the level of free intracellular zinc mediates programmed ...the level of free intracellular zinc...

The Level of Free Intracellular Zinc MediatesProgrammed Cell Death/Cell Survival Decisions inPlant Embryos1[W]

Andreas Helmersson, Sara von Arnold, and Peter V. Bozhkov*

Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish Universityof 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 ofintracellular free zinc are required to protect cells from death, zinc depletion commits cells to death in diverse systems. As inanimals, PCD has a fundamental role in plant biology, but its molecular regulation is poorly understood. In particular, theinvolvement of zinc in the control of plant PCD remains unknown. Here, we used somatic embryos of Norway spruce (Piceaabies) to investigate the role of zinc in developmental PCD, which is crucial for correct embryonic patterning. Staining of theearly embryos with zinc-specific molecular probes (Zinquin-ethyl-ester and Dansylaminoethyl-cyclen) has revealed highaccumulation of zinc in the proliferating cells of the embryonal masses and abrupt decrease of zinc content in the dyingterminally 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 embryonalmasses. This cell death involved the loss of plasma membrane integrity, metacaspase-like proteolytic activity, and nuclear DNAfragmentation. To verify the anti-cell death effect of zinc, we incubated early embryos with increased concentrations of zincsulfate. Zinc supplementation inhibited developmental PCD and led to suppression of terminal differentiation and eliminationof the embryo suspensors, causing inhibition of embryo maturation. Our data demonstrate that perturbation of zinchomeostasis disrupts the balance between cell proliferation and PCD required for plant embryogenesis. This establishes zinc asan important cue governing cell fate decisions in plants.

Zinc homeostasis is paramount for controlling de-velopment and disease in all eukaryotic systems. Thebest-known example of dysregulation of zinc homeo-stasis is zinc deficiency, causing severe developmentaland physiological aberrations in animals and plants(Cakmak, 2000; Hambidge, 2000). At the cellular level,zinc deficiency leads to apoptotic response in animalcells, which is directly related to a decrease in freeintracellular zinc (Truong-Tran et al., 2001; Clegg et al.,2005). Consistent with this, supplementing cells withexogenous zinc decreases susceptibility to apoptoticstimuli, thus establishing zinc as a physiological sup-pressor of cell death in animals.

While the metabolic network underlying zinc ho-meostasis in plants as well as biotechnological ap-proaches to control zinc efficiency are beginning to beunderstood (Grotz and Guerinot, 2006), an instrumen-tal role of zinc in the regulation of programmed celldeath (PCD) in plants remains an open question.

Plants are devoid of key components of apoptoticmachinery, including direct homologues of the cas-pase family of proteases. Furthermore, plant cells aresurrounded by rigid cell walls and plants have no cellsperforming an analogous role to phagocytes, so theformation of apoptotic bodies and their removal viaheterophagocytosis is impossible. Therefore, dyingplant cells must degrade themselves from inside bylytic vacuoles, which are formed de novo via thefusion and growth of autophagosomes, providing anelegant paradigm for autophagic PCD (Bozhkov et al.,2005a; Bassham, 2007; Bozhkov and Jansson, 2007).

The earliest function of PCD in plant ontogenesis isfulfilled during embryo development that relies onestablishment of the embryonal mass (or embryoproper) and the embryo suspensor, two embryonicdomains with contrasting developmental fates. Whilethe embryonal mass develops to mature embryo andthen to plant, the embryo suspensor is a terminallydifferentiated structure committed to death and elim-ination (Meinke, 1995; Bozhkov et al., 2005a). Thus, incommon with animals, plant embryogenesis relies ona strictly coordinated balance between cell death andsurvival.

