Plant Molecular Biology 52: 729–744, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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Proteases associated with programmed cell death of megagametophyte cells after germination of white spruce (Picea glauca) seeds Xu He and Allison R. Kermode∗ Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, B.C., Canada V5A 1S6 (∗ author for correspondence; e-mail [email protected]) Received 9 November 2002; accepted in revised form 17 March 2003

Key words: caspase-like proteases, cysteine endoproteases, megagametophyte, Picea glauca, programmed cell death Abstract During post-germinative seedling growth, the major storage organ of the white spruce (Picea glauca) seed, the megagametophyte, undergoes programmed cell death (PCD). Protease activities in megagametophyte cells that arise post-germinatively were investigated. The accumulation of protease activities can be divided into two phases: the first phase correlated with degradation of storage proteins while the second phase was temporally associated with cell death, although some of the early proteases were also active during the later phase. Proteases induced during PCD were mainly serine and cysteine proteases. One of the PCD-associated cysteine proteases had homology to Cys-EP, a PCD-related cysteine protease of the castor bean endosperm. Transcripts encoding a Cys-EP-related protein were not present in megagametophytes when seeds were imbibed, nor were they present during germination and early post-germinative growth (radicle length ca. 2–5 mm). At a later post-germinative stage (i.e when the seed’s radicle was ca. 15 mm), the Cys-EP-related transcripts (ca. 1.3 kb) became abundant and, at this time, the 48 kDa proform of the enzyme first appeared. The mature form of the Cys-EP (ca. 38 kDa) was predominant at a very late stage of post-germinative growth. Immunocytochemistry showed that the Cys-EPrelated protein was localized to spherical organelles (ca. 2 µm) that may be equivalent to the ‘ricinosomes’ of castor bean endosperm cells. Caspase-like protease (CLP) activities were first detected 3 days after germination with the caspase-specific substrate Ac-DEVD-AMC; maximum activities occurred when the seed’s radicle was ca. 20–25 mm. When germinated seeds were treated with a caspase-3 inhibitor, both the peak of CLP activities and the death of megagametophyte cells were delayed. We propose that the Cys-EP-related protein and CLP activity are involved in PCD of white spruce megagametophyte cells. Abbreviations: Ac-DEVD-AMC, N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin; Ac-DEVD-CMK, Nacetyl-Asp-Glu-Val-Asp-chloromethylketone; Ac-YVAD-AMC, N-acetyl-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin; CLP, caspase-like protease; PCD, programmed cell death; PMSF, phenylmethylsulfonylfluoride

Introduction Programmed cell death (PCD), a triggered event in which the cell kills itself from within, is an active process that is an integral part of the development, differentiation and homeostasis of multicellular organisms (Ellis et al., 1991). Proteases are known to play important roles in PCD. In animal systems, part of the death machinery involves caspases, cysteine pro-

teases that specifically cleave target proteins after an Asp residue. These proteases are not only involved in the initiation phase, but also in the signal transduction and execution phases of apoptosis (Thornberry and Lazebnik, 1998). More specifically, caspases act at several levels to mete out the death process (reviewed in Cohen, 1997). ‘Initiator’ caspases act earlier on in the pathway to pronounce the death sentence; they are activated in response to signals indicating

730 that the cell has been damaged or has received an order to die. Through a complex activation cascade (in which inactive caspase precursors serve as substrates for other caspases), a signal transduction pathway leads to the activation of the executioner caspases. The latter cleave various cellular or nuclear proteins, generally either activating them or inactivating them. Some of the targets are key structural proteins (e.g. nuclear lamins); in the cleaved state, these proteins are no longer able to maintain the integrity of the nucleus. Other targets, inactivated by cleavage, include enzymes involved in DNA repair. Ultimately, there is an orderly dismantling of the cell; some of the more obvious features of this destruction include chromatin condensation, DNA fragmentation and changes to organelles such as mitochondria. Alterations of the cell membrane lead to a fragmentation of the cell and the cellular debris is ingested by phagocytes. In addition to caspases, serine proteases and metalloproteases have also been implicated in animal PCD (Beers et al., 2000). In the mammalian immune system, a specific protease called granzyme B (a serine protease) functions to set up target cells for destruction by killer T cells, effecting this destruction by activating at least six different caspases (reviewed in Cohen, 1997). In plants, little is known about the exact roles of proteases in PCD. However, cysteine proteases, serine proteases, aspartic proteases and caspase-like proteases (also cysteine proteases; see above) are associated with plant PCD in different model systems (reviewed in Beers et al., 2000; Lam and del Pozo, 2000). Among these endopeptidases, cysteine proteases have received the most attention with respect to their putative role in plant PCD. These enzymes have been correlated temporally with leaf and flower senescence (Lohman et al., 1994; Jones et al., 1995; Drake et al., 1996; Griffiths et al., 1997; Guerrero et al., 1998; Stephenson and Rubinstein, 1998; Xu and Chye, 1999), death induced by oxidative stress (Solomon et al., 1999) and death occurring as a preprogrammed event (e.g. tracheary element differentiation and post-germinative aleurone cell death; Minami and Fukuda, 1995; Ye and Varner, 1996; Beers and Freeman, 1997; Swanson et al., 1998; Groover and Jones, 1999). More recently, the accumulation and release of the cysteine endopeptidase, Cys-EP, from ricinosomes (cysteine protease-containing organelles) has been associated with PCD of the endosperm of castor bean seeds following germination (Schmid et al., 1999). The proform of this Cys-EP contains a KDEL sequence at its C-terminus and is localized to an ER-

