The Level of Free Intracellular Zinc Mediates
Programmed Cell Death/Cell Survival Decisions in
Plant Embryos1[W]
Andreas Helmersson, Sara von Arnold, and Peter V. Bozhkov*
Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University
of Agricultural Sciences, SE–750 07 Uppsala, Sweden
Zinc is a potent regulator of programmed cell death (PCD) in animals. While certain, cell-type-specific concentrations of
intracellular free zinc are required to protect cells from death, zinc depletion commits cells to death in diverse systems. As in
animals, PCD has a fundamental role in plant biology, but its molecular regulation is poorly understood. In particular, the
involvement of zinc in the control of plant PCD remains unknown. Here, we used somatic embryos of Norway spruce (Picea
abies) to investigate the role of zinc in developmental PCD, which is crucial for correct embryonic patterning. Staining of the
early embryos with zinc-specific molecular probes (Zinquin-ethyl-ester and Dansylaminoethyl-cyclen) has revealed high
accumulation of zinc in the proliferating cells of the embryonal masses and abrupt decrease of zinc content in the dying
terminally differentiated suspensor cells. Exposure of early embryos to a membrane-permeable zinc chelator N,N,N#,N#tetrakis(2-pyridylmethyl)ethylenediamine led to embryonic lethality, as it induced ectopic cell death affecting embryonal
masses. This cell death involved the loss of plasma membrane integrity, metacaspase-like proteolytic activity, and nuclear DNA
fragmentation. To verify the anti-cell death effect of zinc, we incubated early embryos with increased concentrations of zinc
sulfate. Zinc supplementation inhibited developmental PCD and led to suppression of terminal differentiation and elimination
of the embryo suspensors, causing inhibition of embryo maturation. Our data demonstrate that perturbation of zinc
homeostasis disrupts the balance between cell proliferation and PCD required for plant embryogenesis. This establishes zinc as
an important cue governing cell fate decisions in plants.
Zinc homeostasis is paramount for controlling development and disease in all eukaryotic systems. The
best-known example of dysregulation of zinc homeostasis is zinc deficiency, causing severe developmental
and physiological aberrations in animals and plants
(Cakmak, 2000; Hambidge, 2000). At the cellular level,
zinc deficiency leads to apoptotic response in animal
cells, which is directly related to a decrease in free
intracellular zinc (Truong-Tran et al., 2001; Clegg et al.,
2005). Consistent with this, supplementing cells with
exogenous zinc decreases susceptibility to apoptotic
stimuli, thus establishing zinc as a physiological suppressor of cell death in animals.
While the metabolic network underlying zinc homeostasis in plants as well as biotechnological approaches to control zinc efficiency are beginning to be
understood (Grotz and Guerinot, 2006), an instrumental role of zinc in the regulation of programmed cell
death (PCD) in plants remains an open question.
1
This work was supported by the Forest Tree Breeding Association, the Swedish Research Council, and the Swedish Research
Council for Environment, Agricultural Sciences and Spatial Planning.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Peter V. Bozhkov ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.122598
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Plants are devoid of key components of apoptotic
machinery, including direct homologues of the caspase family of proteases. Furthermore, plant cells are
surrounded by rigid cell walls and plants have no cells
performing an analogous role to phagocytes, so the
formation of apoptotic bodies and their removal via
heterophagocytosis is impossible. Therefore, dying
plant cells must degrade themselves from inside by
lytic vacuoles, which are formed de novo via the
fusion and growth of autophagosomes, providing an
elegant paradigm for autophagic PCD (Bozhkov et al.,
2005a; Bassham, 2007; Bozhkov and Jansson, 2007).
The earliest function of PCD in plant ontogenesis is
fulfilled during embryo development that relies on
establishment of the embryonal mass (or embryo
proper) and the embryo suspensor, two embryonic
domains with contrasting developmental fates. While
the embryonal mass develops to mature embryo and
then to plant, the embryo suspensor is a terminally
differentiated structure committed to death and elimination (Meinke, 1995; Bozhkov et al., 2005a). Thus, in
common with animals, plant embryogenesis relies on
a strictly coordinated balance between cell death and
survival.
Somatic embryogenesis of Norway spruce (Picea
abies) has previously been established as a versatile
model system for studying cellular and molecular
mechanisms regulating and executing PCD in plant
embryos (Bozhkov et al., 2005a). This system enables
the control and manipulation of the entire pathway of
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Role of Zinc in Cell Fate Specification
embryo development in laboratory conditions and the
isolation of large numbers of embryos at specific developmental stages (Von Arnold et al., 2002). Importantly, somatic embryo development of Norway spruce
resembles zygotic embryogenesis, the only difference
being embryo origin, i.e. somatic cells of proembryogenic masses versus zygotes (Filonova et al., 2000b).
