Tree Physiology 33, 1099–1110
doi:10.1093/treephys/tpt082
Research paper
Changes in ATP, glucose-6-phosphate and NAD(P)H cellular
levels during the proliferation and maturation phases of Abies
alba Mill. embryogenic cultures
Jana Krajňáková1,2,5, Alberto Bertolini2, Laura Zoratti2,3, Dušan Gömöry4, Hely Häggman3
and Angelo Vianello2
1Faculty
of Forestry and Wood Technology, Mendel University, Zemědělská 3, 613 00 Czech Republic; 2Department of Agriculture and Environmental Science, University of
Udine, Via delle Scienze 91, Udine 33100, Italy; 3Department of Biology, University of Oulu, PO Box 3000, FI-90014 Oulu, Finland; 4Faculty of Forestry, Technical University
Zvolen, T. G. Masaryka 24, Zvolen SK-960 53, Slovakia; 5Corresponding author ([email protected])
Received May 30, 2013; accepted August 22, 2013; published online November 6, 2013; handling Editor Ron Sederoff
The aim of the present study was to evaluate the adenosine triphospate (ATP), glucose-6-phospate (glu-6P) and reduced form
of nicotinamide adenine dinucleotide phosphate (NAD(P)H) cellular levels during the proliferation and maturation phases of
Abies alba Mill. somatic embryos. For a better understanding of the dynamics of these parameters during the proliferation cycle,
four embryonic cell lines were tested. During the maturation period, three independent experiments were conducted, focused
on the effects of PEG-4000 (5 or 10% (w/v)) and abscisic acid (16, 32 or 64 µM) applied together (Experiments A and B) or with
addition of gibberellic acid (Experiment C) on the dynamics of bio-energetic molecules and on the mean number of cotyledonary
somatic embryos. Our results demonstrated that the cellular levels of bio-energetic molecules strongly depended on the composition of maturation media. Generally, the higher the number of cotyledonary embryos produced, the higher the level of ATP
observed after a 2-week maturation period. The cellular level of ATP, glu-6P and NAD(P)H increased, particularly after the transition from the proliferation to the maturation phase when the differentiation and growth of somatic embryos occurred.
Keywords: abscisic acid, bio-energetic molecules, gibberellic acid, maturation, polyethylene glycol, silver fir.
Introduction
In eukaryotes, cellular energy is provided by adenosine triphospate (ATP), the ubiquitous energy currency of living cells
(Skulachev 2006, Geigenberger et al. 2010). Adenosine triphospate is produced mainly in mitochondria via oxidative
phosphorylation and, in photosynthesizing organisms, in chloroplasts via photo-oxidative phosphorylation (Fernie et al.
2004, Plaxton and Podestà 2006, Wilhelm and Selmar 2011).
The cellular energy state, which is defined by the ATP : adenosine diphosphate (ADP) ratio, the ATP phosphorylation potential
and the adenylate energy charge (AEC) (Atkinson 1977), can
fall to low levels under conditions that either lead to an
increased demand for ATP or that impair the ATP-generating
processes, such as starvation or hypoxia. In microbial and
mammalian cells, only two components of the adenylate pool,
ATP and ADP, are involved in energy metabolism (Ataullakhanov
and Vitvitsky 2002). In plants, the situation is less clear,
because phosphofructokinase and glycolysis, in general, are
not affected by ATP, and there are several non-phosphorylating
bypasses of mitochondrial electron transport that are not subject to feedback regulation by the adenylate energy state
(Plaxton and Podestà 2006, Geigenberger et al. 2010, Wilhelm
and Selmar 2011). The lack of adenylate control of respiration
enables plants to oxidize the reduced form of nicotinamide
adenine dinucleotide phosphate (NAD(P)H) produced in
© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
1100 Krajňáková et al.
v­ arious biosynthetic reactions or during photosynthesis independent of the energy state, preventing over-reduction of the
electron transport chain and generation of harmful oxygen
radicals (Plaxton and Podestà 2006, Geigenberger et al. 2010,
Wilhelm and Selmar 2011).
In mammalian biology, the ATP cellular level is a valuable
determinant of cell death. The cells stay alive as long as a certain ATP level is maintained. When the ATP falls below this level,
programmed cell death (PCD) ensues, provided enough ATP is
still available for the energy-requiring PCD processes. Only
when there is a severe drop in cellular ATP, controlled cell death
ceases and ushers in necrosis (Richter et al. 1996, Tsujimoto
1997, Lemasters 1999, Skulachev 2006). A similar response
was also observed in soya bean suspension cell cultures
(Casolo et al. 2005, Vianello et al. 2007), where even glucose6-phospate (glu-6P) remained high when PCD was induced by
hydrogen peroxide or nitric oxide. Conversely, in the early
stages of necrotic death, the concentration of ATP and glu-6P
decreased significantly (Casolo et al. 2005). Different levels of
ATP could force cell death through apoptosis or necrosis in
mammals (Tsujimoto 1997, Lemasters 1999, Skulachev 2006).
Adenosine triphospate and other nucleoside triphosphates
not only drive energy-dependent reactions inside a cell, but
can also function in the extracellular matrix, where they function as antagonists that can induce diverse physiological
responses without being hydrolysed (Roux and Steinebrunner
2007). In plants, extracellular ATP (eATP, found in extracellular
matrix) has a role in stress and wound responses (Jeter et al.
