Plant Molecular Biology 51: 509–521, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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Nuclease activities and DNA fragmentation during programmed cell death of megagametophyte cells of white spruce (Picea glauca) seeds Xu He and Allison R. Kermode∗ Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6 (∗ author for correspondence; e-mail [email protected]) Received 9 November 2001; accepted in revised form 10 August 2002

Key words: DNA fragmentation, megagametophyte, nucleases, Picea glauca, programmed cell death

Abstract The haploid megagametophyte of white spruce (Picea glauca) seeds undergoes programmed cell death (PCD) during post-germinative seedling growth. Death of the megagametophyte storage parenchyma cells was preceded by reserve mobilization and vacuolation. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling)-positive nuclei indicated that the first megagametophyte cells to die were those closest to the radicle at the micropylar end of the seed as well as those that comprised the most peripheral and innermost layers at the chalazal end of the seed. The death process was accompanied by nuclear fragmentation and internucleosomal DNA cleavage and the sequential activation of several nucleases. The latter comprised at least two groups: those induced relatively early during post-germinative seedling growth, that had pH optima in the neutral range (33, 31, 17 and 15 kDa), and those induced later that had pH optima in the acidic range (73, 62, 48, 43 and 29 kDa). Activities of all of the nucleases were stimulated by Ca2+ , Mg2+ and Mn2+ ; only the nucleases active at neutral pH were inhibited by Zn2+ . The temporal pattern of induction of the neutral and acidic nucleases may suggest that the latter function after tonoplast rupture. Abbreviations: PCD, programmed cell death; PMSF, phenylmethylsulfonyl fluoride; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling Introduction Programmed cell death (PCD) is a genetically controlled process that is essential to the development, differentiation and homeostasis of multicellular organisms (Ellis et al., 1991). Apoptosis is the most characterized form of PCD in animals. The distinctive features of apoptosis include cell shrinkage, membrane blebbing, nuclear fragmentation and the formation of apoptotic bodies (Beers, 1997). The fragmentation of DNA into internucleosomal fragments of about 180 bp is frequently used as a biochemical marker for apoptosis. Regardless of the form of PCD, the cell-death machinery includes a group of proteases, caspases, that initiate and execute the process as well as various other regulators. Once triggered, the process leads

to the orderly disassembly of dying cells (Thornberry and Lazebnik, 1998; Buckner et al., 2000). PCD occurs in various types of plant cells during development and in response to environmental stimuli (Greenburg et al., 1994; Mittler et al., 1997; Mittler, 1998; Pontier et al., 1998; Fath et al., 2000; Fukuda, 2000; Heath, 2000; Quirino et al., 2000; Wu and Cheun, 2000; Young and Gallie, 2000a). A series of orderly cell deaths occur during ovule development and embryogenesis when certain cells degenerate. Death of the starchy endosperm and aleurone layer cells of cereal grains occurs during seed development and post-germinative seedling growth, respectively. Some characteristic features of apoptosis occur during tracheary element differentiation, in tomato root cap cells, in senescing leaves, petals and anthers and in diploid parthenogenesis and early so-

510 matic embryogenesis. PCD also occurs when plants are subjected to biotic or abiotic stresses. Fungal infection can trigger the hypersensitive response in plants, a process characterized by rapid death of plant cells immediately surrounding the site of infection which effectively prevents spread of the pathogen. Comparative studies of PCD in plants and animals reveal both similarities and distinct features. One key difference is the absence of phagocytic cells in plants, which in animal tissues are partially responsible for preventing harmful imflammatory reactions associated with cell lysis. Although the mechanisms of PCD in plants are unknown, nuclear DNA fragmentation and the induction of proteases and nucleases appear to be key events (Aoyagi et al., 1998; Beers et al., 2000; Chen and Foolad, 1997; Pontier et al., 1998; De Jong et al., 2000; del Pozo and Lam, 1998; Delorme et al., 2000; Fath et al., 1999; Koukalova et al., 1997; Lam and del Pozo, 2000; Mittler and Lam, 1995, 1997; Schmid et al., 1999; Solomon et al., 1999; Sugiyama et al., 2000; Vierstra, 1996). The PCD that occurs in seed tissues at a specific post-germinative stage of seedling growth has been most extensively studied in the aleurone layer cells of cereal grains (Wang et al., 1996b, 1998; Bethke et al., 1999; Fath et al., 1999, 2000; Bethke and Jones, 2001). The megagametophyte of white spruce (Picea glauca) seeds is living at seed maturity and is the major storage organ of the seed. Following germination, the megagametophyte cells die after the storage reserves are mobilized. The present study characterizes the programmed death of white spruce megagametophyte cells by examining post-germinative changes in tissue and cellular morphology, induction of nuclease activities and DNA fragmentation.