Autophagic PCD is essential for embryogenesis, first
removing proembryogenic masses after they have
given rise to the embryos and then affecting terminally
differentiated suspensors, which are completely eliminated during late embryogeny (Filonova et al., 2000a;
Fig. 1A). The suspensors of Norway spruce are composed of several layers of elongated cells with increased degree of cellular disassembly, beginning with
the cells committed to PCD in the first layer adjacent
to the embryonal mass and continuing toward the
basal end of the suspensors, where hollow-walled cell
corpses are located. Thus, cells at all stages of PCD can
be observed simultaneously in a single embryo along
its apical-basal axis. Moreover the position of the cell
within the embryo can be used as a marker of the stage
of cell death (Smertenko et al., 2003; Bozhkov et al.,
2005a).
The suspensor cell disassembly encompasses cytoskeleton-mediated autophagocytosis of the cytoplasm
and nuclear degradation as the two major processes
(Filonova et al., 2000a; Smertenko et al., 2003; Bozhkov
et al., 2005a). The activation and cytoplasm-to-nucleus
translocation of the type-II metacaspase mcII-Pa is
critical for the execution of this PCD. McII-Pa directly
participates in nuclear degradation (Bozhkov et al.
2005b) and, like other metacaspases, appears to activate a downstream, as-yet-unidentified protease(s)
with caspase-like activity (Madeo et al., 2002; Bozhkov
et al., 2005b; Watanabe and Lam, 2005). Interestingly,
proteolytic activity of mcII-Pa is highly sensitive to
zinc in in vitro assays, with a 50% inhibition by 1 mM
(Bozhkov et al., 2005b). This observation, together with
the earlier reported inhibitory effect of millimolar
Figure 1. Distribution of intracellular zinc correlates with cell fate specification in the embryos. A, A schematic model of a highly
stereotyped and synchronized pathway of somatic embryo development in Norway spruce (adapted from Filonova et al., 2000a).
The proliferation of proembryogenic masses (PEM; stages I, II, and III) is stimulated by the PGRs auxin and cytokinin. The
development of early somatic embryos (SE) is induced by withdrawal of PGRs. Early embryos are composed of living embryonal
masses (red) and dying suspensors (blue). The late embryogeny and embryo maturation are promoted by ABA. The embryo
suspensors are eliminated during late embryogeny. B to D, Staining with zinc-specific molecular probes reveals high accumulation
of zinc in the embryonal masses (EM) and abrupt decrease of zinc content in the suspensors (S) during both somatic (B and C) and
zygotic (D) embryogenesis. Somatic embryos were sampled at 7 d of ABA treatment, while zygotic embryos were isolated from
seeds at 9 weeks after fertilization. Embryos in B and D were stained with Zinquin, and in C with Dansylaminoethyl-cyclen. The top
and bottom panels display phase contrast and fluorescent microscopy, respectively. Arrowheads in D indicate the remnants of a
megagametophyte attached to a suspensor and yielding strong fluorescent signals. The images show representative samples of
50 somatic and 20 zygotic embryos analyzed. Scale bars 5 100 mm.
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Helmersson et al.
concentrations of zinc on internucleosomal DNA fragmentation in tomato (Solanum lycopersicum) protoplasts (Wang et al., 1996), suggests that zinc may
control PCD in plants.
In this study, we have investigated the role of
intracellular free zinc in the maintenance of a balance
between cell survival and PCD during plant embryo
development. We present evidence showing that zinc
mediates cell fate specification in the embryos through
its strong anti-cell death effect. Accumulation of zinc
in the embryonal masses but not in the suspensors is
required for correct embryonic patterning and embryo
survival. Zinc deficiency is lethal to embryos, whereas
supplementation of extra zinc suppresses terminal
differentiation and death of the suspensors, causing
a delay of suspensor elimination and embryo maturation. Our data suggest that the dual role for zinc in cell
death/survival decisions can be related to its inhibitory effect on metacaspase activity.
RESULTS
Distribution of Intracellular Zinc in the Embryos
Correlates with Cell Fate Specification
Somatic embryos of Norway spruce begin to develop from proembryogenic masses upon withdrawal
of the plant growth regulators (PGRs) auxin and
cytokinin, and subsequently require exogenous abscisic acid (ABA), which promotes late embryogeny
and embryo maturation (Filonova et al., 2000b; Fig.
1A). In the beginning of embryogenesis, the embryos
establish apical-basal polarity. The cells in the basal
part of the embryonal mass divide asymmetrically in
the plane perpendicular to embryo axis; one daughter
cell retains meristematic activity and remains in the
embryonal mass, while its sister cell elongates, undergoes terminal differentiation, and is added to the
suspensor. Thus, suspensor cells in Norway spruce do
not divide but are committed to PCD as soon as they
are formed (Bozhkov et al., 2005a). The suspensors
persist over a period of approximately 2 weeks until
asymmetric cell divisions in the embryonal masses
cease and the whole suspensors become subjected to
elimination (Fig. 1A).
