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First published online May 28, 2008; 10.1104/pp.108.122598

Plant Physiology 147:1158-1167 (2008)
© 2008 American Society of Plant Biologists

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DEVELOPMENT AND HORMONE ACTION

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


   ABSTRACT
TOP
 ABSTRACT
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 
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, 2000Go; Hambidge, 2000Go). 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., 2001Go; Clegg et al., 2005Go). 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, 2006Go), an instrumental role of zinc in the regulation of programmed cell death (PCD) in plants remains an open question. 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., 2005aGo; Bassham, 2007Go; Bozhkov and Jansson, 2007Go).

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, 1995Go; Bozhkov et al., 2005aGo). 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., 2005aGo). This system enables the control and manipulation of the entire pathway of embryo development in laboratory conditions and the isolation of large numbers of embryos at specific developmental stages (Von Arnold et al., 2002Go). 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., 2000bGo). 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., 2000aGo; 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., 2003Go; Bozhkov et al., 2005aGo).


Figure 1
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  		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., 2000aGo). 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 = 100 µm.

 
The suspensor cell disassembly encompasses cytoskeleton-mediated autophagocytosis of the cytoplasm and nuclear degradation as the two major processes (Filonova et al., 2000aGo; Smertenko et al., 2003Go; Bozhkov et al., 2005aGo). 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. 2005bGo) and, like other metacaspases, appears to activate a downstream, as-yet-unidentified protease(s) with caspase-like activity (Madeo et al., 2002Go; Bozhkov et al., 2005bGo; Watanabe and Lam, 2005Go). Interestingly, proteolytic activity of mcII-Pa is highly sensitive to zinc in in vitro assays, with a 50% inhibition by 1 µM (Bozhkov et al., 2005bGo). This observation, together with the earlier reported inhibitory effect of millimolar concentrations of zinc on internucleosomal DNA fragmentation in tomato (Solanum lycopersicum) protoplasts (Wang et al., 1996Go), 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
TOP
 ABSTRACT
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 

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., 2000bGo; 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., 2005aGo). 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 µM 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., 1993Go) and Dansylaminoethyl-cyclen (Koike et al., 1996Go), 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., 2003Go; Bozhkov et al., 2005aGo), 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 zinc-deficient 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., 1995Go; Jankowski-Hennig et al., 2000Go; Hanna et al., 2003Go; Clegg et al., 2005Go). 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 µM to 100 µM was shown to have a strong pro-apoptotic effect on diverse cell types (e.g. McCabe et al., 1993Go; Zalewski et al., 1993Go; Ahn et al., 1998Go; Meerarani et al., 2000Go; Chimienti et al., 2001Go).

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 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 µM), 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 TPEN-treated embryos with Fura 2-acetoxymethylester, which revealed strong accumulation of calcium in the embryonal mass cells (Supplemental Fig. S1B). Thus, TPEN at 1 µM 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, 1999Go; Orrenius et al., 2003Go).


Figure 2
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  		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 TPEN-treated 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 = 100 µm. B, Depletion of zinc leads to increased nuclear DNA fragmentation in the embryos. Data show mean frequency (±SEM) 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 =100 µm. 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 = 100 µm.

 
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., 2005aGo), 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 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 µM and 5 µM 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., 2002Go). As shown in Figure 2D, TPEN-treated 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 µM and 5 µM 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., 2003Go; Supplemental Fig. S3). Although TPEN efficiently chelated zinc in the developmentally arrested line already at 1 µM, 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., 2005aGo). 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, 1994Go; Rojo et al., 2001Go; Smertenko et al., 2003Go; Bozhkov et al., 2004Go; Lukowitz et al., 2004Go; Suarez et al., 2004Go). 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 treatments 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 PGR-free medium supplemented with 100 µM or 300 µM zinc had a significantly lower proportion of TUNEL-positive cells than the control embryos growing with 5 µM zinc (Fig. 3A ). Furthermore, addition of equimolar amounts of copper sulfate could not reproduce this death suppressive effect, and 300 µM copper sulfate was even toxic to the embryos (Supplemental Fig. S2C). However, we could not detect any discernible morphological alterations caused by 100 µM or 300 µM 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.