Materials and methods Plant materials White spruce (Picea glauca) seeds of seed lots 37862 and 39619 were obtained from the Tree Seed Centre in Surrey, B.C., Canada. Seeds were surface-sterilized in 1.5% hydrogen peroxide for 20 min, rinsed several times in sterile distilled water and imbibed in sterile distilled water for 24 h. Seeds were then placed in seed boxes (Hoffman Manufacturing Co., Albany, OR) containing one Kimpak (Hoffman Manufacturing Co.) and one Whatman No. 1 filter paper and 45 ml of sterile distilled water and moist chilled for 3

weeks at 4 ◦ C in darkness. Seeds were then transferred to germination conditions (30 ◦ C days/20 ◦ C nights, 8 h photoperiod; light intensity 25 µmol m−2 s−1 , PAR 400–700 nm). At different time points after germination, seeds were dissected under a dissecting microscope into megagametophytes or embryo axes (radicle, hypocotyl and cotyledons) and the seed parts were used fresh or were immediately frozen in liquid nitrogen or dry ice and stored at −80 ◦ C until use. Protein extraction for determination of nuclease activities Megagametophytes were ground in a glass homogenizer in extraction buffer (150 mM Tris, 1 mM DTT, 0.5 mM PMSF (phenylmethylsulfonyl fluoride), 20 µM leupeptin, pH 6.8; Fath et al., 1999) on ice. The homogenate was centrifuged at 12 000 rpm for 10 min and the supernatant was used for nuclease activity assays as outlined below. Protein was determined by the BioRad Dc protein assay (Alam, 1992) with bovine serum albumin fraction V (BioRad, Mississauga, Canada) as a standard. Viability assay Megagametophytes at different time points following germination were stained with 1% Evans Blue for 1 min, destained with deionized water for 1 h and photographed under a dissecting microscope. In-gel nuclease activity and ribonuclease activity assays In-gel nuclease activity assays were performed essentially according to Thelen et al. (1989) and Mittler and Lam (1995). Briefly, protein extracts were fractionated on 12.5% SDS-PAGE gels containing DNA (50 µg/ml heat-denatured salmon sperm DNA [Sigma Chemical, St. Louis, MO] or native calf thymus DNA [Clontech, Palo Alto, CA]) or RNA (50 µg/ml total RNA extracted from tobacco leaves) and 50 µg/ml bovine fibrinogen (Sigma-Aldrich Canada, Oakville, Canada). After electrophoresis, the gels were washed twice with 25% isopropanol in 50 mM Tris pH 6.8, 1 mM CaCl2 , 1 mM MgCl2 and 10 µM ZnCl2 and twice with buffer (50 mM Tris pH 6.8, 1 mM CaCl2 , 1 mM MgCl2 and 10 µM ZnCl2 ). After overnight incubation at 37 ◦ C, activities were detected by staining the gels in 0.02% toluidine blue followed by destaining in 10 mM Tris pH 7.5. Pre-stained markers (New England Biolabs, Mississauga, Canada) were used to

511 estimate the sizes of the nucleases. To determine the optimum pH for the nuclease activities, the gels were renatured and incubated under the above conditions; however, in this case they were incubated in buffers having different pH values: pH 5.0 (50 mM sodium acetate), pH 6.0 (50 mM MOPS), pH 6.8 (50 mM Tris-HCl) and pH 7.5 (50 mM Tris-HCl). To examine the inhibitory effects of EDTA and EGTA on the nuclease activities, gel slices were incubated in the buffer containing 0.1 mM EDTA or 0.1 mM EGTA. Cation requirements of the nuclease activities were determined by treating gel slices with 0.1 mM EDTA or 0.1 mM EGTA for 45 min and then incubating them overnight at 37 ◦ C in either Ca2+ , Mg2+ , Mn2+ or Zn2+ added to a final concentration of 1 mM.

DNase activity) was added and the solution incubated at 37 ◦ C for 30 min. The samples were extracted with an equal volume of chloroform/isoamyl alcohol (24:1); after centrifugation, the supernatant was removed and the DNA was precipitated by adding 45 µl 7.5 M ammonium acetate and 900 µl of 100% ethanol. After washing the DNA pellet with 70% ethanol, the pellet was dissolved in T10 E1 buffer (10 mM TrisHCl, 1 mM EDTA, pH 8.0). DNA concentrations were determined spectrophotometrically. DNA samples (15 µg) were loaded on a 1.8% agarose gel for analysis of DNA fragmentation. After electrophoresis, the gel was stained with ethidium bromide. The entire experiment was repeated three times (i.e. with different batches of megagametophytes at the various stages after germination).

