels were in
the same range as for the data
shown on the left panels
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anthers and the pistils in fertilized controls. IAA was also
increased in the perianth, although it was decreased in the
peduncle. However, because the peduncle was intensively
growing at the observed period, total free IAA per peduncle
was about 45 pmol during that period, without noticeable
fluctuations. The amount of free IAA in the perianth and
peduncle of depistillated flowers reached about 50% of that
identified in the control. Accumulation of total IAA amide
conjugates followed IAA dynamics in examined flower
parts. Thus, anthers accumulated mostly IAA-Glu, then
IAA-Asp, and low amounts of IAA-Ala and IAA-Val at the
10th day of investigation. Other flower parts such as pistils
and perianth accumulated mostly IAA-Asp at earlier
stages, with an increase in IAA-Glu toward advanced
stages. The perianth of the depistillated flower accumulated
lower amounts of total conjugates than controls, which is in
accordance with the amount of free IAA. Not only the
amount but the pattern of amide conjugates appeared to be
different in depistillated flowers (mostly IAA-Asp has
accumulated in depistillated perianth).
Finally, a comparison of the auxin profile in unfertilized,
depistillated, and intact control flowers was done at the
Green’ stage of development (Fig. 11). Analyzed auxin
(free and amino acid conjugates) was significantly lower in
most of the examined tissues of unfertilized and depistil-
lated flowers, respectively, in comparison to the fertilized
ones. The exception was the perianth of unfertilized
flowers, which contained higher levels of conjugates than
that of fertilized controls. Unfertilized pistils contained
only 0.06% of the total auxin detected in the developing
fruit of the Green flower. The peduncle of unfertilized and
depistillated flowers contained only 12 and 4% of total
auxin, respectively, whereas the perianth contained 84.7
and 45.2% of that found in the fruit-bearing ones. These
results imply that the perianth is capable of synthesizing
more than 50% of its own auxin, independent of the
presence of developing fruits. In contrast, the auxin pool in
the peduncle is mostly dependent on the existence of
developing fruit. It may be hypothesized that more than
90% of auxin in the peduncle is transported from the
developing fruit. This may be supported by the results of
Nishio and others (2010) who suggested that auxin is
transported from young seeds during tomato fruit devel-
opment by SlPIN1 and SlPIN2 and is accumulated in the
peduncles. However, this suggestion needs to be confirmed
in the Helleborus model by future experiments.
Conclusion
Among different plant hormones involved in the regulation
of reproductive plant development, auxins trigger the
flower and the fruit developmental programs (Cheng and
Zhao 2007; Alabadi and others 2009) and participate in the
coordination of processes within and between floral organs
(Sundberg and Østergaard 2009). We investigated the
auxin profile and dynamics as well as their possible coor-
dinating role in the reproductive development of the
Christmas rose (Helleborus niger L.) through pre-anthesis,
anthesis, and post-anthesis. In addition to free IAA, IEt and
seven amino acid conjugates of IAA were identified and
quantified. IEt was accumulated in immature anthers and
sporadically in pistils during pre-anthesis and in develop-
ing seeds during post-anthesis. It probably serves as a pool
for indole-3-acetaldehyde, the immediate precursor for
IAA synthesis in the period of intensive development of
reproductive organs and seeds. In addition to amide con-
jugates described earlier in many plant species (IAA-Asp,
IAA-Glu, IAA-Ala, IAA-Leu) (Bajguz and Piotrowska
2009), some novel conjugates of IAA with Val, Gly, and
Phe were identified and quantified in anthers and devel-
oping seeds according to the recently established method
(Pe
ˇnc
ˇı
´k and others 2009). Although the existence of IAA-
Val, and IAA-Phe as endogenous plant conjugates has been
strongly suggested by Kai and others (2007) based on the
identification of their oxidative metabolites in Arabidopsis,
Fig. 11 Comparison of free IAA (a) and amide conjugates (b)in
floral tissues of fertilized, unfertilized, and depistillated flowers at the
Green developmental stage. Data are arithmetic mean ±SE (n=2).