To investigate the distribution of intracellular free
zinc in the embryos, we collected early somatic embryos with well-developed suspensors grown at a
physiological concentration of zinc (5 mM Zn-EDTA).
For labeling labile pools of intracellular zinc, the
embryos were stained with two membrane-permeant
zinc-specific fluorophores, Zinquin-ethyl-ester (Zinquin;
Zalewski et al., 1993) and Dansylaminoethyl-cyclen
(Koike et al., 1996), and analyzed by fluorescent microscopy. Both molecular probes gave identical results
showing abundant localization of zinc in the living
cells of embryonal masses and abrupt decrease of zinc
content in the suspensor (Fig. 1, B and C). Low intensity of zinc labeling in the suspensor cells was observed
over the whole length of the suspensor, beginning from
the first cellular level adjacent to the embryonal mass.
These cells are in the commitment phase of PCD and
have a large part of the contents preserved (Smertenko
et al., 2003; Bozhkov et al., 2005a), suggesting that zinc
deprivation occurs very early in the PCD pathway.
To verify that the pattern of zinc distribution found
in somatic embryos was not due to the fact that the
embryos were grown in the laboratory, we isolated
zygotic embryos of Norway spruce at a corresponding
developmental stage and assayed them for zinc distribution. As shown in Figure 1D, zinc distribution in
zygotic embryos exhibited the same pattern as observed in somatic embryos, indicating that this is an
innate feature of plant embryogenesis. We next asked
whether this pattern is zinc specific or common for
other divalent cations by staining embryos for calcium
using a Fura 2-acetoxymethylester probe. Contrary to
zinc, the level of calcium in the embryonal masses was
below detection limits, while punctate staining was
often observed in the suspensor cells (Supplemental
Fig. S1A). Taken together, these data show that distribution of intracellular labile zinc correlates with cell
fate specification in the embryos. Zinc content is high
in cells from the embryonal masses, which are destined to survive, and it is low in the suspensor cells,
which are committed to or executing PCD.
Depletion of Intracellular Zinc Induces Ectopic Cell
Death in the Embryos, Causing Embryo Lethality
For animals, the most convincing evidence for a
physiological role of zinc in suppression of cell death
comes from in vivo and in vitro experiments with zincdeficient embryos. In these experiments, the embryos
cultured in the low zinc medium or developed at
maternal zinc deficiency displayed increased rates of
apoptosis in all the structures derived from neural crest
cells, leading to developmental defects and early embryo abortion (Rogers et al., 1995; Jankowski-Hennig
et al., 2000; Hanna et al., 2003; Clegg et al., 2005). A
drop of intracellular zinc content found in the early
Norway spruce embryos upon transition from living
cells in the embryonal masses to the dying terminally differentiated cells in the suspensors (Fig. 1, B–D)
pointed to a possibility that a decrease in the available zinc pool can, as in the case of animal embryos, compromise plant cell viability and trigger cell
death. In animal cell systems, administration of the
cell-permeable zinc chelator N,N,N#,N#-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) in a range of concentrations from 0.5 mM to 100 mM was shown to have
a strong pro-apoptotic effect on diverse cell types
(e.g. McCabe et al., 1993; Zalewski et al., 1993; Ahn
et al., 1998; Meerarani et al., 2000; Chimienti et al.,
2001).
Before analyzing the effect of TPEN on plant embryogenesis, we wanted to make sure that TPEN could
efficiently chelate zinc in the embryos. For this, we
stained early Norway spruce somatic embryos grown
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Role of Zinc in Cell Fate Specification
with or without TPEN with Zinquin and analyzed
them by fluorescent microscopy. As shown in Figure
2A, the level of free intracellular zinc decreased dramatically in the embryonal masses of somatic embryos
grown in the medium supplemented with low concentration of TPEN (1 mM), indicating good penetration of the chelator into the densely packed cells of
the embryonal masses. To ascertain that TPEN does
not chelate other divalent cations, we stained TPENtreated embryos with Fura 2-acetoxymethylester, which
revealed strong accumulation of calcium in the embryonal mass cells (Supplemental Fig. S1B). Thus,
TPEN at 1 mM appears not to chelate divalent cations
other than zinc, but rather stimulates influx of calcium,
known as a primordial mechanism of cell death activation (Groover and Jones, 1999; Orrenius et al., 2003).