Somatic embryogenesis of Norway spruce (Piceaabies) has previously been established as a versatilemodel system for studying cellular and molecularmechanisms regulating and executing PCD in plantembryos (Bozhkov et al., 2005a). This system enablesthe control and manipulation of the entire pathway of

1 This work was supported by the Forest Tree Breeding Associa-tion, the Swedish Research Council, and the Swedish ResearchCouncil for Environment, Agricultural Sciences and Spatial Planning.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed 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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embryo development in laboratory conditions and theisolation of large numbers of embryos at specific de-velopmental stages (Von Arnold et al., 2002). Impor-tantly, somatic embryo development of Norway spruceresembles zygotic embryogenesis, the only differencebeing embryo origin, i.e. somatic cells of proembryo-genic masses versus zygotes (Filonova et al., 2000b).Autophagic PCD is essential for embryogenesis, firstremoving proembryogenic masses after they havegiven rise to the embryos and then affecting terminallydifferentiated suspensors, which are completely elim-inated during late embryogeny (Filonova et al., 2000a;Fig. 1A). The suspensors of Norway spruce are com-posed of several layers of elongated cells with in-creased degree of cellular disassembly, beginning withthe cells committed to PCD in the first layer adjacentto the embryonal mass and continuing toward thebasal end of the suspensors, where hollow-walled cellcorpses are located. Thus, cells at all stages of PCD canbe observed simultaneously in a single embryo along

its apical-basal axis. Moreover the position of the cellwithin the embryo can be used as a marker of the stageof cell death (Smertenko et al., 2003; Bozhkov et al.,2005a).

The suspensor cell disassembly encompasses cyto-skeleton-mediated autophagocytosis of the cytoplasmand nuclear degradation as the two major processes(Filonova et al., 2000a; Smertenko et al., 2003; Bozhkovet al., 2005a). The activation and cytoplasm-to-nucleustranslocation of the type-II metacaspase mcII-Pa iscritical for the execution of this PCD. McII-Pa directlyparticipates in nuclear degradation (Bozhkov et al.2005b) and, like other metacaspases, appears to acti-vate a downstream, as-yet-unidentified protease(s)with caspase-like activity (Madeo et al., 2002; Bozhkovet al., 2005b; Watanabe and Lam, 2005). Interestingly,proteolytic activity of mcII-Pa is highly sensitive tozinc in in vitro assays, with a 50% inhibition by 1 mM

(Bozhkov et al., 2005b). This observation, together withthe 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 highlystereotyped 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. Thedevelopment of early somatic embryos (SE) is induced by withdrawal of PGRs. Early embryos are composed of living embryonalmasses (red) and dying suspensors (blue). The late embryogeny and embryo maturation are promoted by ABA. The embryosuspensors are eliminated during late embryogeny. B to D, Staining with zinc-specific molecular probes reveals high accumulationof zinc in the embryonal masses (EM) and abrupt decrease of zinc content in the suspensors (S) during both somatic (B and C) andzygotic (D) embryogenesis. Somatic embryos were sampled at 7 d of ABA treatment, while zygotic embryos were isolated fromseeds at 9 weeks after fertilization. Embryos in B and D were stained with Zinquin, and in C with Dansylaminoethyl-cyclen. The topand bottom panels display phase contrast and fluorescent microscopy, respectively. Arrowheads in D indicate the remnants of amegagametophyte attached to a suspensor and yielding strong fluorescent signals. The images show representative samples of50 somatic and 20 zygotic embryos analyzed. Scale bars 5 100 mm.

Role of Zinc in Cell Fate Specification

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concentrations of zinc on internucleosomal DNA frag-mentation in tomato (Solanum lycopersicum) proto-plasts (Wang et al., 1996), suggests that zinc maycontrol PCD in plants.

In this study, we have investigated the role ofintracellular free zinc in the maintenance of a balancebetween cell survival and PCD during plant embryodevelopment. We present evidence showing that zincmediates cell fate specification in the embryos throughits strong anti-cell death effect. Accumulation of zincin the embryonal masses but not in the suspensors isrequired for correct embryonic patterning and embryosurvival. Zinc deficiency is lethal to embryos, whereassupplementation of extra zinc suppresses terminaldifferentiation and death of the suspensors, causinga delay of suspensor elimination and embryo matura-tion. Our data suggest that the dual role for zinc in celldeath/survival decisions can be related to its inhibi-tory effect on metacaspase activity.