Figure 3
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  		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 µM Zn-EDTA. A, Supplementation of the embryos with 100 µM or 300 µM zinc sulfate suppresses nuclear DNA fragmentation. Data show a mean frequency (±SEM) 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 = 100 µm. C, Frequency distribution of the four embryo phenotypes in control and zinc-supplemented cultures. The data show a mean frequency (±SEM) 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 (±SEM) 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 = 1 mm (top images); 100 µm (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 = 100 µm.

 
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., 2000bGo; Bozhkov et al., 2002Go). 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., 2000aGo, 2000bGo; Bozhkov et al., 2005aGo). 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 µM Zn-EDTA) and extra-zinc-supplemented (100 µM or 300 µM 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 embryos grown with 100 µM and 300 µM 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 extra-zinc-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 µM) 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., 2004Go) and is directly involved in the execution of PCD in the suspensor (Bozhkov et al., 2005bGo). 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., 2004Go; Watanabe and Lam, 2005Go; Gonzales et al., 2007Go; He et al., 2008Go), has Arg/Lys-specific proteolytic activity (Bozhkov et al., 2005bGo). We have previously reported that 10 µM zinc abrogates activity of mcII-Pa in vitro (Bozhkov et al., 2005bGo). 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-Arg-amido-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., 2005bGo). 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., 2000aGo). Zinc supplementation abrogated this increase; furthermore, the embryos grown with 300 µM 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 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.


Figure 4
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  		Figure 4. Zinc affects metacaspase-like proteolytic activity in vivo. Zinc supplementation suppresses (A) and zinc deprivation strongly up-regulates (B) metacaspase-like 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 Boc-EGR-AMC cleavage represent the mean of three independent experiments ±SEM. 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.

 
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 µM 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
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 LITERATURE CITED
 
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, 1993Go). 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., 2001Go). 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, 2002Go; Golstein and Kroemer, 2005Go; Bozhkov and Jansson, 2007Go). 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, 2005Go; Bassham, 2007Go), 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., 2001Go). 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., 2000Go). 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., 1999Go; Truong-Tran et al., 2001Go). 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., 2000Go). 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., 2004Go; Bozhkov et al., 2005bGo). 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., 2005bGo) 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., 2007Go). 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
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
LITERATURE CITED
 

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 half-strength LP medium supplemented with the PGRs auxin and cytokinin, as described previously (Filonova et al., 2000bGo). 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., 2000aGo). For embryo maturation, the cultures were plated on solidified maturation medium containing ABA (Filonova et al., 2000bGo). Cotyledonary somatic embryos were harvested after 4 to 10 weeks of the maturation treatment.

In the control experiments, 5 µM 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 µM to 25 µM) 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 µM 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 µM Zinquin (Sigma) or 100 µM 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., 2000aGo), with more than 1,000 nuclei (counterstained with 4'-6-diamino-2-phenylindole [Roche]) observed for each sample. For analyzing the integrity of plasma membranes, cells were stained with Evan's blue (Filonova et al., 2000bGo).


Metacaspase-Like Activity Assay

Norway spruce cell extracts were prepared and assayed for metacaspase-like proteolytic activity using 50 µM Boc-EGR-AMC substrate (Bachem) as described (Bozhkov et al., 2005bGo). 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 –80°C.


Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Distribution of calcium in control and TPEN-treated embryos.
Supplemental Figure S2. Unlike zinc, copper does not suppress cell death in the embryos.
Supplemental Figure S3. Chelation of zinc by 1 µM and 5 µM 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.


   FOOTNOTES
 
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. Back

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 (peter.bozhkov{at}vbsg.slu.se).

[W] The online version of this article contains Web-only data. Back

www.plantphysiol.org/cgi/doi/10.1104/pp.108.122598

* Corresponding author; e-mail peter.bozhkov{at}vbsg.slu.se.


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