To assess the effect of zinc depletion on cell death,
we compared the frequency of cells with nuclear DNA
fragmentation, a biochemical hallmark of plant embryonic PCD (Bozhkov et al., 2005a), in TPEN-treated
and control embryos using terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling (TUNEL)
assay. Zinc-deprived embryos had increased frequency
of TUNEL-positive cells compared with control embryos (Fig. 2B). This effect seemed to be zinc specific,
as copper chelation had no effect on the level of cell
death in embryos (Supplemental Fig. S2, A and B).
While in the control embryos, TUNEL-positive cells
were mainly located in the suspensors, TPEN treatment induced DNA fragmentation in the embryonal
masses (Fig. 2C). To quantify this effect of TPEN, we
estimated the frequency of embryos containing two
Figure 2. Depletion of intracellular zinc induces ectopic cell death in the embryos, leading to embryo lethality. Zinc chelator
TPEN was added to embryogenic cultures 4 d after withdrawal of PGRs and then after the following 3 d, concomitant with the
start of ABA-stimulated embryo maturation. Control embryos were grown without TPEN. The control and TPEN-treated embryos
were collected for analyses after being grown for 7 d with ABA. A, TPEN prevents zinc accumulation in the embryonal masses
(EM), as revealed by Zinquin staining. Note the much weaker zinc-specific fluorescent signal in the embryonal masses of TPENtreated embryos as compared with control embryos. The top and bottom panels display phase contrast and fluorescent
microscopy, respectively. The images are representative examples of 20 embryos stained with Zinquin. S, Suspensor. Scale bars 5
100 mm. B, Depletion of zinc leads to increased nuclear DNA fragmentation in the embryos. Data show mean frequency (6SEM) of
TUNEL-positive cells estimated using three samples per treatment, with 400 nuclei observed for each sample. C, Depletion of zinc
leads to ectopic cell death in the embryos, as revealed by in situ DNA fragmentation analysis. Note the abnormal accumulation of
TUNEL-positive cells in the embryonal mass (EM) of a TPEN-treated embryo. The images are representative examples of 20
embryos stained with Zinquin. Scale bars 5100 mm. D, Massive cell death in the embryonal masses of TPEN-treated embryos, as
revealed by Evan’s blue staining. Note the absence of Evan’s blue staining in the embryonal mass (EM) of the control embryo.
Scale bars 5 100 mm.
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Helmersson et al.
or more TUNEL-positive cells in the embryonal
masses by observing 200 embryos for each treatment.
The frequency increased from 12% in the control
embryos to 42% and 60% in the embryos treated
with 1 mM and 5 mM TPEN, respectively. These results
demonstrate that zinc deprivation induces ectopic cell
death, which affects embryonal masses. Activation of
ectopic cell death was further confirmed by staining
embryos with Evan’s blue, the vital dye excluded by
living cells but readily penetrating through compromised plasma membrane into dead and dying cells
(Bozhkov et al., 2002). As shown in Figure 2D, TPENtreated embryos exhibited unrestricted staining of
both embryonal masses and suspensors, in contrast
to control embryos, in which Evan’s blue staining was
restricted to suspensors only. Zinc-depletion-induced
cell death in the embryonal masses had a lethal effect;
no cotyledonary embryos were regenerated from
TPEN-treated cultures (data not shown).
Is the observed lethal effect of zinc depletion on
Norway spruce embryos attributed to a general cytotoxic effect of low concentrations of TPEN on plant
cells? To answer this question, we tested the effect of
1 mM and 5 mM TPEN on the level of intracellular zinc
and cell death in the developmentally arrested cell line
of Norway spruce, which is unable to form embryos
and proliferates as proembryogenic masses regardless
of treatment (Smertenko et al., 2003; Supplemental Fig.
S3). Although TPEN efficiently chelated zinc in the
developmentally arrested line already at 1 mM, this
effect was not accompanied by increased DNA fragmentation, indicating that less differentiated cells are
much less sensitive to zinc deprivation compared with
embryonic cells. Taken together, our data suggest that
free intracellular zinc sustains cell viability in the
embryonal masses, which is necessary for the normal
progression of embryogenesis.
Zinc Supplementation Impairs Embryo Patterning via
Suppression of Suspensor Cell Specification
Terminal differentiation and PCD are two interrelated processes underlying suspensor cell identity
during plant embryogenesis (Bozhkov et al., 2005a).
Genetic or pharmacological dysregulation of embryonic pattern formation is often associated with the
loss of suspensor cell identity, leading to developmental aberrations and sometimes embryonic lethality
(Vernon and Meinke, 1994; Rojo et al., 2001; Smertenko
et al., 2003; Bozhkov et al., 2004; Lukowitz et al., 2004;
Suarez et al., 2004). The abrupt decline of zinc content
detected by specific fluorophores in Norway spruce
suspensor cells (Fig. 1, B–D) might indicate that zinc
deprivation is important for establishing suspensor
cell identity and embryonic patterning.