RESULTS

Distribution of Intracellular Zinc in the Embryos

Correlates with Cell Fate Specification

Somatic embryos of Norway spruce begin to de-velop from proembryogenic masses upon withdrawalof the plant growth regulators (PGRs) auxin andcytokinin, and subsequently require exogenous ab-scisic acid (ABA), which promotes late embryogenyand embryo maturation (Filonova et al., 2000b; Fig.1A). In the beginning of embryogenesis, the embryosestablish apical-basal polarity. The cells in the basalpart of the embryonal mass divide asymmetrically inthe plane perpendicular to embryo axis; one daughtercell retains meristematic activity and remains in theembryonal mass, while its sister cell elongates, un-dergoes terminal differentiation, and is added to thesuspensor. Thus, suspensor cells in Norway spruce donot divide but are committed to PCD as soon as theyare formed (Bozhkov et al., 2005a). The suspensorspersist over a period of approximately 2 weeks untilasymmetric cell divisions in the embryonal massescease and the whole suspensors become subjected toelimination (Fig. 1A).

To investigate the distribution of intracellular freezinc in the embryos, we collected early somatic em-bryos with well-developed suspensors grown at aphysiological concentration of zinc (5 mM Zn-EDTA).For labeling labile pools of intracellular zinc, theembryos were stained with two membrane-permeantzinc-specific fluorophores, Zinquin-ethyl-ester (Zinquin;Zalewski et al., 1993) and Dansylaminoethyl-cyclen(Koike et al., 1996), and analyzed by fluorescent mi-croscopy. Both molecular probes gave identical resultsshowing abundant localization of zinc in the livingcells of embryonal masses and abrupt decrease of zinccontent in the suspensor (Fig. 1, B and C). Low inten-sity of zinc labeling in the suspensor cells was observed

over the whole length of the suspensor, beginning fromthe first cellular level adjacent to the embryonal mass.These cells are in the commitment phase of PCD andhave a large part of the contents preserved (Smertenkoet al., 2003; Bozhkov et al., 2005a), suggesting that zincdeprivation occurs very early in the PCD pathway.

To verify that the pattern of zinc distribution foundin somatic embryos was not due to the fact that theembryos were grown in the laboratory, we isolatedzygotic embryos of Norway spruce at a correspondingdevelopmental stage and assayed them for zinc dis-tribution. As shown in Figure 1D, zinc distribution inzygotic embryos exhibited the same pattern as ob-served in somatic embryos, indicating that this is aninnate feature of plant embryogenesis. We next askedwhether this pattern is zinc specific or common forother divalent cations by staining embryos for calciumusing a Fura 2-acetoxymethylester probe. Contrary tozinc, the level of calcium in the embryonal masses wasbelow detection limits, while punctate staining wasoften observed in the suspensor cells (SupplementalFig. S1A). Taken together, these data show that distri-bution of intracellular labile zinc correlates with cellfate specification in the embryos. Zinc content is highin cells from the embryonal masses, which are des-tined to survive, and it is low in the suspensor cells,which are committed to or executing PCD.