Determination of nuclease activities in solution The assay is based on the capacity of DNases to degrade DNA into acid-soluble products (Thelen and Northcote, 1989). Protein extract (5 µl) was added to 150 µl buffer (50 mM Tris pH 6.8, 1 mM CaCl2 , 1 mM MgCl2 and 10 µM ZnCl2 ) containing 100 µg/ml heatdenatured salmon sperm DNA. After a 15 min incubation at 37 ◦ C, the reaction was stopped by adding 20 µl 20% TCA. After incubation on ice for 30 min, the samples were centrifuged at 12 000 rpm for 10 min and the O.D. of the supernatant was measured at 260 nm. The relative nuclease activity for each sample was calculated by determining the difference in O.D. (at 260 nm) between samples with substrate and those without substrate. DNA isolation and electrophoresis Genomic DNA was extracted from megagametophytes according to the protocol of Dellaporta (1983) with some modifications. Megagametophytes (ca. 0.1 g) were ground to a fine powder with a mortar and pestle in liquid N2 . Extraction buffer (0.8 ml of 100 mM Tris-HCl pH 8.0, 50 mM EDTA, 500 mM NaCl, 1% SDS and 7 µL/ml 2-mercaptoethanol) was added, followed by 0.8 ml of phenol and chloroform (1:1, v/v). Samples were centrifuged at 12,000 rpm for 10 min and the supernatant was further extracted with an equal volume of chloroform/isoamyl alcohol (24:1). Total DNA was precipitated by adding 1/10 vol 7.5 M ammonium acetate and 2/3 vol isopropanol to the supernatant. After centrifugation (10 min at 12 000 rpm), the pellet was washed in 70% ethanol and dissolved in 500 µl of T50 E10 buffer (50 mM Tris-HCl, 10 mM EDTA, pH 8.0). RNAse (5 µl, 10 mg/ml, free of

Light microscopy and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay Megagametophytes at different stages were fixed in FAA solution (10% formaldehyde: 5% acetic acid/45% ethanol) at 4 ◦ C overnight. Dehydration was performed with a graded series of ethanol: 30%, 50%, 70%, 80%, 90%, 95%, and 100%; 1 h each step. Megagametophytes were incubated in 100% ethanol overnight at 4 ◦ C and then taken through a graded series of Hemo-De (Fisher Scientific, Pittsburgh, PA) in ethanol: 30%, 50%, 70%, 85%, 95%, 100%; 1 h each step. Finally, megagametophytes were embedded in paraffin (TissuePrep 2; Fisher Scientific, Pittsburgh, PA) with a graded series of paraffin: 30%, 50%, 70%, 85%, 95%, 100%; 2 h each step at 60 ◦ C. The megagametophytes were infiltrated with 100% paraffin overnight at 60 ◦ C and later sectioned with a rotary microtome to a thickness of 7 µm. The sections were mounted on Superfrost Plus slides (Fisher Scientific) and incubated overnight at 45 ◦ C. Paraffin was removed from sections using a graded series of Hemo-De in ethanol: 30%, 50%, 70%, 85%, 95% and 100%; 2 min each step; rehydration was achieved through a graded series of ethanol solutions: 100%, 95%, 85%, 70%, 50%, 30%; 2 min per step. The sections were washed in PBS buffer and then digested with protease K (20 µg/ml) for 20 min at 30 ◦ C. The labeling of fragmented DNA was performed according the manufacturer’s instructions (Apoptosis detection system, fluorescein; Promega, Madison, WI). For the negative control, terminal transferase was omitted in the reaction. As a positive control, the sections

512 were first treated with DNase I for 10 min after protease K digestion. The sections were counterstained with 1 µg/ml of 4,6-diamidino-2-phenylindole (DAPI, Sigma Chemical) for 15 min at room temperature, washed with PBS, mounted in anti-FADE medium (n-propyl-gallate, 6.15 mg/ml in PBS buffer containing 50% glycerol) and examined under a fluorescence microscope (Olympus Vanox AHBS3; Olympus Optical Co., Tokyo). A standard fluorescence filter set was used to view the fluorescence from fluorescein staining (520±20 nm; green) and from DAPI staining (450±20 nm; blue).

Results Morphological changes of the megagametophyte after seed germination After imbibition and during the early stages of postgerminative growth of the embryo, the megagametophyte was white and shiny, but its appearance gradually changed during later post-germinative growth, first becoming transparent and then brownish (Figure 1A I, II and III). Eventually, the tissue was diminished to the extent that it comprised only a thin layer detached from the cotyledons. Morphological changes at the cellular level were observed using light microscopy (Figure 1C and 1D). The megagametophyte cells surrounding the radicle at the micropylar end collapsed just after the completion of germination (radicle emergence) (data not shown). Soon after germination, when the seed’s radicle was ca. 2 mm long, most megagametophyte cells were full of storage organelles (protein bodies and oil bodies) (Figure 1C I and 1D a). However, the cells innermost to the embryo as well as the outermost layer of megagametophyte cells adjacent to the nucellus became vacuolated (Figure 1C I and 1D b and c). As post-germinative seedling growth continued, there was a progressive decline of storage organelles across the megagametophyte tissue (Figure 1C II), indicating mobilization of the lipid and protein reserves. More cells became vacuolated at this stage. Loss of megagametophyte cell structure was evident at later stages of seedling growth (Figure 1C III and D d). Progressive cell death was indicated by viability staining of megagametophytes with Evans Blue dye. Living cells are able to exclude the dye (and thus cells remain unstained), while any dead cells lose membrane integrity and stain blue. As shown in Figure 1E I,