To investigate the physiological significance of the
low zinc level in the suspensor, we attempted to counteract the naturally occurring decline of zinc in the
suspensor by growing embryos with increased concentrations of zinc sulfate. Zinc supplementation treat-
ments were applied continuously, throughout all stages
of embryogenesis, beginning with early embryo development induced by withdrawal of PGRs until cotyledonary embryo formation stimulated by ABA (Fig.
1A). The early embryos induced to develop in PGRfree medium supplemented with 100 mM or 300 mM
zinc had a significantly lower proportion of TUNELpositive cells than the control embryos growing with
5 mM zinc (Fig. 3A). Furthermore, addition of equimolar amounts of copper sulfate could not reproduce this
death suppressive effect, and 300 mM copper sulfate
was even toxic to the embryos (Supplemental Fig.
S2C). However, we could not detect any discernible
morphological alterations caused by 100 mM or 300 mM
zinc over the 7-d period of early embryo development.
Further increase of zinc concentration to 1 mM suppressed embryogenesis (data not shown) and led to a
slightly elevated frequency of TUNEL-positive cells
(Fig. 3A), demonstrating that this supraphysiological
concentration of zinc is toxic to the embryos.
Early embryos require a regular supply of exogenous ABA to enter late embryogeny when primary
shoot and root meristems are set off and to accomplish
maturation (Filonova et al., 2000b; Bozhkov et al.,
2002). Once embryos enter late embryogeny, the asymmetric cell divisions in the basal regions of the embryonal masses cease and dead suspensors are gradually
eliminated (Fig. 1A; Filonova et al., 2000a, 2000b;
Bozhkov et al., 2005a). We investigated whether high
zinc-mediated suppression of embryonic PCD (Fig.
3A) had a developmental effect on the transition from
early to late embryogeny. For this, we analyzed the
frequency distribution of all embryos in the control
(5 mM Zn-EDTA) and extra-zinc-supplemented (100 mM
or 300 mM zinc sulfate) cultures at 7 d after addition of
ABA into four major phenotypes distinguished by
developmental stage and apical-basal pattern (Fig. 3B):
phenotype i, small polarized embryos in the beginning
of early embryogeny, composed of less than 16 cells;
phenotype ii, morphologically normal early embryos
with well-delineated rounded embryonal masses and
massive suspensors; phenotype iii, aberrant early embryos possessing elongated embryonal masses and a
lack of strict border between embryonal mass and
suspensor; and phenotype iv, late embryos without
suspensors or possessing remnants of suspensors. The
aberrant phenotype iii was caused by equal instead of
asymmetric cell divisions in the basal part of the
embryonal masses. As a consequence, both halves of
the daughter cells acquired meristematic identity and
were incorporated into the embryonal masses, thus
suppressing suspensor cell specification. This phenotype was very rarely observed among embryos from
control cultures (less than 3%), which were mainly
represented by equal proportions of morphologically
normal early embryos (phenotype ii) and late embryos
without suspensors (phenotype iv; Fig. 3C). However,
the frequency of the abnormal early embryos increased dramatically in the extra-zinc-supplemented
cultures, accounting for 34% and 40% of all the em-
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Role of Zinc in Cell Fate Specification
Figure 3. Zinc supplementation impairs embryo patterning via suppression of suspensor cell specification. Zinc sulfate was added to
embryogenic cultures at the time of withdrawal of PGRs and thereafter every 2 weeks during embryo maturation. Control embryos
were grown with 5 mM Zn-EDTA. A, Supplementation of the embryos with 100 mM or 300 mM zinc sulfate suppresses nuclear DNA
fragmentation. Data show a mean frequency (6SEM) of TUNEL-positive cells assessed at 4 d after withdrawal of PGRs using five
samples per treatment, with 200 nuclei observed for each sample. B, Phase contrast images of the four embryo phenotypes (i–iv)
constituting control and zinc-supplemented cultures in the beginning of the embryo maturation treatment (7 d with ABA). EM,
Embryonal mass; S, suspensor. Scale bars 5 100 mm. C, Frequency distribution of the four embryo phenotypes in control and zincsupplemented cultures. The data show a mean frequency (6SEM) of embryos of a given phenotype assessed in seven samples per
treatment by counting 100 embryos in each sample. D, Zinc supplementation inhibits embryo maturation. The data show mean
number (6SEM) of cotyledonary somatic embryos per plate (10 plates per treatment) formed over 10 weeks on ABA-containing
medium. E, Delay of embryo maturation in zinc-supplemented cultures observed after 6 weeks on ABA-containing medium.