derived organelle, the ‘ricinosome’. The mature form is released to the cytosol from the disrupted ricinosomes during endosperm cell death (Schmid et al., 2001; Gietl and Schmid, 2001). The involvement of caspase-like protease (CLP) activities in plant PCD was first reported during the hypersensitive-response induced by pathogen attack of tobacco leaves (del Pozo and Lam, 1998). More recently, they have been linked to chemical- and stressinduced death of suspension cells and protoplasts (Sun et al., 1999; de Jong et al., 2000). Apoptosis-like cleavage of poly (ADP-ribose) polymerase (PARP), carried out by plant cell extracts in vitro, and changes in purified nuclei have been observed (D’Silva et al., 1998; Zhao et al., 1999). However, to date, no caspase-like proteases or CLP-encoding genes have been isolated from plants. The megagametophyte of white spruce seeds is a living tissue at seed maturity, but undergoes PCD after germination. The present study investigates some of the proteases associated with this process. In addition to characterizing the protease activities of white spruce megagametophyte cells, the induction, processing and localization of a Cys-EP-related protein was examined. Finally, the effects of a caspase-3 inhibitor on CLP activities and death of megagametophyte cells were investigated. 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 night, 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 embryos (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.

731 Protein extraction for SDS-PAGE analysis of soluble and insoluble proteins Proteins were extracted from megagametophytes at different stages by grinding in extraction buffer (50 mM Tris-HCl pH 7.4, 0.5 mM phenylmethylsulfhonylfluoride [PMSF], 20 µM leupeptin, 10 µM pepstatin A, 1 mM EDTA) on ice. The supernatant (soluble protein fraction) was removed after centrifugation at 4 ◦ C (10 min, 12 000 rpm). The pellet was solubilized with the above buffer containing 2% SDS and 10% glycerol and boiled for 5 min (Gifford et al., 1982; Misra and Green, 1990); after centrifugation (10 min, 12 000 rpm), the supernatant contained the insoluble protein fraction. Protein amount was determined by the BioRad Dc protein assay (Alam, 1992) with bovine serum albumin fraction V (BioRad, Mississauga, Canada) as a standard. Protease activity assays Ten megagametophytes excised from seeds at different stages after germination were ground in a glass homogenizer in extraction buffer (50 mM Tris-HCl pH 7.5, 20 mM NaCl, 1 mM DTT, 0.1% Triton X-100) on ice. The homogenate was centrifuged at 12 000 rpm for 10 min and the supernatant was used for protease activity assays. In-solution assays of protease activities were performed with azocasein as substrate (Stephenson and Rubinstein, 1998). Of the extract 10 µl was added to 150 µl buffer (50 mM sodium acetate pH 5.5) containing 500 µg/ml azocasein. After 4 h of incubation at 37 ◦ C, the reaction was stopped by adding 50 µl 20% TCA. After 20 min on ice the reaction mixture was centrifuged for 10 min at 12 000 rpm and the optical density of the supernatant determined at 340 nm (A340). Relative activity was determined by the formula:
A340 = A340 (enzyme extract plus substrate) – A340 (enzyme extract without substrate) – A340 (substrate without enzyme extract). Each assay was carried out in duplicate on 3 sets of megagametophytes at each stage. In-gel activity assays were carried out according to Ye and Varner (1996) and Bethke et al. (1996). Briefly, protein extracts were fractionated on 10% SDS-PAGE gels containing 0.08% gelatin or 0.03% acidified hemoglobin in the separating gel. After electrophoresis, the gels were washed twice (30 min each wash) with 50 mM sodium acetate pH 5.5 containing 0.1% Triton X-100. After two washes with the above buffer without Triton X-100, the gels were incubated at 37 ◦ C overnight. The activity was detected by staining the gels with Coomassie