Depletion of Intracellular Zinc Induces Ectopic CellDeath in the Embryos, Causing Embryo Lethality

For animals, the most convincing evidence for aphysiological role of zinc in suppression of cell deathcomes from in vivo and in vitro experiments with zinc-deficient embryos. In these experiments, the embryoscultured in the low zinc medium or developed atmaternal zinc deficiency displayed increased rates ofapoptosis in all the structures derived from neural crestcells, leading to developmental defects and early em-bryo abortion (Rogers et al., 1995; Jankowski-Henniget al., 2000; Hanna et al., 2003; Clegg et al., 2005). Adrop of intracellular zinc content found in the earlyNorway spruce embryos upon transition from livingcells in the embryonal masses to the dying termi-nally differentiated cells in the suspensors (Fig. 1, B–D)pointed to a possibility that a decrease in the avail-able zinc pool can, as in the case of animal em-bryos, compromise plant cell viability and trigger celldeath. In animal cell systems, administration of thecell-permeable zinc chelator N,N,N#,N#-tetrakis(2-pyri-dylmethyl)ethylenediamine (TPEN) in a range of con-centrations from 0.5 mM to 100 mM was shown to havea strong pro-apoptotic effect on diverse cell types(e.g. McCabe et al., 1993; Zalewski et al., 1993; Ahnet al., 1998; Meerarani et al., 2000; Chimienti et al.,2001).

Before analyzing the effect of TPEN on plant em-bryogenesis, we wanted to make sure that TPEN couldefficiently chelate zinc in the embryos. For this, westained early Norway spruce somatic embryos grown

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with or without TPEN with Zinquin and analyzedthem by fluorescent microscopy. As shown in Figure2A, the level of free intracellular zinc decreased dra-matically in the embryonal masses of somatic embryosgrown in the medium supplemented with low con-centration of TPEN (1 mM), indicating good penetra-tion of the chelator into the densely packed cells ofthe embryonal masses. To ascertain that TPEN doesnot chelate other divalent cations, we stained TPEN-treated embryos with Fura 2-acetoxymethylester, whichrevealed strong accumulation of calcium in the em-bryonal mass cells (Supplemental Fig. S1B). Thus,TPEN at 1 mM appears not to chelate divalent cationsother than zinc, but rather stimulates influx of calcium,known as a primordial mechanism of cell death acti-vation (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 DNAfragmentation, a biochemical hallmark of plant em-bryonic PCD (Bozhkov et al., 2005a), in TPEN-treatedand control embryos using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)assay. Zinc-deprived embryos had increased frequencyof TUNEL-positive cells compared with control em-bryos (Fig. 2B). This effect seemed to be zinc specific,as copper chelation had no effect on the level of celldeath in embryos (Supplemental Fig. S2, A and B).While in the control embryos, TUNEL-positive cellswere mainly located in the suspensors, TPEN treat-ment induced DNA fragmentation in the embryonalmasses (Fig. 2C). To quantify this effect of TPEN, weestimated the frequency of embryos containing two

Figure 2. Depletion of intracellular zinc induces ectopic cell death in the embryos, leading to embryo lethality. Zinc chelatorTPEN was added to embryogenic cultures 4 d after withdrawal of PGRs and then after the following 3 d, concomitant with thestart of ABA-stimulated embryo maturation. Control embryos were grown without TPEN. The control and TPEN-treated embryoswere 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 fluorescentmicroscopy, 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) ofTUNEL-positive cells estimated using three samples per treatment, with 400 nuclei observed for each sample. C, Depletion of zincleads to ectopic cell death in the embryos, as revealed by in situ DNA fragmentation analysis. Note the abnormal accumulation ofTUNEL-positive cells in the embryonal mass (EM) of a TPEN-treated embryo. The images are representative examples of 20embryos stained with Zinquin. Scale bars 5100 mm. D, Massive cell death in the embryonal masses of TPEN-treated embryos, asrevealed 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.





or more TUNEL-positive cells in the embryonalmasses by observing 200 embryos for each treatment.The frequency increased from 12% in the controlembryos to 42% and 60% in the embryos treatedwith 1 mM and 5 mM TPEN, respectively. These resultsdemonstrate that zinc deprivation induces ectopic celldeath, which affects embryonal masses. Activation ofectopic cell death was further confirmed by stainingembryos with Evan’s blue, the vital dye excluded byliving cells but readily penetrating through compro-mised plasma membrane into dead and dying cells(Bozhkov et al., 2002). As shown in Figure 2D, TPEN-treated embryos exhibited unrestricted staining ofboth embryonal masses and suspensors, in contrastto control embryos, in which Evan’s blue staining wasrestricted to suspensors only. Zinc-depletion-inducedcell death in the embryonal masses had a lethal effect;no cotyledonary embryos were regenerated fromTPEN-treated cultures (data not shown).