2004, Song et al. 2006, Chivasa et al. 2009), pathogen
response (Chivasa et al. 2009) and in growth (Tang et al.
2003, Wu et al. 2007). The physiological source of eATP has
not yet been clarified, and might involve ATP-release by
wounded cells, ATP transport or ATP release by the secretory
pathway (Roux and Steinebrunner 2007, Clark and Roux 2009,
Geigenberger et al. 2010, Tanaka et al. 2010).
Cellular ATP concentration or AEC has been used in many different physiological studies to investigate the responses of
plants to stress conditions such as: anoxia (Igamberdiev and
Kleczkowski 2011, Edwards et al. 2012); high-frequency, lowamplitude electromagnetic field (Roux et al. 2008); disease
development (Yi et al. 2008); seed development and viability
(Borisjuk et al. 2003, Rolletschek et al., 2003, 2004, Kibinza
et al. 2006, Pasquini et al. 2012); organ senescence (Azad et al.
2008); and morphogenesis (Murch et al. 1997). Also, some
stress conditions, related to in vitro cultivation, e.g., hyperhydricity (Saher et al. 2005), temporary immersion (Martre et al.
2001) and fusion of mitochondria in tobacco suspension cultures (Wakamatsu et al. 2010), have been assessed using bioenergetic molecules (ATP and ATP : ADP ratio).
Somatic embryogenesis (SE), the process by which somatic
cells differentiate into somatic embryos, became a model for
studying different developmental, molecular and biochemical
Tree Physiology Volume 33, 2013
processes (von Arnold et al. 2002, Stasolla and Yeung 2003,
Cairney and Pullman 2007). In particular, the SE of Picea abies
L. Karst. was used to study embryology in conifers, providing a
well-characterized sequence of developmental stages, which
can be synchronized by specific treatments, making it possible
to collect a large number of somatic embryos at specific developmental stages (Bozhkov et al. 2002, 2005, von Arnold
2008, Vestman et al. 2011). Despite the fact that the SE of
Abies species has not reached a high level of synchronization
and is still far from representing a model system, it has been
recently used in investigations related to energy metabolism. In
our first study, Petrussa et al. (2008), a method of isolation
and purification of mitochondria from embryogenic cell masses
(ECMs) of two conifers (P. abies and Abies cephalonica Loud.)
is described. The mitochondrial bio-energetics linked to PCD
during SE of Abies alba Mill. was studied in Petrussa et al.
(2009). Fulvic acid affected the proliferation and maturation
phases in A. cephalonica embryogenic cultures, based on ATP
and glu-6P analyses (Zancani et al. 2011). We have also
­examined the same bio-energetic parameters (ATP and glu6P) ­during cryopreservation and recovery of A. cephalonica
embryogenic cell lines (Krajňáková et al. 2011).
In conifer zygotic embryos, the nutrients and energy are partly
provided by the megagametophyte, which is lacking in somatic
embryos. The aim of the present work was to evaluate the ATP,
glu-6P and NAD(P)H cellular levels during the proliferation and
maturation phases of A. alba SE. For a better understanding of
the dynamics of the bio-energetic molecules during the proliferation and maturation phases, the proliferation rate, proportion of
pro-embryogenic cell aggregates, as well as the number and
quality of developing somatic embryos were monitored.
Materials and methods
Plant material
Embryogenic cultures of A. alba, silver fir, were initiated as
described by Krajňáková et al. (2013). Proliferating embryogenic cultures were cultivated on medium composed of halfstrength Murashige and Skoog (MS) medium with full-strength
vitamins (Murashige and Skoog 1962), supplemented with
58 mM sucrose, 4.44 µM benzyl adenine, 0.1% (w/v) casein
hydrolysate, 3.4 mM l-glutamine (filter sterilized and added
into the cooled media) and solidified with 0.3% (w/v) gellan
gum (Phytagel™, Sigma, St. Louis, MO, USA). The pH of the
medium was adjusted to 5.7. The proliferating cultures were
subcultured every 2 weeks and maintained at 24 °C in the
dark. Four embryogenic cell lines (A1, A3, A4/ST4 and B2),
derived from cryopreserved stock (Krajňáková et al. 2013),
were cultured under standard conditions for 3 months prior to
the onset of the experiments.
For determining the proliferation rates (Wi/W0), the embryogenic cell lines (A1, A3, A4/ST4 and B2) were grown on the
ATP, glucose-6 phosphate and NAD(P)H during somatic embryogenesis 1101
MS proliferation medium. For each cell line, five ECM pieces
were weighed (200 ± 25 mg fresh weight (FW), W0) and
placed equidistant from each other on each Petri dish. After a
period of proliferation, ECMs were picked up and re-weighed
(Wi), and the ratio Wi/W0 was used as a measure of the proliferation rate. Sampling was done after 3, 7, 14 and 21 days.
The experiment was carried out in three replicates.
The different developmental pro-ECMs (PEMs, at stages I, II
and III) and early somatic embryos, as described by Filonova
et al. (2000) for P. abies, by Petrussa et al. (2009) for A. alba
and by Zancani et al. (2011) for A. cephalonica, were monitored using a double staining technique with acetocarmine and
Evan’s blue (Gupta and Holmstrom 2005) on each sampling
day. The morphological evaluation of cell aggregates was
based on at least 500 individual determinations.