the few megagametophyte cells surrounding the radicle at the micropylar end died first when the seed’s radicle was ca. 2 mm long (i.e. just after the completion of germination), while most cells were alive. The proportion of dead megagametophyte cells did not increase significantly until the length of the seed’s radicle and hypocotyl reached ca. 15 mm; at this time, only the inner layers of the megagametophyte tissue (i.e. those closest to the embryo) were dead (Figure 1E II). Extensive cell death occurred at a later stage of post-germinative growth, when the length of the radicle, hypocotyl and cotyledons was >30 mm (Figure 1E III). Thus, not surprisingly, extensive cell death did not occur until well after reserve mobilization was completed; once reserves were depleted, death of the megagametophyte cells rapidly ensued. DNA fragmentation in post-germinative megagametophyte cells Nuclear DNA fragmentation is a distinct feature of PCD of animal and plant cells and can be detected in situ by the TUNEL assay. The assay is based on labeling of fragmented nuclear DNA at the 3 -OH end by using terminal deoxynucleotidyl transferase and dUTP conjugated to a fluorochrome. The extent of green fluoresence reflects DNA degradation. The same sections were counterstained with DAPI; blue fluoresence indicates the presence of nuclei. After moist chilling of mature white spruce seeds, but prior to germination, no labeled nuclei were detected in megagametophyte cells (compare Figure 2a and b), despite staining of all nuclei in the positive control (i.e. those sections treated with DNase I) (compare Figure 2c and d). TUNEL-positive nuclei were first detected soon after germination (when the seed’s radicle was ca. 2 mm) in the megagametophyte cells closest to the radicle at the micropylar end of the seed as well as those that comprised the most peripheral and innermost layers at the chalazal end of the seed (Figure 2e and g); the nuclei of the remaining (TUNEL-negative) cells stained only with DAPI (Figure 2f and h). At later stages after germination (i.e. in seeds in which the radicle and hypocotyl was 10–15 mm), TUNEL-positive nuclei appeared in most cells at the chalazal end of the seed (Figure 2i), but were not yet detected to any significant extent in the megagametophyte cells comprising the central region of the seed (Figure 2k). TUNELpositive nuclei were observed in all the megagametophyte cells at a late post-germinative stage when the seed’s radicle, hypocotyl and cotyledons were ca.

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Figure 1. Morphological changes and viability staining of the megagametophyte during different stages following seed germination. In A, B, C and E, megagametophytes taken from seeds with a radicle of ca. 2 mm (stage I), from seeds with a radicle and hypocotyl of ca. 15 mm (stage II) and from seeds with radicle, hypocotyl and cotyledons >30 mm (stage III). A. Changes in the general appearance of the megagametophyte at the different stages. Bar represents 1 mm. B. Seedlings at the three stages examined. C. Light micrographs of the megagametophyte at the chalazal end of the seed during the different stages after germination. Megagametophytes were fixed and sectioned with a microtome; the sections were observed with differential interference-contrast (DIC) optics. Arrow in micrograph at stage III indicates mucous mass (largely pectinaceous material) resulting from cell wall hydrolysis of the dead megagametophyte cells. Bar represents 50 µm. D. Megagametophyte cells at post-germinative stages shown at a higher magnification using DIC optics. a. Megagametophyte cells full of storage organelles (protein bodies/protein storage vacuoles and oil bodies). b. Megagametophyte cells becoming vacuolated. Arrows indicate larger vacuoles that may result from the coalescence of protein storage vacuoles. c. Megagametophyte cells in a highly vacuolated state. d. Megagametophyte cells showing loss of cell structure. Bar represents 10 µm. E. Viability staining of megagametophytes at stages I–III. Bar represents 1 mm.