SEs on the panel bwere in the same range as for data shown on the
panel a
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they are confirmed in the Christmas rose. Recently, IAA-
Val has been found in moss (Physcomitrella patens) grown
on medium supplemented with IAA (Ludwig-Mu
¨ller and
others 2009).
Results of our research showed that reproductive organs,
pistils and anthers before fertilization, and then developing
fruit at post-anthesis are rich sources of auxin. Based on the
data obtained in unfertilized and depistillated flowers, one
may conclude that the peduncle is dependent on mostly
auxin supplied from other flower parts. Upon emasculation
or depistillation at the early bud stage, the auxin content of
the peduncle decreased to 50% at anthesis and then to 4%
at post-anthesis development in comparison to intact con-
trols. During pre-anthesis and anthesis, when the peduncle
is significantly elongated, auxin necessary for peduncle
elongation seems to be supplied mostly by the anthers at
pre-anthesis and anthesis, until their abscission. It is in
agreement with results obtained on Cymbidium flowers
(Ohno and Kako 1991), which suggested that the peduncle
is supplied mostly by auxins from the developing flower
buds, especially from the anthers. Furthermore, selective
removal of the floral parts in tulip and narcissus showed
that gynoecium controls stem extension (Hanks and Rees
1977).
In the post-anthesis period developing fruits, mostly
seeds, become tremendously rich in free and amide-con-
jugated IAA, causing the increase of free IAA in the
peduncle. This is supported by the results of the expression
analysis of the auxin efflux carriers in tomato during
reproductive development (Nishio and others 2010). The
authors suggested that auxin is likely transported from
young seeds by SlPIN1 and SlPIN2 and accumulated in the
peduncles. One of the possible roles of auxin transported
from young seeds to peduncles during tomato fruit devel-
opment is the prevention of abscission of fertilized flowers
and young developing fruits. In Helleborus plants, unfer-
tilized or depistillated flowers remain attached to the
mother plant although sepal greening and peduncle elon-
gation were impaired. Investigation of the vascular cross-
section anatomy of the peduncle showed a noticeable
deposition of lignin in the sclerenchyma surrounding vas-
cular bundles in the peduncle of fertilized controls, whereas
this process was arrested in the ‘‘auxin-poor’’ peduncle of
depistillated flowers. It was shown earlier that the lignifi-
cation level was markedly reduced in a uro ‘‘soft stem’
Arabidopsis mutant in which auxin content and signaling
were found to be abnormal (Guo and others 2004; Yuan
and others 2007). Although auxin is the primary signaling
compound necessary for regulation of lignification and
overall vascular tissue development, tight crosstalk with
other plant hormones such as cytokinins and gibberellins is
required (Aloni and others 1990; Aloni 2004; Dettmer and
others 2009). Our previous research demonstrated that
cytokinins (Salopek-Sondi and others 2002; Tarkowski and
others 2006) and gibberellins (Ayele and others 2010), in
addition to the herein investigated auxin, are highly
involved in flower and fruit development of the Christmas
rose. Although cytokinins are mostly responsible for pro-
moting sepal greening, gibberellins are shown to be
involved in the greening process, but, also in coordination
with auxin, in the regulation of peduncle elongation.
Therefore, tight crosstalk between these and probably other
plant hormones coordinates flower and fruit development
of this ornamental plant. Details of the mechanism of the
hormonal crosstalk during the reproductive development of
the Christmas rose, with the particular role in peduncle
elongation and vascular development, need to be eluci-
dated in the future work.
Acknowledgment This manuscript is dedicated to Dr. Sc. Volker
Magnus, who was involved in the research of Christmas rose devel-
opment for many years. This work was supported by research grants
no. 098-0982913-2829, 098-0982913-2838, 073-0731674-0841, and
073-0731674-1673 (Croatian Ministry of Science, Education and
Sports), by grant MSM6198959216 (Ministry of Education, Youth
and Sports of the Czech Republic) and by grant KAN200380801
(Academy of Sciences of the Czech Republic). We thank the staff of
Pharmaceutical Botanical Garden ‘‘Fran Kus
ˇan’’ who made their
Christmas rose collections available for our experiments, Dr. Sc.