Is the observed lethal effect of zinc depletion onNorway spruce embryos attributed to a general cyto-toxic effect of low concentrations of TPEN on plantcells? To answer this question, we tested the effect of1 mM and 5 mM TPEN on the level of intracellular zincand cell death in the developmentally arrested cell lineof Norway spruce, which is unable to form embryosand proliferates as proembryogenic masses regardlessof treatment (Smertenko et al., 2003; Supplemental Fig.S3). Although TPEN efficiently chelated zinc in thedevelopmentally arrested line already at 1 mM, thiseffect was not accompanied by increased DNA frag-mentation, indicating that less differentiated cells aremuch less sensitive to zinc deprivation compared withembryonic cells. Taken together, our data suggest thatfree intracellular zinc sustains cell viability in theembryonal masses, which is necessary for the normalprogression of embryogenesis.

Zinc Supplementation Impairs Embryo Patterning viaSuppression of Suspensor Cell Specification

Terminal differentiation and PCD are two interre-lated processes underlying suspensor cell identityduring plant embryogenesis (Bozhkov et al., 2005a).Genetic or pharmacological dysregulation of embry-onic pattern formation is often associated with theloss of suspensor cell identity, leading to developmen-tal aberrations and sometimes embryonic lethality(Vernon and Meinke, 1994; Rojo et al., 2001; Smertenkoet al., 2003; Bozhkov et al., 2004; Lukowitz et al., 2004;Suarez et al., 2004). The abrupt decline of zinc contentdetected by specific fluorophores in Norway sprucesuspensor cells (Fig. 1, B–D) might indicate that zincdeprivation is important for establishing suspensorcell identity and embryonic patterning.

To investigate the physiological significance of thelow zinc level in the suspensor, we attempted to coun-teract the naturally occurring decline of zinc in thesuspensor by growing embryos with increased concen-trations of zinc sulfate. Zinc supplementation treat-

ments were applied continuously, throughout all stagesof embryogenesis, beginning with early embryo devel-opment induced by withdrawal of PGRs until cot-yledonary embryo formation stimulated by ABA (Fig.1A). The early embryos induced to develop in PGR-free medium supplemented with 100 mM or 300 mM

zinc had a significantly lower proportion of TUNEL-positive cells than the control embryos growing with5 mM zinc (Fig. 3A). Furthermore, addition of equimo-lar amounts of copper sulfate could not reproduce thisdeath suppressive effect, and 300 mM copper sulfatewas even toxic to the embryos (Supplemental Fig.S2C). However, we could not detect any discerniblemorphological 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 sup-pressed embryogenesis (data not shown) and led to aslightly elevated frequency of TUNEL-positive cells(Fig. 3A), demonstrating that this supraphysiologicalconcentration of zinc is toxic to the embryos.