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Figure 2. In situ detection of nuclear DNA degradation by TUNEL assay. Five stages of the megagametophyte are shown, including those of mature seed following moist chilling (a–d); seed with a radicle of ca. 2 mm (e–h); seed with a radicle and hypocotyl of 10–15 mm (i–l); seed with radicle, hypocotyl and cotyledons of 20–25 mm (m–p), and seed with radicle, hypocotyl and cotyledons of >30 mm (q–t). a, TUNEL-labeled section (em, embryo; mg, megagametophyte); b, section a counterstained with DAPI; c, TUNEL-positive control (section treated with DNase I) (ct, cotyledons; mg, megagametophyte); d, section c counterstained with DAPI; e, TUNEL-labeled section, showing megagametophyte at chalazal end of seed (ct, cotyledons; mg, megagametophyte); f, DAPI staining of section e; g, TUNEL-labeled section showing megagametophyte at micropylar end of seed; h, DAPI staining of section g; I, TUNEL-labeled section, showing megagametophyte at chalazal end of seed; j, DAPI staining of section i; k, TUNEL-labeled section showing central region of megagametophyte; l, DAPI staining of section k; m, TUNEL-labeled section, showing megagametophyte at chalazal end of seed; n, DAPI staining of section m; o, TUNEL-labeled section showing a megagametophyte at the micropylar end of the seed; p, DAPI staining of section o; q, TUNEL-labeled megagametophyte; r, DAPI staining of section q (arrows indicate the mucous mass, largely pectinaceous material resulting from cell wall hydrolysis of the dead megagametophyte cells); s, higher magnification of TUNEL-labeled nuclei in cells at the latest stage examined; t, DAPI staining of section s. Bars represent 50 µm.

515 25 mm (Figure 2m and o). At this time, the innermost cells had already collapsed and contained no nuclei. Later, the nuclei were further degraded as indicated in Figure 2 (q and r) (i.e. at this stage, most of the cells did not contain nuclei). The TUNEL-positive nuclei appeared before cell collapse, but after storage reserves were mobilized and the cells were highly vacuolated (compare Figure 1C with Figure 2). No fragmentation of nuclei was evident at any stage (Figure 2s and t); in contrast, fragmentation of nuclei is a common characteristic of apoptosis of animal cells. The temporal and spatial degradation of megagametophyte nuclei indicates that the process of nuclear DNA degradation is a tightly controlled process. Fragmentation of nuclear DNA into ca. 180 bp fragments has generally been viewed as the hallmark of apoptosis. To examine this in white spruce megagametophyte cells, DNA was isolated from megagametophytes at different stages after germination. DNA laddering was observed when megagametophytes were derived from seeds in which the radicle and hypocotyl length was ≥15 mm (Figure 3). Increased nuclease activities accompany DNA degradation Increased nuclease activities are associated with nuclear DNA fragmentation in plant and animal PCD; thus we investigated the induction of nuclease activities in megagametophyte cells after seed germination. Before germination, nuclease (DNase) activities were barely detectable (Figure 4, numerical data in lower panel, stage 1). After germination, nuclease activities increased (on a per megagametophyte basis) and remained at a similar level until the seed’s radicle and hypocotyl were ca. 10 mm; thereafter, nuclease activities increased again reaching a peak at a stage when the seedling (radicle, hypocotyl and cotyledons) was ca. 20 mm (Figure 4, numerical data in lower panel, stage 6). The increase in DNase activities was correlated with the decrease in DNA amount (Figure 4, numerical data in lower panel). To further investigate qualitative changes in nuclease activities associated with the process of nuclear DNA fragmentation, in-gel nuclease activity assays were carried out (Figure 4, upper panels). As shown in Figure 4, the nuclease activities became more abundant or were induced in the megagametophyte when the length of the seed’s radicle and hypocotyl was ca. 15 mm (stage 5), which was consistent with the rise in activities determined by the in-solution assay.

Figure 3. Agarose gel electrophoresis of megagametophyte DNA to show fragmentation. Lanes 1–5, DNA isolated from megagametophytes taken from seeds at different stages after germination as follows; lane 1, mature seeds after moist chilling; lane 2, seeds with radicle 2 mm; lane 3, seeds with radicle and hypocotyl ca. 15 mm; lane 4, seeds with radicle, hypocotyl and cotyledons ca. 25 mm; lane 5, seeds with radicle, hypocotyl and cotyledons >30 mm; M, 1 kb DNA markers.

Moreover, the nucleases active at an acidic pH appeared much later than those active at a more neutral pH (pH 6.8) (Figure 4A and B). The nucleases active at the neutral pH likely function at an earlier stage, i.e. before the cytoplasm becomes acidified or before tonoplast rupture, while the nucleases active at more acidic pHs may augment DNA fragmentation, particularly after tonoplast burst when the cytosol becomes acidic. Characteristics of the nuclease activities Ca2+ - and Mg2+ -dependent nucleases have been implicated in animal and plant PCD; therefore we examined the characteristics of the nucleases induced