From this step onwards, the cell line A4/ST4 was selected
based on its good maturation ability (Krajňáková et al. 2013).
Cultures were transferred to a maturation medium 2 weeks
after the last proliferation sub-culture. At the beginning of the
maturation, 4 g of fresh ECMs were transferred to sterile Falcon
flasks with 20 ml of liquid proliferation medium without cytokinin. A suspension was created by vortexing and allowed to
settle. After removal of the supernatant, 1 ml of suspension
containing ~250 mg of ECMs (FW) was plated onto sterile filter paper (Whatman No. 2, diameter 7 cm) on the MS-based
maturation medium. The basic maturation medium composed
of half-strength MS (Murashige and Skoog 1962) supplemented with 0.05% (w/v) casein hydrolysate and 1.7 mM
l-glutamine, pH 5.7. The medium was solidified with 0.25%
(w/v) gellan gum (Phytagel™) and supplemented with
83.3 mM maltose. This basic medium was supplemented with
either 5 or 10% (w/v) polyethylene glycol (PEG-4000), and
three different concentrations (16, 32 and 64 µM) of (±)
abscisic acid (ABA) were tested in the presence or in the
absence of 10 µM gibberellic acid (GA3). Eight different maturation media divided into three independent experiments were
used: Experiment A (EXP A), Experiment B (EXP B) and
Experiment C (EXP C) (Table 1). Sub-culturing was done every
2 weeks and maturation took up to 12 weeks. Cultures were
monitored at the time of sub-culturing when the sampling for
bio-energetic molecules took place.
Cellular ATP, glu-6P and NAD(P)H measurement
For the analysis of the cellular metabolites, ECMs at different
developmental stages (sampling days or maturation weeks for
proliferation and maturation phase, respectively) were first frozen with liquid N2, then ground to a fine powder, re-suspended
in 1 ml of 50 mM Tris–HCl (pH 7.5) and 0.05% (w/v) Triton
X-100, and immediately boiled for 2 min. After a brief centrifugation, aliquots of supernatant were used for the following assays.
Adenosine triphospate, glu-6P and NAD(P)H contents of
ECM were determined by means of the luciferin–luciferase
luminometric assay, reduction of β-NADP+ catalysed by glu-6P
dehydrogenase and the total NAD(P)H pool was determined
by spectrophotometry, respectively, as described by Petrussa
et al. (2009). For each experiment, a separate calibration curve
was performed for ATP, glu-6P and NAD(P)H against known
concentrations of these molecules. The value of each measured observation was then calculated by intrapolation from
the calibrated curves.
The actual values were expressed as nmoles ATP, glu-6P or
NAD(P)H per gram FW.
Experimental design and data analysis
Four embryogenic cell lines (A1, A3, A4/ST4 and B2) were
tested during the proliferation period (four sampling days: 3, 7,
14 and 21), according to their proliferation activities, formation
of different morphological aggregates and determination of
cellular levels of ATP, glu-6P and NAD(P)H (nmol g−1 FW) in
three independent repetitions. Proliferation rates (Wi/W0) were
analysed by two-way analysis of variance (ANOVA; both effects
of cell line and sampling day were considered fixed), pairwise
contrasts were tested using Duncan’s tests. The GLM (SAS
2009) procedure was used.
Table 1. ​Design of maturation experiments and composition of maturation media.
Experiment
Maturation media
Maturation week
PEG 4000 (%) (w/v)
ABA, μM
GA3, μM
A
A1
A2
A3
A4
B1
1–12
1–12
1–12
1–12
1–6
6–12
1–4
4–12
1–6
6–12
1–6
6–12
5
5
10
10
10
0
10
0
10
10
10
10
16
32
16
32
64
32
64
32
64
32
64
32
0
0
0
0
0
0
0
0
50
50
0
50
B
B2
C
C1
C2
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1102 Krajňáková et al.
The effects of cell line and sampling date on the ATP, glu-6P
and NAD(P)H contents were assessed by two-way ANOVA. In
the case of NAD(P)H, only two cell lines were monitored (A1
and A4/ST4). The GLM (SAS 2009) procedure was used for
the calculations.
The embryogenic cell line A4/ST4 was used in three independent maturation experiments (EXP A, EXP B, EXP C) and
the numbers of Petri dishes tested were between 10 and 12
per maturation medium; the total number of different maturation
media was eight (Table 1). Each maturation experiment was
repeated twice.
In EXP A, the effect of two different PEG-4000 concentrations (5 or 10%) (w/v), applied together with 16 or 32 µM
ABA (Table 1, maturation media A1, A2, A3 and A4), on the
response of ECMs was studied; the cellular levels of ATP, glu6P, NAD(P)H were determined and the number of developing
somatic embryos was counted.
In EXP B, the effect of 10% (w/v) PEG-4000 and 64 µM
ABA applied either for 6 or 4 weeks (Table 1, maturation media
B1 and B2) during the maturation phase on the response of
ECMs was studied. In EXP C, the effect of 10 µM GA3 either
during the whole maturation period or during the last 6 weeks
of maturation (Table 1, maturation media C1and C2) on the
response of ECMs was examined.
Maturing ECMs were monitored six times, at 2-week intervals,
using the material from three independent Petri dishes, and the
data of cellular levels of ATP, glu-6P and NAD(P)H were analysed.