Early embryos require a regular supply of exoge-nous ABA to enter late embryogeny when primaryshoot and root meristems are set off and to accomplishmaturation (Filonova et al., 2000b; Bozhkov et al.,2002). Once embryos enter late embryogeny, the asym-metric cell divisions in the basal regions of the embry-onal masses cease and dead suspensors are graduallyeliminated (Fig. 1A; Filonova et al., 2000a, 2000b;Bozhkov et al., 2005a). We investigated whether highzinc-mediated suppression of embryonic PCD (Fig.3A) had a developmental effect on the transition fromearly to late embryogeny. For this, we analyzed thefrequency 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 ofABA into four major phenotypes distinguished bydevelopmental stage and apical-basal pattern (Fig. 3B):phenotype i, small polarized embryos in the beginningof early embryogeny, composed of less than 16 cells;phenotype ii, morphologically normal early embryoswith well-delineated rounded embryonal masses andmassive suspensors; phenotype iii, aberrant early em-bryos possessing elongated embryonal masses and alack of strict border between embryonal mass andsuspensor; and phenotype iv, late embryos withoutsuspensors or possessing remnants of suspensors. Theaberrant phenotype iii was caused by equal instead ofasymmetric cell divisions in the basal part of theembryonal masses. As a consequence, both halves ofthe daughter cells acquired meristematic identity andwere incorporated into the embryonal masses, thussuppressing suspensor cell specification. This pheno-type was very rarely observed among embryos fromcontrol cultures (less than 3%), which were mainlyrepresented by equal proportions of morphologicallynormal early embryos (phenotype ii) and late embryoswithout suspensors (phenotype iv; Fig. 3C). However,the frequency of the abnormal early embryos in-creased dramatically in the extra-zinc-supplementedcultures, accounting for 34% and 40% of all the em-

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Figure 3. Zinc supplementation impairs embryo patterning via suppression of suspensor cell specification. Zinc sulfate was added toembryogenic cultures at the time of withdrawal of PGRs and thereafter every 2 weeks during embryo maturation. Control embryoswere grown with 5 mM Zn-EDTA. A, Supplementation of the embryos with 100 mM or 300 mM zinc sulfate suppresses nuclear DNAfragmentation. Data show a mean frequency (6SEM) of TUNEL-positive cells assessed at 4 d after withdrawal of PGRs using fivesamples 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 zinc-supplemented cultures. The data show a mean frequency (6SEM) of embryos of a given phenotype assessed in seven samples pertreatment by counting 100 embryos in each sample. D, Zinc supplementation inhibits embryo maturation. The data show meannumber (6SEM) of cotyledonary somatic embryos per plate (10 plates per treatment) formed over 10 weeks on ABA-containingmedium. 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 embry-ogeny (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 zinclocalization. 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 panelsdisplay phase contrast and fluorescent microscopy, respectively. The images are representative examples of 20 embryos stained withZinquin after 7 d of the ABA treatment from both the control and zinc-supplemented cultures. Scale bars 5 100 mm.





bryos grown with 100 mM and 300 mM zinc sulfate,respectively (Fig. 3C). Concomitantly, the fraction oflate embryos decreased from 48% to 13% and 6%,respectively, indicating that abnormal early embryosare arrested at the transition to late embryogeny. As aresult, the yield of cotyledonary embryos in the extra-zinc-supplemented cultures decreased 2.5- to 5-foldcompared with the control (Fig. 3D). Interestingly, apartfrom cotyledonary embryos, there were a large numberof early embryos in the extra-zinc-supplemented cul-tures at the end of the embryo maturation treatment(Fig. 3E) as a consequence of zinc-stimulated accumu-lation of small embryos (phenotype i) at the earlier timepoint (Fig. 3, B and C).

We conclude that zinc supplementation to a certainlevel (up to 300 mM) greatly enhances cell survival,which can impair embryonic pattern formation. Stain-ing of abnormal early embryos (phenotype iii) withZinquin revealed abundant accumulation of zinc notonly in the embryonal masses but also in the suspensorcomposed of a mixture of densely cytoplasmic andvacuolated cells (Fig. 3F). Therefore, an abnormalembryo phenotype described in our study is a directconsequence of enhanced zinc accumulation, whichsuppresses terminal differentiation and PCD of theembryo suspensor.