blue followed by de-staining in 25% methanol solution containing 0.25% acetic acid. Pre-stained markers (Sigma Chemical, St. Louis, MO) were used to estimate the sizes of the proteases. To determine the optimum pH for the protease activities, the gels were renatured and incubated under the above conditions except the incubation buffers were at pH 3.5 (50 mM sodium succinate), pH 4.5 (50 mM sodium acetate), pH 5.5 (50 mM sodium acetate), pH 6.5 (50 mM Tris-HCl), and pH 7.5 (50 mM Tris-HCl). Immunocytochemical detection of the cysteine protease, Cys-EP 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%, and 100%, 1 h each step. Finally, megagametophytes were embedded in paraffin (TissuePrep 2; Fisher Scientific) with a graded series of paraffin: 30%, 50%, 70%, 85%, 95%, and 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 with 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 ethanol solutions: 100%, 95%, 85%, 70%, 50%, and 30%, 2 min per step. Immunolabelling of the sections was carried out as described by Schmid et al. (1999). Sections were permeabilized in 5% Tween-20 in phosphate-buffered saline (PBS) for 1 h at room temperature, and rinsed in PBS. Sections were then incubated in 2% BSA (in PBS containing 0.05% Tween-20) for 1 h to block nonspecific sites. The primary antibody raised against the Cys-EP (kindly provided by C. Gietl; diluted 1:100 in blocking solution) was applied to the sections overnight at 4 ◦ C. Pre-immune serum was used for the negative controls. Sections were washed three times in PBS containing 0.05% Tween-20 and incubated with a goat anti-rabbit secondary antibody conjugated to FITC (Sigma, 1:80 dilution in the blocking solution)

732 for 3 h in the dark at room temperature. After rinsing in PBS three times, sections were mounted in anti-FADE medium (n-propyl gallate 6.15 mg/ml in PBS buffer containing 50% glycerol) and observed under a fluorescent microscope. Images were captured and analyzed with a Sony 3 CCD Color Video Camera (Sony, Tokyo, Japan) and the Northern Eclipse software (Empix Imaging, Mississauga, Canada). Western blot analysis Proteins were extracted from megagametophytes at different stages as described above (SDS-PAGE analysis of soluble proteins). Samples with equal protein (20 µg) were fractionated on 10% SDS-PAGE gels (Laemmli, 1970) and then electroblotted onto nitrocellulose membranes. Membranes were blocked with 5% skim milk powder in PBS containing 0.05% Tween20 (1 h at room temperature) and then incubated in the primary antibody (anti-Cys-EP or anti-KDEL [Stressgen, Vancouver, Canada], both diluted 1:1000 in 3% skim milk powder) for 2 h at room temperature. The blots were then washed three times in PBS containing 0.05% Tween-20 (each 20 min) and incubated in goat anti-rabbit secondary antibody conjugated to alkaline phosphatase (BioRad). After three washes (20 min each) in PBS containing 0.05% Tween-20, immunodetection was achieved with NBT and BCIP as substrates. Northern blot analysis Total RNA was extracted from megagametophytes of white spruce seeds at different stages before and after germination according to the method of Wang et al. (2000). Total RNA (10 µg) was loaded into each lane and RNA samples were fractionated on 1.0% agarose formaldehyde gels. After transfer onto Hybond-N nylon membrane (Amersham Life Science, Buckinghamshire, UK), RNA was fixed onto the membrane by UV cross-linking. Membranes were hybridized with a 32 P-labeled Cys-EP cDNA probe (cDNA kindly provided by C. Gietl); labeling was achieved with the RTS RadPrime DNA Labelling System (Life Technologies, Gaithersburg, MD) with [α-32 P]-dCTP. Blots were rehybridized with an 18S rDNA probe (a 401 bp cDNA clone from Sitka spruce) to standardize equal amounts of RNA loading.