Three-way ANOVA (effects of PEG, ABA and maturation period
were all considered fixed) was used for the evaluation of EXP A,
whereas two-way ANOVA (with fixed effects of maturation
medium and maturation period) was used for EXP B and EXP C,
with subsequent Duncan’s tests for pairwise comparisons.
After the 12 weeks of maturation, the presence of developing
somatic embryos in the ECMs was also documented. Somatic
embryos at different developmental stages (early precotyledonary, early cotyledonary and cotyledonary) were counted and
recalculated per 1 g of FW of ECMs, whereby early cotyledonary and cotyledonary embryos were pooled into one category.
Differences in the average numbers of somatic embryos
between experimental variants were tested using non-parametric
Kruskal–Wallis tests. The frequencies of precotyledonary and
cotyledonary embryos were calculated and modelled as linear
response functions using weighted least-squares analysis (procedure CATMOD) (SAS 2009).
Results
ATP, glu-6P, NAD(P)H cellular levels during
proliferation phase
Cell lines and sampling days significantly affected the cellular
levels of ATP and glu-6P (in the case of ATP, the interaction
term was also significant, Table 2). The NAD(P)H cellular levels
were significantly affected only by the sampling day. It was
mainly the cell line A1, which showed consistent and significant
superiority in the levels of cellular ATP and glu-6P over the
other cell lines (Figure 1A and B).
Figure 1. Cellular levels of ATP (A) and glu-6P (B) in different embryogenic cell lines of A. alba. Columns with different letters are significantly different (Duncan’s test; P < 0.05).
Table 2. ​Analysis of variance of the effects of A. alba embryogenic cell lines (A1, A3, A4/ST4 and B2), sampling days (3, 7, 14 and 21) on the
­cellular levels of ATP, glu-6P and NAD(P)H and on the proliferation rate.
Source
Cellular levels of
ATP
Cell line
Sampling day
Cell line × sampling day
Error
Proliferation rate
Glu-6P
NAD(P)Ha
DF
F value
DF
F value
DF
F value
DF
F value
3
3
9
74
14.43***
11.04***
2.45**
3
3
9
75
13.09***
4.73***
0.75 NS
1
3
3
26
0.29 NS
3.57**
0.02 NS
3
3
9
508
32.77***
704.96***
8.14***
aOnly
cell lines A1 and A4/ST4 were assessed.
Significance labels (used also in subsequent tables): ***P < 0.001, **P < 0.01, *P < 0.05, NS P ≥ 0.05 (non-significant). DF, degrees of freedom.
Tree Physiology Volume 33, 2013
ATP, glucose-6 phosphate and NAD(P)H during somatic embryogenesis 1103
The levels of ATP and glu-6P were relatively stable among the
first three sampling days (Figure 2A and B). The reduced form of
nicotinamide adenine dinucleotide phosphate levels reached the
highest value at day 7 (26.4 nmol g−1 FW) (Figure 2C). A drop
of all biochemical parameters was visible after a 21-day-long
proliferation.
Proliferation rates (analysed by ANOVA) among embryogenic cell lines of A. alba during the four sampling days showed
that both cell line and, as expected, the sampling day significantly affected the proliferation rate (Table 2). The interaction
term was also significant, indicating that the temporal course of
the proliferation rate differed between cell lines (see Figure S1
available as Supplementary Data at Tree Physiology Online).
A longer lag phase was observed with cell line B2 after transfer to the fresh proliferation medium, but after 21 days of cultivation, the proliferation rate reached the highest level compared
with the other cell lines. The cell line A4/ST4 demonstrated
good proliferation growth during the whole testing period (see
Figure S1 available as Supplementary Data at Tree Physiology
Online). Proliferation of cell line A1 slowed down after 14 days.
The percentage composition of PEMs (at stage I, II and III) and
early somatic embryos were determined at four sequential
sampling days during the proliferation cycle (see Figure S2
available as Supplementary Data at Tree Physiology Online).
With increasing duration of proliferation, the percentage of
PEM I decreased (being 15.4 and 6.6% in sampling days 3
and 21, respectively) and formation of PEM III and SE increased
(20.4% of PEM III and 8.92 of SE in sampling day 3 and 30.8%
of PEM III and 17.3% of SE in sampling day 21). Interestingly,
cell lines A1 and B2 formed a high percentage of PEM II stages
(62.2 and 59.2, respectively) and, very rarely, morphological
structures described as early SE (the percentage of early SE
was only 1.0 and 0.57 for cell lines A1 and B2, respectively)
(data not shown).
ATP, glu-6P, NAD(P)H cellular levels during
the maturation phase
In the different experiments, the ECMs of embryogenic cell line
A4/ST4 were transferred to maturation media at the end of the
proliferation cycle (sampling day 14), and their levels of ATP,
glu-6P and NAD(P)H at that time were found to be 0.76, 55.4
and 22.1 nmol g−1 FW, respectively. These values are used as
base values against which all measurements in the subsequent
maturation experiments are assessed.