Zinc Affects Metacaspase-Like Proteolytic Activityin the Embryos

The type-II metacaspase mcII-Pa is essential forNorway spruce embryogenesis, as it is required forestablishing 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 proces-sive Cys-dependent protease and, like other members ofthe metacaspase family proteases from plants, fungiand protozoa (Vercammen et al., 2004; Watanabe andLam, 2005; Gonzales et al., 2007; He et al., 2008), hasArg/Lys-specific proteolytic activity (Bozhkov et al.,2005b). We have previously reported that 10 mM zincabrogates activity of mcII-Pa in vitro (Bozhkov et al.,2005b). In this study, we asked whether zinc-mediatedregulation of cell death in the embryos correlates withthe 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 haspreviously been shown to be one of the preferredsubstrates for mcII-Pa both in vitro and in vivo(Bozhkov et al., 2005b). Zinc supplementation orTPEN treatments were applied simultaneously withwithdrawal of PGRs, and cell lysates were preparedfrom control (untreated) and treated cells at 0, 24, 48,96, and 168 h. As shown in Figure 4, A and B, normalembryo development in the control cultures was ac-companied by an increase in metacaspase-like activitybetween 96 and 168 h, coinciding with the period ofapical-basal pattern formation associated with termi-nal differentiation and death of the embryo suspensorcells (Filonova et al., 2000a). Zinc supplementationabrogated this increase; furthermore, the embryosgrown with 300 mM zinc sulfate displayed a moderatedecrease in metacaspase-like activity between 48 h to168 h (Fig. 4A). Consistent with this observation,chelation of free intracellular zinc by TPEN led to apronounced increase in metacaspase-like activity overthe initial period of 48 h, followed by a decrease to

Figure 4. Zinc affects metacaspase-like proteolytic ac-tivity in vivo. Zinc supplementation suppresses (A) andzinc deprivation strongly up-regulates (B) metacaspase-like activity in the embryos. Zinc sulfate or TPEN wasadded simultaneously with the withdrawal of PGRs,and samples were collected after 0, 24, 48, 96, and168 h. Metacaspase-like activity was measured usingan AMC-conjugated Boc-EGR peptide. Data for Boc-EGR-AMC cleavage represent the mean of three in-dependent experiments 6SEM. C, First-order kineticsof Boc-EGR-AMC cleavage by cell lysates preparedfrom control and zinc sulfate-treated embryos, whichwere collected after being grown for 7 d with ABAand microsurgically separated into suspensors (S) andembryonal masses (EM). Dotted lines denote 95%confidence limits.

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below control level (Fig. 4B). Evan’s blue stainingconfirmed that all cells were already dead at 168 h, sothe loss of metacaspase activity at the end of TPENtreatment was not surprising.

We next microsurgically separated embryos grownfor 7 d on ABA-containing medium into two parts,embryonal masses and suspensors, and measuredmetacaspase-like activity in the corresponding celllysates. Metacaspase-like activity was approximately2-fold higher in the lysates prepared from the suspen-sors compared with those from embryonal masses (Fig.4C). This activity was reduced in the both fractions by15% if the embryos were grown on the mediumsupplemented with 300 mM zinc sulfate. Collectively,our results establish metacaspase activation as one ofthe potential targets of zinc during zinc-mediatedsuppression of PCD in embryos.

DISCUSSION

Zinc has diverse functions in eukaryotic cells, whichextend from structural and/or catalytic roles of poorlyexchangeable zinc within metalloenzymes and zincfinger proteins to transient interactions of kineticallylabile zinc with cellular signaling pathways (Valleeand Falchuk, 1993). Since apoptosis of animal cells isreadily influenced by zinc deprivation or supplemen-tation, it has been postulated that it is more labile poolsof zinc that are involved in cell death regulation(Truong-Tran et al., 2001). This study has extended therole of zinc in the regulation of eukaryotic cell death toplants, and our results have three major implications.