Although zinc supplementation did not prevent embryo maturation (top images), it suppressed transition from early to late embryogeny (bottom images). Apart from large degrading proembryogenic masses, zinc-supplemented cultures contained early embryos
(bottom images). Scale bars 5 1 mm (top images); 100 mm (bottom images). F, Zinc supplementation affects the pattern of zinc
localization. The embryos growing at increased zinc concentrations show high accumulation of zinc in both embryonal masses (EM)
and suspensors (S), while in the control embryos zinc accumulation is restricted to embryonal masses. The top and bottom panels
display phase contrast and fluorescent microscopy, respectively. The images are representative examples of 20 embryos stained with
Zinquin after 7 d of the ABA treatment from both the control and zinc-supplemented cultures. Scale bars 5 100 mm.
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Helmersson et al.
bryos grown with 100 mM and 300 mM zinc sulfate,
respectively (Fig. 3C). Concomitantly, the fraction of
late embryos decreased from 48% to 13% and 6%,
respectively, indicating that abnormal early embryos
are arrested at the transition to late embryogeny. As a
result, the yield of cotyledonary embryos in the extrazinc-supplemented cultures decreased 2.5- to 5-fold
compared with the control (Fig. 3D). Interestingly, apart
from cotyledonary embryos, there were a large number
of early embryos in the extra-zinc-supplemented cultures at the end of the embryo maturation treatment
(Fig. 3E) as a consequence of zinc-stimulated accumulation of small embryos (phenotype i) at the earlier time
point (Fig. 3, B and C).
We conclude that zinc supplementation to a certain
level (up to 300 mM) greatly enhances cell survival,
which can impair embryonic pattern formation. Staining of abnormal early embryos (phenotype iii) with
Zinquin revealed abundant accumulation of zinc not
only in the embryonal masses but also in the suspensor
composed of a mixture of densely cytoplasmic and
vacuolated cells (Fig. 3F). Therefore, an abnormal
embryo phenotype described in our study is a direct
consequence of enhanced zinc accumulation, which
suppresses terminal differentiation and PCD of the
embryo suspensor.
Zinc Affects Metacaspase-Like Proteolytic Activity
in the Embryos
The type-II metacaspase mcII-Pa is essential for
Norway spruce embryogenesis, as it is required for
establishing apical-basal pattern (Suarez et al., 2004)
and is directly involved in the execution of PCD in the
suspensor (Bozhkov et al., 2005b). McII-Pa is a processive Cys-dependent protease and, like other members of
the metacaspase family proteases from plants, fungi
and protozoa (Vercammen et al., 2004; Watanabe and
Lam, 2005; Gonzales et al., 2007; He et al., 2008), has
Arg/Lys-specific proteolytic activity (Bozhkov et al.,
2005b). We have previously reported that 10 mM zinc
abrogates activity of mcII-Pa in vitro (Bozhkov et al.,
2005b). In this study, we asked whether zinc-mediated
regulation of cell death in the embryos correlates with
the level of metacaspase activity.
For measuring metacaspase activity in cell lysates,
we used the fluorogenic substrate Boc-Glu-Gly-Argamido-4-methylcoumarin (Boc-EGR-AMC), which has
previously been shown to be one of the preferred
substrates for mcII-Pa both in vitro and in vivo
(Bozhkov et al., 2005b). Zinc supplementation or
TPEN treatments were applied simultaneously with
withdrawal of PGRs, and cell lysates were prepared
from control (untreated) and treated cells at 0, 24, 48,
96, and 168 h. As shown in Figure 4, A and B, normal
embryo development in the control cultures was accompanied by an increase in metacaspase-like activity
between 96 and 168 h, coinciding with the period of
apical-basal pattern formation associated with terminal differentiation and death of the embryo suspensor
cells (Filonova et al., 2000a). Zinc supplementation
abrogated this increase; furthermore, the embryos
grown with 300 mM zinc sulfate displayed a moderate
decrease in metacaspase-like activity between 48 h to
168 h (Fig. 4A). Consistent with this observation,
chelation of free intracellular zinc by TPEN led to a
pronounced increase in metacaspase-like activity over
the initial period of 48 h, followed by a decrease to
Figure 4. Zinc affects metacaspase-like proteolytic activity in vivo. Zinc supplementation suppresses (A) and
zinc deprivation strongly up-regulates (B) metacaspaselike activity in the embryos. Zinc sulfate or TPEN was
added simultaneously with the withdrawal of PGRs,
and samples were collected after 0, 24, 48, 96, and
168 h. Metacaspase-like activity was measured using
an AMC-conjugated Boc-EGR peptide. Data for BocEGR-AMC cleavage represent the mean of three independent experiments 6SEM. C, First-order kinetics
of Boc-EGR-AMC cleavage by cell lysates prepared
from control and zinc sulfate-treated embryos, which
were collected after being grown for 7 d with ABA
and microsurgically separated into suspensors (S) and
embryonal masses (EM). Dotted lines denote 95%
confidence limits.