Kroata Hazler-Pilepic
´for constructive discussions connected to vas-
cular development, and Dr. Sc. Marija Mary Sopta for critical reading
of manuscript.
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... This example demonstrates the importance of selecting appropriate analytical methods for auxin analysis which will give the most credible results. In recent years, for auxin analysis more sensitive and selective liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) analysis has been used [34,39,48,53,54]. This is the best currently available analytical technology that is capable of determining, besides free IAA, its auxin precursors, conjugates (reversible/irreversible), and IAA catabolites. ...
... Recently modern analytical approaches for unambiguous conjugate identification and quantification have been developed. Usually they include the use of an internal standard, immunoaffinity extraction [53,54], and the use of modern analytical technology based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), which is capable of determining both IAA and its amino acid conjugates [45]. ...
Chapter
As sessile organisms, plants are often exposed to unfavorable environmental conditions (abiotic stress). Considerable losses in crop productivity all over the world are caused by abiotic stresses such as water, temperature, and salinity stresses. Phytohormones are crucial for the ability of plants to adapt to abiotic stress by mediating a wide range of adaptive responses such as modification of photosynthesis, increased antioxidant activities, secondary metabolites accumulation, changes in protein profile and activity, as well as gene expression. In recent years emerging evidence suggests that phytohormone auxin acts as a common player in the majority of hormonal interactions in stress conditions. Auxin, ROS, and antioxidants, such as ascorbate, GSH, and related proteins, have been proposed to form a redox-signaling module that links plant development with environmental cues. Research on a variety of plant species demonstrated that auxin homeostasis directly links growth regulation with stress adaptation responses. Here, we summarize the recent findings on the auxin as a part of complex signaling network. We discuss on how plants utilize auxin signaling and transport to modify growth plasticity when responding to diverse abiotic stresses such as temperature, water stress and increased salinity. Furthermore, we focus on the importance of the crosstalk between auxin and stress hormones, such as abscisic acid, jasmonate, salicylic acid, and ethylene, in stress responses and potential adaptation.
... The increase in chlorophyll a and b during flower development was significant not only for H. niger, but also for H. odorus. An increase in chlorophyll after fertilisation was also shown in the study of Brcko et al. [34], while in the ripe stage, the concentrations of chlorophyll gradually declined. This way, the photosynthetic capacity of sepals and other green parts of the flowers contributes to the process of seed ripening. ...
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Helleborus niger is an evergreen species, while H. odorus is an herbaceous understorey species. They both develop flowers before the forest canopy layer closes. Their sepals remain after flowering and have multiple biological functions. To further elucidate the functions of sepals during flower development, we examined their optical and chemical properties, and the photochemical efficiency of photosystem II in the developing, flowering, and fruiting flowers. Sepals of the two species differed significantly in the contents of photosynthetic pigments and anthocyanins, but less in the UV-absorbing substances’ contents. Significant differences in photosynthetic pigment contents were also revealed within different developmental phases. The sepal potential photochemical efficiency of photosystem II was high in all developmental phases in H. odorus, whereas in H. niger, it was initially low and later increased. In the green H. odorus sepals, we obtained typical green leaf spectra with peaks in the green and NIR regions, and a low reflectance and transmittance in the UV region. On the other hand, in the white H. niger sepals in the developing and flowering phases, the response was relatively constant along the visible and NIR regions. Pigment profiles, especially chlorophylls, were shown to be important in shaping sepal optical properties, which confirms their role in light harvesting. All significant parameters together accounted for 44% and 34% of the reflectance and transmittance spectra variability, respectively. These results may contribute to the selection of Helleborus species and to a greater understanding of the ecological diversity of understorey plants in the forests.
... Auxin is also effective in the growth and seed development of oilseed crops, subsequently increasing the production of oils from seeds. Among different plant hormones that play a role in regulating reproductive plant growth, auxins trigger flower and fruit development programs that are closely related to flower and fruit development [84]. Villacorta et al. [85] reported that IAA is the most abundant plant hormone and is associated with both vegetative and reproductive development in hop (Humulus lupulus) plants. ...