Experiment A
In EXP A, cellular ATP, glu-6P and NAD(P)H levels were determined in ECMs of A. alba exposed to four maturation media
(A1, A2, A3, A4) after a 12-week maturation period. Among the
tested factors (maturation period, ABA and PEG concentrations), only the duration of the maturation period significantly
affected the cellular levels of ATP (Table 3). In contrast, the
levels of glu-6P were affected by the maturation period, PEG
and ABA concentrations, while the effects of ABA concentration and ABA × PEG combination also depended on the maturation period (Table 3). Interestingly, even the maturation
period did not significantly affect the cellular levels of NAD(P)H
(Table 3). In ECMs cultivated on maturation media s­ upplemented
Table 3. ​Analysis of variance of the effects of maturation period (2, 4,
6, 8, 10 and 12 weeks), ABA concentrations (16 and 32 µM) and
concentrations of PEG (5 and 10%) on the average cellular levels of
ATP, glu-6P and NAD(P)H determined in ECMs of A. alba (cell line A4/
ST4) in EXP A during maturation period.
Source
Figure 2. Cellular levels of ATP (A), gluc-6P (B) and NADP(H) (C) in A.
alba embryogenic cell lines during the proliferation cycle. Columns with
different letters are significantly different (Duncan’s test; P < 0.05).
Maturation period
PEG
ABA
PEG × ABA
PEG × maturation period
ABA × maturation period
PEG × ABA × maturation
period
Error DF
DF
5
1
1
1
5
5
5
Cellular levels of
ATP
Glu-6P
NAD(P)Ha
F-test
F-test
F-test
32.99***
2.08 NS
0.69 NS
0.0 NS
1.68 NS
0.42 NS
0.82 NS
17.68***
6.11**
4.22**
0.03 NS
1.93 NS
2.39*
2.43*
0.37 NS
186
174
26
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1104 Krajňáková et al.
with higher ABA concentration (32 µM ABA), significantly
higher levels of glu-6P (107.1 nmol g−1 FW) were detected
than in ECMs cultivated on media with 16 µM ABA (glu-6P was
92.2 nmol g−1 FW).
Pairwise contrasts for cellular levels of ATP and glu-6P
determined in ECMs of A. alba during six sequential maturation
periods are presented in Figure 3A and B. Within the first 6
weeks of maturation, the levels of ATP and glu-6P were low
(0.56–0.69 nmol g−1 FW for ATP and 81.9–84.5 nmol g−1 FW
for glu-6P). At the eighth week, there was a significant increase
in the ATP level, reaching 0.97 nmol g−1 FW. This value was
even significantly higher at 10 weeks and was 1.38 nmol g−1
FW. The increasing trend, after 6 weeks of maturation, was
even more evident for glu-6P, where at eigh weeks of maturation 140.8 nmol g−1 of glu-6P was recorded. At 12 weeks, both
ATP and glu-6P had decreased. Cellular NAD(P)H levels
Figure 3. Cellular levels of ATP (A) and glu-6P (B) in A. alba ECMs during the maturation period (2, 4, 6, 8, 10 and 12 weeks), EXP A.
Columns with different letters are significantly different (Duncan’s test;
P < 0.05).
decreased from 50.01 to 29.64 nmol g−1 FW of ECMs between
maturation weeks 4 and 12, respectively.
After the first 6 weeks on maturation media, the first early
precotyledonary embryos developed (see Figure S3a available as Supplementary Data at Tree Physiology Online).
Subsequently, early cotyledonary embryos were observed
(see Figure S3b available as Supplementary Data at Tree
Physiology Online) and after 10–12 weeks on maturation
medium, the cotyledonary embryos became pre-dominant
(see Figure S3c available as Supplementary Data at Tree
Physiology Online). The mean number of somatic embryos per
gram FW of ECMs and the relative proportion of developed
early precotyledonary and cotyledonary somatic embryos are
presented in Table S1 available as Supplementary Data at Tree
Physiology Online (EXP A). The differences in the mean numbers of early precotyledonary somatic embryos among the
tested maturation media (A1, A2, A3 and A4) were not significant (Kruskal–Wallis test). On the other hand, the tested media
significantly differed for the mean number of cotyledonary
embryos. Generally, a higher proportion of cotyledonary
embryos was found on media containing 10% (w/v) of PEG4000 (see Table S1 available as Supplementary Data at Tree
Physiology Online, EXP A). This was also confirmed by the
weighted least-squares analysis of frequencies, i.e., only PEG
concentrations significantly affected the proportion of early
precotyledonary and cotyledonary somatic embryos (see
Table S2 available as Supplementary Data at Tree Physiology
Online, EXP A).
Experiment B
In EXP B, the effect of two maturation media (B1 and B2,
Table 1) on the cellular levels of ATP and glu-6P was studied.
Both media, B1 and B2, were supplemented with 10% (w/v)
PEG-4000 and 64 µM ABA. The PEG supplement was omitted
from the medium and the ABA concentration was reduced to
32 µM either after 6 weeks (medium B1) or after 4 weeks
(medium B2) maturation period. Among the tested factors, the
duration of the maturation period, maturation medium and
interactions between them significantly affected the cellular
Table 4. ​Analysis of variance of the effects of maturation period (2, 4, 6, 8, 10 and 12 weeks) and maturation media (B1 and B2, EXP B, and C1
and C2, EXP C) on the average cellular levels of ATP and glu-6P determined in ECMs of A. alba during maturation period.