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Role of Zinc in Cell Fate Specification
below control level (Fig. 4B). Evan’s blue staining
confirmed that all cells were already dead at 168 h, so
the loss of metacaspase activity at the end of TPEN
treatment was not surprising.
We next microsurgically separated embryos grown
for 7 d on ABA-containing medium into two parts,
embryonal masses and suspensors, and measured
metacaspase-like activity in the corresponding cell
lysates. Metacaspase-like activity was approximately
2-fold higher in the lysates prepared from the suspensors compared with those from embryonal masses (Fig.
4C). This activity was reduced in the both fractions by
15% if the embryos were grown on the medium
supplemented with 300 mM zinc sulfate. Collectively,
our results establish metacaspase activation as one of
the potential targets of zinc during zinc-mediated
suppression of PCD in embryos.
DISCUSSION
Zinc has diverse functions in eukaryotic cells, which
extend from structural and/or catalytic roles of poorly
exchangeable zinc within metalloenzymes and zinc
finger proteins to transient interactions of kinetically
labile zinc with cellular signaling pathways (Vallee
and Falchuk, 1993). Since apoptosis of animal cells is
readily influenced by zinc deprivation or supplementation, it has been postulated that it is more labile pools
of zinc that are involved in cell death regulation
(Truong-Tran et al., 2001). This study has extended the
role of zinc in the regulation of eukaryotic cell death to
plants, and our results have three major implications.
First, distribution of zinc in plant embryos is developmentally regulated and correlates with cell fate
specification during apical-basal pattern formation.
We show that accumulation of zinc in the embryonal
masses and abrupt decline in the suspensors are tightly
controlled and hallmark zinc homeostasis at early
stages of plant embryogenesis (Fig. 1, B–D). Dysregulation of zinc homeostasis causes developmental defects or lethality associated with the disruption of the
proper balance between cell survival (in the embryonal
masses) and PCD (in the suspensors; Figs. 2 and 3).
Further studies are required to unravel the molecular
mechanisms of unequal redistribution of zinc in the
two daughter cells resulting from asymmetric cell
divisions during apical-basal pattern formation and,
in particular, the role of zinc transporters.
Second, this is the first study in which the role of zinc
in the control of autophagic PCD has been addressed.
Being a bona fide route of physiological cell death in
plants, autophagic PCD is an evolutionarily ancient and
phylogenetically conserved type of PCD from which
animal-specific apoptosis is believed to have evolved
(Zhivotovsky, 2002; Golstein and Kroemer, 2005;
Bozhkov and Jansson, 2007). Finding a potent role for
zinc in the regulation of autophagic PCD establishes
zinc deprivation as a universal cell death signal, regardless of which route of degradation—apoptotic or
autophagic —is chosen by cells. Since autophagy has
a dual role in cell death and survival (Baehrecke,
2005; Bassham, 2007), it appears especially interesting
to determine whether zinc-deficiency-associated autophagy in the embryo suspensor implicates release of
free zinc from catabolized metalloenzymes and zinc
finger proteins in order to adjust and control the rate of
cell death.
Third, inactivation of several members of caspase
family proteases (primarily caspases 3, 6, and 9) by
zinc is thought to be the main process underlying the
anti-apoptotic effect of zinc in mammalian cells
(Truong-Tran et al., 2001). Although the exact biochemical mechanisms of caspase inhibition by zinc are
unknown, it has been demonstrated that zinc blocks
the process of pro-caspase-3 activation rather than the
already activated enzyme (Truong-Tran et al., 2000). It
is proposed that zinc binds reversibly to the thiol
group of catalytic Cys-163, inhibiting activation of the
proenzyme and protecting the essential residue from
oxidation (Maret et al., 1999; Truong-Tran et al., 2001).
Plants do not have close homologues of caspases but
possess the metacaspase family of Cys proteases,
which have been proposed to be the ancestors of
metazoan caspases (Uren et al., 2000). Despite the fact
that metacaspases and caspases display low sequence
similarity and have distinct substrate specificities,
they have a conserved catalytic diad of His and Cys,
and both require Cys-dependent processing of zymogen for enzyme activation (Vercammen et al., 2004;
Bozhkov et al., 2005b). Here, we demonstrate that, like
activity of mammalian caspases, plant metacaspase
activity is suppressed by zinc in vivo (Fig. 4), suggesting that metacaspases could be one of the targets for
zinc during zinc-mediated regulation of plant PCD.