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... YUCCA regulates the initiation of floral meristems and lateral organs during vegetative and reproductive development (Gallavotti et al., 2008). The tissues of unfertilized and depistillated flowers significantly accumulated with lower levels of auxin (Brcko et al., 2012). Several studies have reported an increase in the auxin concentration in the ovary after GA treatment (Sastry and Muir, 1963;Niu et al., 2014). ...
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Vrste roda Helleborus se u ukrasnoj hortikulturi koriste kao vrtne biljke, lončanice za primjenu u vanjskom prostoru, kao vrste za uređenje interijera, a sve se više traže kao cvjetna vrsta za rez tijekom zime. Cilj ovog rada je opisati osnovne morfološke značajke i uvjete uzgoja vrsta roda Helleborus te dati pregled asortimana vrsta i kultivara kasnojesenske i zimske cvatnje. Pregledom literature utvrđeno je da Helleborus niger L., crni kukurijek, na tržištu postaje sve traženija ukrasna biljka, a prate ga i križanci između vrste H. orientalis i drugih vrsta ovoga roda (H. × hybridus). Iako tržišno slabo zastupljene u ukrasnoj hortikulturi se primjenjuju i druge vrste (H. viridis, H. foetidus, H. purpurascens, H. tibethanus, H. vesicarius te H. multifidus) te sve veći broj kultivara. Za cvatnju početkom zime, posebno u vrijeme Božića, najpoznatija je vrsta Helleborus niger L., a sve se više komercijaliziraju i međuvrsni križanci. Pri tome osobit značaj imaju križanci između vrsta H. niger, H. argutifolius Viv. i H. lividus Aiton. Razmnožavanje je moguće sjemenom, dijeljenjem te kulturom tkiva. Uzgaja se u uzgojnim posudama 12- 14 cm promjera u koje se sadi od 50. do 18. tjedna. Potreban mu je supstrat pH od 5,8 do 6,0, ne podnosi visoku razinu soli u tlu, a za prodaju početkom zime uzgaja se u zaštićenim prostorima od sredine listopada. Osjetljiv je na visoku vlagu, koja uz visoke temperature potencira pojavu bolesti (Fusarium, Pythium i Phytophtora).
Article
The effect of gibberellic acid (GA) on retarding loss of persimmon firmness and fruit coloration has been previously reported. Nevertheless, information about the effect of this treatment on calyx senescence is lacking. In this study, chlorophyll fluorescence imaging (CFI) was used to evaluate calyx senescence in persimmon fruit treated with GA. At three harvest times, physico-chemical parameters measured on persimmon fruit and CFI parameters (Fo, Fm and Fv/Fm) on calyx sepals were evaluated on the fruit treated once or twice with GA, and also on untreated fruit (CTL). A decline in the chlorophyll fluorescence parameters correlated with calyx senescence and progressed during fruit ripening. Spatial images heterogeneity in the Fm/Fv measurements illustrates senescence and necrosis dynamics, which began in the apical area of sepals and progressed to the basal area. Besides retarding fruit ripening, GA treatments delayed the calyx senescence process, and hence improved external fruit quality maintenance. The CFI parameters measured on the calyx correlated with both external color evolution and firmness loss during fruit ripening. Consequently, these chlorophyll fluorescence parameters could act as a potential non-intrusive tool to determine persimmon harvesting time.
Article
Covering: up to 2018 Tryptophol (indole-3-ethanol) is a metabolite produced by plants, bacteria, fungi and sponges. This review reports on the natural occurrence, bioactivity and various synthetic approaches to the preparation of tryptophol and its derivatives. Syntheses of various naturally occurring tryptophol derivatives known for their enhanced pharmacological profile are also presented.