First, distribution of zinc in plant embryos is devel-opmentally regulated and correlates with cell fatespecification during apical-basal pattern formation.We show that accumulation of zinc in the embryonalmasses and abrupt decline in the suspensors are tightlycontrolled and hallmark zinc homeostasis at earlystages of plant embryogenesis (Fig. 1, B–D). Dysregu-lation of zinc homeostasis causes developmental de-fects or lethality associated with the disruption of theproper balance between cell survival (in the embryonalmasses) and PCD (in the suspensors; Figs. 2 and 3).Further studies are required to unravel the molecularmechanisms of unequal redistribution of zinc in thetwo daughter cells resulting from asymmetric celldivisions during apical-basal pattern formation and,in particular, the role of zinc transporters.

Second, this is the first study in which the role of zincin the control of autophagic PCD has been addressed.Being a bona fide route of physiological cell death inplants, autophagic PCD is an evolutionarily ancient andphylogenetically conserved type of PCD from whichanimal-specific apoptosis is believed to have evolved(Zhivotovsky, 2002; Golstein and Kroemer, 2005;Bozhkov and Jansson, 2007). Finding a potent role forzinc in the regulation of autophagic PCD establisheszinc deprivation as a universal cell death signal, re-gardless of which route of degradation—apoptotic or

autophagic —is chosen by cells. Since autophagy hasa dual role in cell death and survival (Baehrecke,2005; Bassham, 2007), it appears especially interestingto determine whether zinc-deficiency-associated au-tophagy in the embryo suspensor implicates release offree zinc from catabolized metalloenzymes and zincfinger proteins in order to adjust and control the rate ofcell death.

Third, inactivation of several members of caspasefamily proteases (primarily caspases 3, 6, and 9) byzinc is thought to be the main process underlying theanti-apoptotic effect of zinc in mammalian cells(Truong-Tran et al., 2001). Although the exact bio-chemical mechanisms of caspase inhibition by zinc areunknown, it has been demonstrated that zinc blocksthe process of pro-caspase-3 activation rather than thealready activated enzyme (Truong-Tran et al., 2000). Itis proposed that zinc binds reversibly to the thiolgroup of catalytic Cys-163, inhibiting activation of theproenzyme and protecting the essential residue fromoxidation (Maret et al., 1999; Truong-Tran et al., 2001).Plants do not have close homologues of caspases butpossess the metacaspase family of Cys proteases,which have been proposed to be the ancestors ofmetazoan caspases (Uren et al., 2000). Despite the factthat metacaspases and caspases display low sequencesimilarity and have distinct substrate specificities,they have a conserved catalytic diad of His and Cys,and both require Cys-dependent processing of zymo-gen for enzyme activation (Vercammen et al., 2004;Bozhkov et al., 2005b). Here, we demonstrate that, likeactivity of mammalian caspases, plant metacaspaseactivity is suppressed by zinc in vivo (Fig. 4), suggest-ing that metacaspases could be one of the targets forzinc during zinc-mediated regulation of plant PCD.Suppression of metacaspase activity by zinc found invivo (this study) and in vitro (Bozhkov et al., 2005b)suggests another mechanism of posttranslational reg-ulation of metacaspases, in addition to the recentlyidentified S-nitrosylation of a catalytic Cys of Arabi-dopsis (Arabidopsis thaliana) metacaspase-9 (Belenghiet al., 2007). However, it remains to be investigatedwhether zinc binds to a critical Cys residue of meta-caspases to keep the enzyme in the unprocessedinactive form and/or to inactivate the already activeenzyme.

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 half-

strength 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 me-

dium for 1 week to induce embryo development (Filonova et al., 2000a). For

embryo maturation, the cultures were plated on solidified maturation me-

dium containing ABA (Filonova et al., 2000b). Cotyledonary somatic embryos

were harvested after 4 to 10 weeks of the maturation treatment.





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 proce-

dures for copper chelation and supplementation are provided in Supplemen-

tal 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 Labo-

ratories) 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#-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., 2000b).

Metacaspase-Like Activity Assay

Norway spruce cell extracts were prepared and assayed for metacaspase-

like 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 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 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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