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Figure 4. Upper panels (A and B). In-gel assay of nuclease activities at pH 6.8 and pH 5.0. Protein (5 µg) was loaded in each lane. After electrophoresis, the gels were renatured and incubated in 50 mM Tris-HCl buffer (pH 6.8, 1 mM CaCl2 , 1 mM MgCl2 and 10 µM ZnCl2 ) (A), or in 50 mM sodium acetate buffer (pH 5.0, 1 mM CaCl2 , 1 mM MgCl2 and 10 µM ZnCl2 ) (B). Lanes 1–9: protein extracts from megagametophytes taken from seeds at different stages after germination (1, mature seeds after moist chilling; 2, seeds with a radicle of 2 mm; 3, seeds with a radicle of ca. 5 mm; 4, seeds with a radicle and hypocotyl of ca. 10 mm; 5, seeds with a radicle and hypocotyl of ca. 15 mm; 6, seeds with radicle, hypocotyl and cotyledons of ca. 20 mm; 7, seeds with radicle, hypocotyl and cotyledons of ca. 25 mm; 8, seeds with radicle, hypocotyl and cotyledons of ca. 30 mm; 9, seeds with radicle, hypocotyl and cotyledons ≥35 mm; M, pre-stained protein markers (kDa). Lower panel (numerical data). Nuclease activities (in-solution assay) and extractable DNA content of megagametophytes at the corresponding stages on a per megagametophyte basis. Data are based on the average of 3 replicates ± S.D. N/A, extractable DNA beyond stage 7 was extremely low; meg, megagametophyte.

517 Table 1. Characteristics of the nuclease activities in the megagametophyte of white spruce seeds at late post-germinative stages.a Nuclease (kDa)

73 62 48 43 29 33 31 17 15

Substrateb

Cation restores activityc

Inhibition

ss DNA

ds DNA

RNA

EDTA

EGTA

Zn2+

Ca2+

Mg2+

Mn2+

+ + ++ + + ++ ++ + +

± − + − + ± + ± +

− ± − + + − + + +

+ + + + + + + + +

+ + + + + + + + +

− − − − − + + + +

± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

+ + + + + + + + +

pH optimum

5.0–5.5 5.0–5.5 5.0 5.0 5.0 6.8–7.5 6.8–7.5 6.8–7.5 6.8–7.5

a radicle, hypocotyl and cotyledons ≥25 mm. b ++ preferred; ± weak. c ± partial restoration of activity after EDTA

inhibition; + full restoration of activity after EDTA inhibition.

in megagametophyte cells with respect to their ion requirements. At least nine nuclease activities were detected in the megagametophyte at an advanced postgerminative stage (Table 1). The pH optima of the nuclease activities were determined by measuring their activities at four pHs (5.0, 6.0, 6.8 and 7.5) as noted in Materials and methods. Five nucleases (ca. 73, 62, 48, 43 and 29 kDa) were most active at pH 5.0, showing no activity at pH ≥6.0. The other nucleases (ca. 33, 31, 17 and 15 kDa) had pH optima of 6.8–7.5, showing very little activity at pH 5.0. EDTA and EGTA inhibited all the nuclease activities (Table 1), while Ca2+ and Mg2+ stimulated them (data not shown). After 0.1 mM EDTA or EGTA treatment, partial activities of the nucleases could be restored by incubation with 1 mM Ca2+ or Mg2+ ; however, 1 mM Mn2+ led to complete restoration of the activities (Table 1). Zn2+ was not inhibitory at 10 µM (data not shown), but inhibited some nuclease activities (33, 31, 17 and 15 kDa) at ≥100 µM (Table 1). RNA or double-stranded DNA was also used in in-gel activity assays to investigate the substrate specificity of the detected nuclease activities. Three nucleases, with sizes ca. 48, 33 and 31 kDa, preferred single-stranded DNA as a substrate, but could also digest double-stranded DNA. Two nucleases (ca. 29 and 15 kDa) showed no significant substrate preference, digesting double-stranded DNA, single-stranded DNA and RNA. Western blot analyses were conducted to investigate whether any of the nucleases of post-germinative white spruce megagametophyte cells was related to other PCD-related nucleases, particularly that impli-

cated in PCD of barley aleurone cells (nuclease I) (Fath et al., 1999). The monoclonal antibody to barley nuclease I (Brown et al., 1986) recognized a ca. 29 kDa protein at a late post-germinative stage of white spruce megagametophyte cells (when seedlings were ca. 30 mm; data not shown). This correlates temporally with detection of a ca. 29 kDa nuclease on the activity gel (Figure 4B); thus, the ca. 29 kDa white spruce nuclease may be related to barley nuclease I.