Article
The rachis, the structural framework of the grapevine (Vitis vinifera L.) inflorescence (and subsequent bunch), consists of a main axis and one or more orders of lateral branches with the flower-bearing pedicels at their fine tips. The rachis is crucial both for support, and transport from the shoot. Earlier suggestions that the flowers per se affect normal rachis development are investigated further in this study. Different percentages (0, 25, 50, 75 or 100) of flowers were removed manually one week before anthesis on field-grown vines. Treatment effects on subsequent rachis development (curvature, vitality, anatomy, starch deposit) were assessed. Sections, both fixed and embedded, and fresh hand-cut were observed by fluorescence and bright-field optics after appropriate staining. Emphasis was on measurement of changes in cross-sectional area of secondary xylem and phloem, and on maturation of fibres and periderm. Specific defects in rachis development were dependent on the percent and location of flower removal one week prior to anthesis. The rachises curved inwards where most of the flowers were removed. When fully de-flowered, they became progressively necrotic from the laterals back to the primary axes and from the distal to the proximal end of those axes, with a concurrent disorganisation of their anatomy. A few remaining groups of flowers prevented desiccation and abscission of the rachis axes proximal to the group, but not distally. Flower removal (50%) reduced rachis elongation, while 75% removal reduced xylem and phloem area and delayed phloem fibre and periderm development. 75% flower removal did not affect starch present in the rachis during berry development. Developing flowers affect the growth and vitality of the rachis and the development of its vascular and support structures. The extent of these effects depends on the cultivar and the number and position of flowers remaining after some are removed one week before anthesis.
Article
Chlorophyll fluorescence analysis has become one of the most powerful and widely used techniques available to plant physiologists and ecophysiologists. This review aims to provide an introduction for the novice into the methodology and applications of chlorophyll fluorescence. After a brief introduction into the theoretical background of the technique, the methodology and some of the technical pitfalls that can be encountered are explained. A selection of examples is then used to illustrate the types of information that fluorescence can provide.
Article
The geotropic response of a poppy flower stalk was studied and the following results were obtained. • After formation, the stalk first grows upright showing negative geotropic behavior, then positive behavior by growing downward, and finally after about 10 days negative behavior by standing upright followed by the opening of the flower. • The bending zone of the stalk showing negative geotropic curvature moves acropetally from the base as the stalk ages, and finally the whole stalk acquires negative geotropic behavior. • Curvature occurs due to enhanced elongation of the convex (upper) side of the stalk compared with that of the concave (lower) side, and this appears to be due to differential rates of cell elongation between the upper and lower sides. The epidermal cell wall was loosened more in the upper side according to stress-relaxation analysis. • When the curvature disappears and the stalk grows upright again, the concave (lower) side of the zones of curvature elongates faster than the convex (upper) side. • If the flower bud is decapitated, the stalk quickly becomes straight, standing upright. However, if a lanolin paste containing IAA (1 mg/plant) is applied to the cut end, the growth and movements proceed in a manner similar to that of the control plants having flower buds. On the other hand, with GA (3 mg/plant) application, the curvature is not retained although elongation of the decapitated stalk is restored more than 50%.
Article
The role of oxidative stress during petal senescence in rose was investigated. Two cut-rose (Rosa hybrida L.) cultivars, 'Grand Gala' and 'First Red' were obtained from a commercial grower. Petals were harvested from seven different whorls, outermost-to-innermost in flowers of both cultivars. H 2O2 production was determined throughout flower bud senescence, and the H2O2-scavenging enzyme system was studied. The activities of catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APOD) increased up to Stage-5 of flower bud development (petals completely unfolded) and declined thereafter. The highest level of glutathione reductase (GR) activity was observed at Stage-4 (sepals completely opened, petal starting to unfold), followed by a significant decrease at later stages, coincident with higher levels of ethylene production. This limits the role of APOD from Stage-4 onward and is responsible for the failure of free radical scavenging in rose petals. In this context, the role of POD becomes important in protecting the flower from free radicals. Thus, an increase in endogenous H2O2 levels and a decrease in anti-oxidant enzyme activities may be partly responsible for initiating senescence in rose petals.