Source
Maturation period
Maturation medium
Medium × period
Error DF
DF
5
1
5
Tree Physiology Volume 33, 2013
Exp B
Exp C
Cellular levels of
Cellular levels of
ATP
Glu-6P
ATP
Glu-6P
F-test
F-test
F-test
F-test
20.34***
11.80**
7.87***
35
5.05**
0.24 NS
3.12*
34
34.26***
3.06 NS
1.39 NS
33
1.95 NS
17.89***
1.22 NS
33
ATP, glucose-6 phosphate and NAD(P)H during somatic embryogenesis 1105
levels of ATP (Table 4). In contrast, the levels of glu-6P were
affected by the maturation period and interaction maturation
period × medium (Table 4).
Cellular levels of ATP determined in ECMs maturing on
medium B1 (1.99 nmol g−1 FW) were significantly higher than
the levels of ATP detected in the tissues on B2 medium
(1.63 nmol g−1 FW) (Figure 4A). Tissues on B1 and B2 media
did not differ in cellular levels of glu-6P (Figure 4B) and
reached a value of 199.12 nmol g−1 FW. Throughout the whole
maturation period, the levels of ATP and glu-6P were low only
during the first 2 weeks of maturation (0.77 nmol g−1 FW for
ATP and 151.32 nmol g−1 FW for glu-6P) (Figure 5A and B).
Between the fourth and 10th week of maturation, the ATP level
varied between 1.68 and 2.06 nmol g−1 FW. At the 12th week,
the ATP level increased significantly, reaching 2.45 nmol g−1
Figure 4. Cellular levels of ATP (A) and glu-6P (B) in A. alba ECMs
during maturation in different maturation media of EXP B. Columns
with different letters are significantly different (Duncan’s test;
P < 0.05).
Figure 5. Cellular levels of ATP (A) and glu-6P (B) in A. alba ECMs
during the maturation period (2, 4, 6, 8, 10 and 12 weeks), EXP B.
Columns with different letters are significantly different (Duncan’s test;
P < 0.05).
FW (Figure 5A). Cellular levels of glu-6P showed two significant peaks at 4 and 12 weeks (Figure 5B).
The mean number of somatic embryos per gram FW of ECMs
and the relative proportion of developed early precotyledonary
and ­cotyledonary somatic embryos are presented in Table S1
available as Supplementary Data at Tree Physiology Online,
EXP B. The differences in the mean numbers of cotyledonary
somatic embryos and the sum of all embryos between maturation media B1 and B2 were significant (Kruskal–Wallis test, see
Table S1 available as Supplementary Data at Tree Physiology
Online, EXP B). The highest mean number of cotyledonary
embryos and the significantly higher proportion of precotyledonary and cotyledonary embryos (see Tables S1 and S2 available as Supplementary Data at Tree Physiology Online, EXP B)
were obtained on maturation medium B1, supplemented with
10% (w/v) PEG-4000 and 64 µM ABA for 6 weeks.
Experiment C
In EXP C, the effect of 10 µM GA3 on the cellular level of ATP
and glu-6P was studied. GA3 was applied either during the
whole maturation period (maturation medium C1, Table 1) or
only from the sixth week until the end of maturation (medium
C2, Table 1). Of the tested factors (maturation period and
medium), only the duration of the maturation period significantly
increased the cellular levels of ATP and the level of glu-6P was
affected significantly only by the maturation medium (Table 4).
Cellular levels of ATP in ECMs maturing on medium C2
(2.17 nmol g−1 FW) were significantly higher than the levels of
ATP detected on C1 medium (1.63 nmol g−1 FW) (Figure 6A).
Interestingly, the cellular content of glu-6P was significantly
higher on C1 medium (212.51 nmol g−1 FW) than on C2
medium (146.27 nmol g−1 FW) (Figure 6B). Within the whole
maturation, the levels of ATP and glu-6P were low only during
the first 2 weeks of maturation (0.77 nmol g−1 FW for ATP and
135.88 nmol g−1 FW for glu-6P) (Figure 7A and B). The ATP
level remained relatively stable between the fourth and 10th
week of maturation (from 2.12 to 2.48 nmol g−1 FW). At the
12th week, the ATP content increased significantly, ­reaching
2.86 nmol g−1 FW (Figure 7A). Cellular levels of g
­ lu-6P were
the highest at the second week of maturation (Figure 7B).
Figure 6. Cellular levels of ATP (A) and glu-6P (B) in A. alba ECMs during maturation in different maturation media of EXP C. Columns with
different letters are significantly different (Duncan’s test; P < 0.05).
Tree Physiology Online at http://www.treephys.oxfordjournals.org
1106 Krajňáková et al.
Figure 7. Cellular levels of ATP (A) and glu-6P (B) in A. alba ECMs during
the maturation period (2, 4, 6, 8, 10 and 12 weeks), EXP C. Columns
with different letters are significantly different (Duncan’s test; P < 0.05).
The mean number of somatic embryos per gram FW of ECMs
and the relative proportion of early precotyledonary and cotyledonary somatic embryos are presented in Table S1 available as
Supplementary Data at Tree Physiology Online (EXP C). The differences in the mean numbers of early precotyledonary, cotyledonary and all somatic embryos on C1 and C2 maturation
media were statistically significant (Kruskal–Wallis test, see
Table S1 available as Supplementary Data at Tree Physiology
Online, EXP C). The highest mean number of cotyledonary
embryos and the highest proportion of cotyledonary somatic
embryos were obtained on maturation medium C2 (see
Table S1 available as Supplementary Data at Tree Physiology
Online, EXP C). The latter also confirmed, by weighted leastsquares analysis, that the levels of glu-6P were significantly
affected only by the maturation medium (see Table S2 available
as Supplementary Data at Tree Physiology Online, EXP C).