Discussion Unlike the endosperm of angiosperm seeds, the nutritive storage tissue of conifer seeds, the megagametophyte, is a haploid tissue. The megagametophyte develops from one functional megaspore derived from meiosis of the megaspore mother cell; the remaining three megaspores closest to the micropyle degenerate. The early stage of megagametophyte development involves mitosis of the megaspore without cell wall formation. When the free nuclei accumulate to about 2000, the cell walls begin to form (Raven et al., 1999). After fertilization, during the expansion stage of seed and embryo development, the megagametophyte cells begin to accumulate reserves, primarily lipids and proteins (Owens et al., 1992). Some of the megagametophyte cells die during embryo maturation leaving a cavity between the embryo and the megagametophyte (Owens et al., 1992; Tillman-Sutela and Kauppi, 2000). The remaining megagametophyte cells are living at seed maturity and serve as a food reserve for the growing axis during post-germinative seedling

518 growth. We investigated the death of the megagametophyte cells of white spruce seeds after germination. The megagametophyte tissue underwent distinct morphological changes; its white and shiny appearance was lost and the tissue was diminished to a dry thin layer. Viability staining indicated that the megagametophyte cells surrounding the radicle at the micropylar end of the seed died shortly after germination (radicle emergence), while the majority of the cells of the megagametophyte died at a very late post-germinative stage (i.e. in seedlings in which the length of the radicle, hypocotyl and cotyledons was >25 mm). Consistent with other plant systems (Bethke et al., 1999; Fath et al., 1999, 2000; Schmid et al., 1999; Fukuda, 2000; Filonova et al., 2000), vacuolation of megagametophyte cells occurred before death. DNA degradation is an integral part of PCD in both plants and animals. During apoptosis, DNA degradation appears to involve two sequential steps: nuclear DNA is first cleaved into 300–50 kb fragments; these fragments are then broken down further into ca. 180 bp internucleosomal fragments (Walker et al., 1993; Walker and Sikorska, 1994). Although internucleosomal DNA cleavage is not always observed during PCD of animal and plant cells (Mittler and Lam, 1997; Sikorska and Walker, 1998; Fath et al., 1999, 2000; Fukuda, 2000), degradation of nuclear DNA into high molecular weight fragments is a consistent finding. Internucleosomal DNA laddering has been widely reported during PCD of seed tissues, for example, in maize and wheat endosperm cells during seed development (Young et al., 1997; Young and Gallie, 1999, 2000a, b) and in barley aleurone cells following germination (Wang et al., 1996b, 1998; but see also Fath et al., 1999). It has also been reported in developing anthers (Wang et al., 1999), during carpel and petal senescence (Orzaez and Granell, 1997; Xu and Hanson, 2000), in mannose-induced cell death of maize cell cultures (Stein and Hansen, 1999), during somatic embryo formation (Filonova et al., 2000), and during plant defense responses to biotic and abiotic stimuli (Ryerson and Heath, 1996; Wang et al., 1996a; Koukalova et al., 1997). More interestingly, random and internucleosomal DNA cleavage are observed during different treatments of embryogenic cultures of carrot; the same cultures being responsive or nonresponsive to death-inducing signals depending upon their physiological states (LoSchiavo et al., 2000). In the present study, nuclear DNA of megagametophyte cells underwent degradation as demonstrated by both TUNEL assays and agarose gel electrophore-

sis. TUNEL-positive nuclei were first detected in the megagametophyte cells closest to the radicle at the micropylar end of the seed as well as those that comprised the most peripheral and innermost layers at the chalazal end of the seed. The central regions of the megagametophyte were the last to exhibit TUNELpositive nuclei. TUNEL-positive nuclei occurred in cells only after their reserves were mobilized and the cells appeared highly vacuolated, but prior to a loss of cell membrane integrity. The temporal and spatial pattern of storage reserve mobilization, followed by nuclear DNA degradation, indicates that the death of megagametophyte cells is a tightly controlled process. Internucleosomal DNA cleavage occurred in megagametophyte cells during a post-germinative stage when cell death was also obvious by viability staining (in seeds with a radicle/hypocotyl length of ca. 15 mm). Thus it is not entirely clear whether internucleosomal DNA cleavage occurred as an integral part of the cell death mechanism or as a part of ‘corpse’ removal. TUNEL-positive nuclei appeared earlier than DNA laddering, suggesting that the former arise as a result of the labeling of the free 3 -OH ends of high molecular weight DNA fragments. Alternatively, the internucleosomal DNA fragments at an earlier stage represent only a small fraction of the total DNA such that DNA laddering is not visible on the agarose gel until a later stage. In animal PCD, three classes of nucleases have been implicated in nuclear DNA fragmentation (reviewed in Counis and Torriglia, 2000 and references therein). The first group comprises Ca2+ - and Mg2+ dependent nucleases with diverse molecular weights; they require Ca2+ and Mg2+ for maximal activity, have pH optima in the neutral range and are also activated by Mn2+ and inhibited by Zn2+ . The second group comprises Mg2+ -dependent DNases, which include caspase-activated DNases (CAD). Acid endonucleases or cation-independent DNases constitute the third group. The nucleases so far characterized in plant PCD have been classed as Ca2+ , Mg2+ -dependent or Zn2+ -dependent (Sugiyama et al., 2000). The former function at a more neutral pH, while the latter function at pH 5.0–5.5. BEN 1 (barley endonuclease 1, also referred to as barley nuclease I) and ZEN 1 (zinnia endonuclease 1) are Zn2+ -dependent nucleases associated with PCD of barley aleurone layer cells and death of differentiating tracheary elements, respectively (Thelen and Northcote, 1989; Fath et al., 1999). The genes encoding these two nucleases have been cloned (Aoyagi et al., 1998); the deduced amino acid