Suppression of metacaspase activity by zinc found in
vivo (this study) and in vitro (Bozhkov et al., 2005b)
suggests another mechanism of posttranslational regulation of metacaspases, in addition to the recently
identified S-nitrosylation of a catalytic Cys of Arabidopsis (Arabidopsis thaliana) metacaspase-9 (Belenghi
et al., 2007). However, it remains to be investigated
whether zinc binds to a critical Cys residue of metacaspases to keep the enzyme in the unprocessed
inactive form and/or to inactivate the already active
enzyme.
MATERIALS AND METHODS
Embryogenesis System
Two morphologically similar embryogenic cell lines of Norway spruce
(Picea abies), 95.88.22 and 95.76.04, were used in this study. The cell lines were
stored in liquid nitrogen, thawed, and allowed to proliferate on solidified halfstrength LP medium supplemented with the PGRs auxin and cytokinin, as
described previously (Filonova et al., 2000b). Proliferating cell suspension
cultures were initiated 3 weeks prior to the onset of the experiments and were
maintained by weekly subculture before being transferred to PGR-free medium for 1 week to induce embryo development (Filonova et al., 2000a). For
embryo maturation, the cultures were plated on solidified maturation medium containing ABA (Filonova et al., 2000b). Cotyledonary somatic embryos
were harvested after 4 to 10 weeks of the maturation treatment.
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Helmersson et al.
In the control experiments, 5 mM Zn-EDTA was used as the sole source of
zinc during all stages of somatic embryogenesis. For depleting cells of
intracellular zinc, the cultures were treated with different concentrations
(1 mM to 25 mM) of the membrane-permeable zinc chelator TPEN (Sigma) in
Zn-EDTA-free medium beginning from 4 d after withdrawal of PGRs.
Thereafter, TPEN was added to the fresh maturation medium at every
2-week subculture. In the zinc supplementation experiments, the cultures
were treated with 100 mM to 1 mM ZnSO4 at the time of the withdrawal of PGRs
and during the following subculturing onto maturation medium. The procedures for copper chelation and supplementation are provided in Supplemental Materials and Methods S1.
In Situ Zinc Localization
The early somatic or zygotic Norway spruce embryos were stained with
25 mM Zinquin (Sigma) or 100 mM Dansylaminoethyl-cyclen (Dojindo Laboratories) in the liquid culture medium devoid of PGRs and Zn-EDTA for
30 min in the dark on a gyratory shaker at room temperature and then washed
three times with the medium. The samples were observed under a Nikon
Microphot-FXA fluorescence microscope using a standard UV2-A set of filters
(excitation filter, 340–380 nm; dichroic mirror, 400 nm; barrier filter, 420 nm).
Images were taken with a NIKON Digital Sight-L1 camera. The procedure for
intracellular calcium localization is described in Supplemental Materials and
Methods S1.
Cell Death Assays
Nuclear DNA fragmentation was assessed by a whole mount TUNEL (In
Situ Cell Death Detection Kit, TMR red; Roche) as described previously
(Filonova et al., 2000a), with more than 1,000 nuclei (counterstained with 4#-6diamino-2-phenylindole [Roche]) observed for each sample. For analyzing the
integrity of plasma membranes, cells were stained with Evan’s blue (Filonova
et al., 2000b).
Metacaspase-Like Activity Assay
Norway spruce cell extracts were prepared and assayed for metacaspaselike proteolytic activity using 50 mM Boc-EGR-AMC substrate (Bachem) as
described (Bozhkov et al., 2005b). To separate the embryonal masses from the
suspensors, the embryos collected at 7 d of maturation treatment were cut
transversally using surgical blades (No. 11; Feather) in droplets of zinc-free
culture medium viewed under a binocular microscope (Zeiss). The embryonal
masses and the suspensors were then transferred to individual Eppendorf
tubes using Dumont tweezers (size 5/45). All the samples were thoroughly
washed by three aliquots of zinc-free culture medium to remove unbound
zinc, the medium aspirated, and the samples snap-frozen in liquid nitrogen
prior to storage at 280°C.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Distribution of calcium in control and TPENtreated embryos.
Supplemental Figure S2. Unlike zinc, copper does not suppress cell death
in the embryos.
Supplemental Figure S3. Chelation of zinc by 1 mM and 5 mM TPEN does
not activate nuclear DNA fragmentation in the developmentally
arrested cell line.
Supplemental Materials and Methods S1. Procedures for in situ calcium
localization and for copper chelation and supplementation.
ACKNOWLEDGMENTS
The authors are grateful to Shyam Kumar Gudey and Mahmudur
Rahman for technical assistance and to David Clapham for critical reading
of the manuscript.
Received May 5, 2008; accepted May 22, 2008; published May 28, 2008.
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