Discussion
In the present study, we have used the SE developmental pathway of A. alba to study the cellular levels of bio-energetic molecules (ATP, glu-6 and NAD(P)H), and their dynamics during
the proliferation and maturation phases. Similar results were
obtained in our earlier studies with A. cephalonica (Krajňáková
et al. 2011, Zancani et al. 2011).
ATP and glu-6P cellular levels during the
proliferation phase
It is well known that the genetic background of embryogenic
cell lines affects the success of SE (von Arnold et al. 2002,
Tree Physiology Volume 33, 2013
Bonga et al. 2010). We found differences in the cellular levels
of ATP, glu-6P, NAD(P)H and growth rate among tested
embryogenic cell lines of A. alba during the proliferation phase.
The cell line A1 reached the highest values for ATP and for glu6P. However, this embryogenic cell line did not have the highest proliferation rate among the tested lines and did not
produce somatic embryos on any maturation medium tested by
Krajňáková et al. (2013). A variety (genotype)-dependent
reduction in the ATP cellular content was also observed by
Saher et al. (2005), who tested four varieties of carnation
shoots for hyperhydricity.
In accordance with our previous data dealing with the proliferation phase of A. cephalonica (Zancani et al. 2011), relatively
stable levels of ATP and glu-6P were found during the first
sampling days, but the decrease in ATP and glu-6P after
21 days of growth was less profound in the present study with
A. alba compared with A. cephalonica.
Although data on the cellular levels of ATP and glu-6P in
embryogenic tissues are still rare in the literature, we can compare our data with those of Belmonte et al. (2003) and Ashihara
et al. (2000), who performed a series of tracer experiments
with labelled inosine, adenine and adenosine, intermediates of
purine metabolism during proliferation and maturation of white
spruce (Picea glauca (Moench) Voss.) somatic embryos. They
showed that during embryo proliferation in liquid medium de
novo synthesis, salvage synthesis and degradation pathways
were operative (Ashihara et al. 2000, Belmonte et al. 2003).
These authors concluded that de novo synthesis of purine
nucleotides may be required to increase the endogenous ADP
and ATP pool during the exponential growth phase of white
spruce embryogenic cells. The present data with A. alba and
our previous data (Zancani et al. 2011) with A. cephalonica
related to cellular ATP and glu-6P levels are in accordance with
the results of Belmonte et al. (2003). According to Zancani
et al. (2011), the high levels of cellular ATP and glu-6P during
the proliferation phase indicate a high requirement for energy
for the initial growth. The general decrease in ATP and glu-6P,
after 3 weeks of growth, might be due to decreasing microand/or macro-nutrients in the medium or due to the increase in
ethylene in the airspace above the culture media (George and
de Klerk 2008, Thorpe et al. 2008).
Increased concentrations of ABA and PEG strongly
affect the cellular ATP and glu-6P levels
A combined application of ABA and PEG (high molecular mass
osmoticum) is a routine method to induce somatic embryo maturation in some genera of Coniferales (Bozhkov and von Arnold
1998, von Arnold et al. 2002, Stasolla and Yeung 2003),
including A. alba (Hristoforoglu et al. 1995, Vooková and
Kormut’ák 2009, Krajňáková et al. 2013). In the present study,
the induction of embryo development was triggered by transferring ECMs to maturation media supplemented with d
­ ifferent
ATP, glucose-6 phosphate and NAD(P)H during somatic embryogenesis 1107
concentrations of PEG-4000, ABA and GA3 applied together for
a certain time or throughout the maturation period (Table 1).
The dynamics of ATP and glu-6P cellular levels during the
whole maturation period, observed in EXP A, are in agreement
with our previous findings with A. cephalonica (Zancani et al.
2011), except for the decreased ATP level at the end of the
maturation period. Also, the highest mean number of cotyledonary somatic embryos per 1 g FW of ECM observed in this
study (maturation medium A4) corresponds to the values previously achieved with A. cephalonica (Zancani et al. 2011).
In EXP B and C, where the higher concentrations of PEG4000 and ABA were tested, the measured ATP and glu-6P
levels were ~40–44% higher than the averaged values of ATP
in EXP A. The levels of ATP and glu-6P were low only during
the first 2 weeks of maturation (Figure 7A and B) and were
similar to the value at the end of the proliferation period. The
tested components of the maturation media (EXP B and EXP
C) were able to: (i) increase the levels of ATP and glu-6P (averaged values) significantly above those attained in EXP A; (ii)
increase the glu-6P level within the first 2 weeks of matur­ation
threefold above the level attained at the end of proliferation.
Embryo development and histo-differentiation are biological
processes accompanied by profound alternations in purine and
pyrimidine nucleotide biosynthesis (Stasolla and Thorpe
2004). Studies of Ashihara et al. (2001a, b), Stasolla et al.