519 sequences of the plant nucleases show significant similarities to each other, but exhibit no homology to the nucleases involved in animal PCD. Some types of plant PCD appear to involve Ca2+ , Mg2+ -dependent nucleases (Mittler and Lam, 1995, 1997; Stein and Hansen, 1999; Xu and Hanson, 2000). However, generally several nuclease activities are induced in each system, an exception being mannose-induced cell death of cultured maize cells, in which only one nuclease is found (Stein and Hansen, 1999). Whether there is a conservation of some of the nucleases associated with different types of plant PCD is presently unknown. Reduced DNA content during post-germinative death of the megagametophyte of white spruce was strongly correlated with increased activities of nucleases which presumably are responsible for the breakdown of nuclear DNA. Very little nuclease activity was present in mature moist-chilled seed. However, following the transfer of seeds to germination conditions, several nuclease activities were sequentially induced, some arising early and increasing in abundance during later post-germinative stages, others being induced during relatively late stages. While most of the nucleases may be synthesized de novo after germination, there could be some activation of pre-existing enzymes. The nuclease activities of the post-germinative megagametophyte were all inhibited by EDTA or EGTA (i.e. they require certain cations for their activity). All were stimulated by Ca2+ , Mg2+ and Mn2+ , and some of them, those active at neutral pH, were strongly inhibited by Zn2+ at ≥100 µM. The results suggest that these nucleases belong to the Ca2+ , Mg2+ -dependent class. Other nucleases were active at a more acidic pH; however, these were not stimulated by Zn2+ . Despite this, their pH preference and other characteristics (e.g. a distinct preference for singlestranded DNA and RNA over double-stranded DNA), may indicate that they belong to the Zn2+ -dependent group of nucleases (see review by Sugiyama et al., 2000). There was a temporal separation of the induction of the activities of the neutral and acidic nucleases, with the former induced earlier than the latter. We propose that degradation of genomic DNA of megagametophyte cells involves both groups of nucleases, with the earlier group leading to the detection of the first TUNEL-positive nuclei. Although TUNELpositive nuclei appeared early, the marked decline of DNA occurred at a later post-germinative stage, which is consistent with the synergistic increase of nuclease activities. Acidification of the cytosol would ac-

company tonoplast rupture at a later post-germinative stage; thus more nucleases are activated at this time to assist in further DNA degradation. A subject of controversy is which specific forms of PCD in plants can be classed as apoptosis. It is also unclear what events of the DNA fragmentation process are conserved between different types of plant PCD and between plant and animal PCD. The notion that the internucleosomal phase of DNA fragmentation is a conserved or essential step in apoptosis is no longer supported (Sikorska and Walker, 1998). Even a consideration of apoptosis alone reveals that different cell types have different death signaling pathways; furthermore, different pathways exist even in the same type of cell death (Sikorska and Walker, 1998). Plant cells have unique subcellular structures as compared to animal cells, including the presence of a semi-rigid cell wall, vacuoles and plastids. Thus processes that are integral to the active process of the initiation and execution of cell death as well as to the removal of the ‘corpse’ are likely to be somewhat unique. As with other key processes in life, cell death will likely also have some conserved key players. To what extent plant and animal PCD are alike remains to be determined; both are active processes. Plant PCD based on cytological features has been grouped into three categories (Fukuda, 2000): (1) apoptosis-like PCD; (2) PCD, in which vacuole plays a central role (e.g. death of tracheary elements) and (3) senescence (e.g. leaf senescence). The features of PCD of white spruce megagametophyte cells include vacuolation, nuclear DNA fragmentation and internucleosomal DNA cleavage, and sequential activation of several nucleases. Protease activation, caspase-like protease activities, and poly (ADP-ribose) polymerase (PARP) cleavage are also integral components of this process (He and Kermode, unpublished data). Thus, the post-germinative death of haploid megagametophyte cells of a gymnosperm seed shares some common characteristics with the PCD of other plant cells.

Acknowledgements We appreciate the help in obtaining mature seed of white spruce from Dave Kolotelo of the Tree Seed Centre (B.C. Ministry of Forests, Surrey, B.C.). We are also grateful to T.-H. David Ho (Washington University) for providing the nuclease I antiserum. This research was supported by a Natural Sciences and En-

520 gineering Research Council of Canada (NSERC) grant awarded to A.R.K.

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