Article
Kernels of lea mays on an intact plant accumulate indole-3-acetic acid (IAA) at the rate of 190 ng g(-1) fresh weight h(-1). Of the IAA synthesized, 97% is in the esterified form and less than 3% remains as the free acid. The site of biosynthesis of the IAA, whether synthesized in the leaf and transported to the kernel, or in the kernel and remaining in the kernel, has not been established. In an attempt to determine the locus of synthesis, we grew isolated kernels on agar media not containing tryptophan or other possible aromatic precursors of IAA and observed IAA synthesis of 99 ng g(-1) fresh weight h(-1), approximately 52% of the in situ rate. Thus, the kernel contains all of the enzymes required for de novo aromatic biosynthesis of IAA and its ester conjugates. Furthermore, endosperm cells in suspension culture, grown on hormone-free media and in the absence of aromatic precursors, are able to synthesize IAA at a rate of 9.2 ng g(-1) fresh weight h(-1), or 4.8% of the in situ rate. This finding establishes that all of the enzymes of IAA biosynthesis occur in the endosperm and that the endosperm is a site of IAA biosynthesis. Isolated endosperm, prepared from developing kernels, synthesized IAA from labeled anthranilate at a rate of 8.6 ng g(-1) fresh weight h(-1), or 4.5% of the in situ rate. Frozen endosperm preparations maintained the ability to synthesize labeled IAA from labeled anthranilate. The identity of the synthesized IAA was established by mass spectral analysis. We suggest that endosperm preparations of Z. mays are suitable for study of the mechanism(s) of IAA biosynthesis because they (a) have high rates of synthesis; (b) show stability to freezing, enabling enzyme storage; (c) provide a system with a known rate of in situ synthesis; and (d) are available in large amounts for use as an enzyme source.
Article
Diffusates from flower buds, flower fruits, and scape segments, and extracts of flower stalks of Narcissus pseudonarcissus contain an auxin active in the Avena geo-curvature test. The auxin behaved like indole-3-acetic acid (IAA) in thin-layer chromatography (TLC) with neutral and basic solvents on different adsorbents. After TLC, the auxin of the extracts showed chromogenic reactions identical with those of IAA; in gas-liquid chromatography on two different columns, the purified substance, after methylation, appeared at the retention time of IAA methyl ester. The auxin content of the extracts has been estimated to be equivalent to ca. 10 μg IAA kg(-1) fresh weight. Diffusates, collected at the basal end of excised flowering apices and of scape segments at different developmental stages, showed highest auxin activity when collected from old buds and young flowers, and from the basal, rapidly elongating scape regions. The diffusible auxin obtained from scape segments was very likely produced by the segments themselves. Thus, the shoot of Narcissus appears to possess two different sites of auxin production, namely, the apical region represented by the flower bud, the flower or the fruit, and the scape.
Article
Abstract Plant secondary growth is of tremendous importance, not only for plant growth and development but also for economic usefulness. Secondary tissues such as xylem and phloem are the conducting tissues in plant vascular systems, essentially for water and nutrient transport, respectively. On the other hand, products of plant secondary growth are important raw materials and renewable sources of energy. Although advances have been recently made towards describing molecular mechanisms that regulate secondary growth, the genetic control for this process is not yet fully understood. Secondary cell wall formation in plants shares some common mechanisms with other plant secondary growth processes. Thus, studies on the secondary cell wall formation using Arabidopsis may help to understand the regulatory mechanisms for plant secondary growth. We previously reported phenotypic characterizations of an Arabidopsis semi-dominant mutant, upright rosette (uro), which is defective in secondary cell wall growth and has an unusually soft stem. Here, we show that lignification in the secondary cell wall in uro is aberrant by analyzing hypocotyl and stem. We also show genome-wide expression profiles of uro seedlings, using the Affymetrix GeneChip that contains approximately 24 000 Arabidopsis genes. Genes identified with altered expression levels include those that function in plant hormone biosynthesis and signaling, cell division and plant secondary tissue growth. These results provide useful information for further characterizations of the regulatory network in plant secondary cell wall formation.