(2001a, b, c) with P. glauca and Belmonte et al. (2011) with
Brasica napus SE revealed that fluctuations in nucleotide synthesis and degradation occur during morphogenesis, including
embryogenesis. Studies on purine salvage during white spruce
somatic embryos development have shown that both adenine
and adenosine were easily salvaged and utilized for ATP and
nucleic acid synthesis during all stages of embryo development
(Ashihara et al. 2001a, b). Alterations in salvage activity may
represent a metabolic switch, which terminates proliferation
and initiates embryonic development (Stasolla and Yeung
2003). Another plausible interpretation of the data (Stasolla
and Thorpe 2004) is that a reduction in salvage activity,
observed at the onset of embryo development, may not be a
true reflection of the actual rate of purine salvage in different
tissue types of the embryo. With the formation of new tissues,
in fact, it cannot be excluded that high salvage activity may still
be present, but restricted to the specific regions or tissues of
the developing embryos where cell division is occurring.
In the present study, the highest cellular levels of ATP and glu6P were observed in tissues on media C2 and B1, where also
the highest mean number of somatic embryos was observed.
The difference between these two media was only the addition
of GA3 to the C2 medium. The role of gibberellins during SE was
reviewed by Jimenez (2005), Moshkov et al. (2008) and Rose
et al. (2010). The effect of exogenously applied gibberellins
(GAs) on induction of SE is highly variable and depends on the
species, tissues or endogenous levels of GAs (Jimenez and
Bangerth 2001). Somatic embryogenesis is stimulated by exogenous gibberellic acid (GA3), for example in Medicago sativa L.
tissue cultures (Rudus et al. 2002), several rose cultivars
(Kintzios et al. 1999, Li et al. 2002), Gossypium species (Sun
et al. 2006), Cocos nucifera (L.) (Montero-Cortes et al. 2010)
and Magnolia obovata Thunb. (Park et al. 2012). However, it
appears that, for many species, SE is inhibited by exogenous
GAs, for example, in Arabidopsis (Ezura and Harberd 1995), linseed (da Cunha and Ferreira 1997), Geranium (Hutchinson et al.
1997), Centaurium erythraea Gillib. (Subotic et al. 2009) and
wheat (Miroshnichenko et al. 2009). With coniferous SE, Pullman
et al. (2005) found an improvement in the initiation of SE in
several conifers using paclobutrazol (an inhibitor of gibberellins
synthesis).
There is evidence that a transcriptional factor LEAFY
COTYLEDON (LEC) can induce SE independently of hormones
and that LEC can affect the balance of ABA to GA during the
totipotency and maturation phase (Braybrook and Harada 2008,
Rose et al. 2010). Vestman et al. (2011) studied the global
changes in gene expression during early stages of somatic
embryo development of P. abies. These authors recognized the
changes in the expression of genes involved in regulating auxin
biosynthesis and the auxin response, gibberellin-mediated signalling, signalling between the embryo and the female gametophyte,
tissue specification including the formation of boundary regions,
and the switch from embryogenic to vegetative development.
Our work shows the importance of the timing of exogenous gibberellin application during the maturation phase. In media C1,
10 µM GA3 was added at the beginning of the maturation phase,
and despite the high cellular level of ATP and glu-6P, a negative
effect of this plant hormone on the development of somatic
embryos was observed. On this medium, the mean number of
cotyledonary somatic embryos per 1 g FW ECMs was reduced to
1.7. In comparison, on medium C2 to which GA3 was added only
for the last 6 weeks of maturation, 44.5 cotyledonary somatic
embryos were produced per 1 g FW ECMs.
Elhiti et al. (2011), working with transgenic cells of Brassica
napus (cv. Topas DH4079), reported that the increase in embryo
number and improvement in quality affected by the over-­
expression of SHOOTMERISTEMLESS (STM) was connected to
increased pyrimidine and purine salvage activity during the early
phases of embryogenesis and to the enlargement of the adenylate pool (ATP + ADP). Our results demonstrated a strong effect
of the maturation media on the level of bio-energetic molecules.
Generally, the higher the number of cotyledonary embryos produced, the higher the level of ATP observed after a 2-week-long
maturation period. The main bio-energetic molecules (ATP, glu6P and NAD(P)H) increased, particularly after the transition
from the proliferation to the maturation phase when the differentiation and growth of somatic embryos occurred.
The current methods to monitor cellular ATP do not provide
spatial or temporal localization of ATP in single cells in r­ eal-time,
Tree Physiology Online at http://www.treephys.oxfordjournals.org
1108 Krajňáková et al.
or they display imperfect specificity to ATP (Ando et al. 2012,
Zadran et al. 2013). However, recently developed biosensors
for mammalian cells allow visualization of ATP in real time
(Imamura et al. 2009, Zadran et al. 2013). This technology
could also help plant scientists to better understand the role of
bio-energetic molecules at the single-cell or in small-cell aggregates. For these kinds of studies, the plant embryogenic cell
cultures and development of somatic embryos might be an
excellent platform.
Supplementary data
Supplementary data for this article are available at Tree
Physiology Online.
Acknowledgments
Dr Jan Bonga is acknowledged for his valuable comments on
manuscript.
Funding
The research was funded by the University of Udine, Department
of Agricultural and Environmental Science, Italy and by SoMoPro
programme, financed by the European Community within the
Seventh Framework Programme (FP/2007-2013) under Grant
Agreement No. 229603 and was co-financed by the South
Moravian Region.
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