Caspase-like protease activity assay Caspase-like protease (CLP) activity assays were performed according to Talanian et al. (1997) and del Pozo and Lam (1998) with some modifications. Proteins were extracted from megagametophytes (10 megagametophytes/stage) by grinding in a glass homogenizer with extraction buffer (50 mM sodium acetate pH 5.5, 20% glycerol, 1 mM EDTA, 1 mM DTT, 0.2% BSA and 1 mM PMSF) on ice. Of the enzyme extract 25 µl was added to 25 µl 150 µM AC-DEVD-AMC (caspase-3 substrate) dissolved in the above buffer. The reaction was stopped by adding 20 µl of 1 M HCl after 4 h of incubation at 30 ◦ C. The fluorescence was measured with a Hoefer TKO 100 Fluorometer at an excitation/emission wavelength of 365/460 nm. The relative activities of CLP were determined by the formula:
λ460 = λ460 (enzyme extract plus substrate) – λ460 (enzyme extract without substrate) – λ460 (substrate plus extraction buffer without enzyme extract). Each assay was carried out in duplicate with 3 sets of megagametophytes at each stage. To determine inhibition of CLP activities by different inhibitors (aprotinin, pepstatin, E-64, leupeptin, Ac-DEVD-CHO, or Ac-YVAD-CHO), CLP activity assays were conducted in the presence of 20 µM inhibitor (final concentration). To determine the optimal pH of the CLP activities, proteins were extracted from megagametopytes using buffers at different pHs and the assay was carried out at the corresponding pH (buffers included: 50 mM sodium succinate pH 3.5 and 4.5; 50 mM sodium acetate pH 5.0, 5.5 and 6.0; 50 mM Tris-HCl pH 6.5, 7.0 and 7.5). To avoid the inhibitory effect of salt concentration on CLP activities (Korthout et al., 2000), 50 mM HEPES pH 7.5 and 50 mM MOPS pH 6.0 were also tested. No significant differences were observed between the Tris-HCl and HEPES buffers (pH 7.5) and between the MOPS and sodium acetate buffers (pH 6.0). Treatment of germinated seeds with caspase-3 inhibitor Germinated seeds (with radicle lengths of ca. 2 mm) were treated with water, or 100 µM Ac-DEVD-CMK (a cell permeable caspase-3 inhibitor); the solution was replaced every 2 days. Megagametophytes were sampled at different times following treatment and analyzed immediately (viability, DNA fragmentation and CLP activity assays).

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Figure 1. Post-germinative stages and the timing of programmed cell death of megagametophyte cells of white spruce seeds. A. White spruce seed at an early post-germinative stage (radicle ca. 4 mm). B. Viability staining (Evans Blue) of longitudinal sections of megagametophytes from seeds at different stages (a, seed with radicle ca. 2 mm; b, seed with radicle and hypocotyl ca. 15 mm; c, seed with radicle, hypocotyl and cotyledons ca. 25 mm; d, seed with radicle, hypocotyl and cotyledons ca. 35 mm). Dead cells stain blue as a result of a lack of membrane integrity. C. Internucleosomal DNA cleavage of genomic DNA extracted from megagametophytes of seeds at different post-germinative stages. Stages: 1, seeds with radicle ca. 2 mm; 2, seeds with radicle and hypocotyl 10–15 mm; 3, seeds with radicle, hypocotyl and cotyledons 20–25 mm; 4, seeds with radicle, hypocotyl and cotyledons 25–30 mm. Experiments did not include any later post-germinative stages because the DNA content decreased about 7-fold. M = DNA markers.

Viability assays Cross sections of megagametophytes at different time points following water or caspase-3 inhibitor treatment were stained with 1% Evans Blue for 1 min and destained with deionized water for 1 h. Isolation and electrophoresis of DNA to assess fragmentation Genomic DNA was extracted from megagametophytes by 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/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/iso-amyl 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). A 5 µl portion of RNAse (10 mg/ml, free of 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 100% ethanol. After washing the DNA pellet with 70% ethanol, the pellet was dissolved in T10 E1 buffer (10 mM Tris-HCl, 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. Results Megagametophyte viability and internucleosomal cleavage of nuclear DNA upon seed germination The haploid megametophyte of white spruce seeds is living at seed maturity and is the major storage tissue

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Figure 3. Analysis of protease activities (using azocasein as a substrate) in the megagametophyte of white spruce seeds before and after germination. Proteins were extracted from megagametophytes of seeds at different stages (1, before germination; 2, radicle ca. 2 mm; 3, radicle ca. 5 mm; 4, radicle ca. 10 mm; 5, radicle and hypocotyl ca. 15 mm; 6, radicle, hypocotyl and cotyledons ca. 20 mm; 7, radicle, hypocotyl and cotyledons ca. 25 mm; 8, radicle, hypocotyl and cotyledons ca. 30 mm; 9, radicle, hypocotyl and cotyledons ≥35 mm). Data are based on the mean of three replicates ± SD.

Protease activities increase upon germination of white spruce seeds

Figure 2. Coomassie blue-stained SDS-PAGE profiles of proteins from megagametophytes of white spruce seeds before and after germination. A. Soluble proteins. B. Insoluble proteins. Proteins were extracted from megagametophytes of seeds at different stages (1, before germination; 2, radicle ca. 2 mm; 3, radicle ca. 5 mm; 4, radicle ca. 10 mm; 5, radicle and hypocotyl ca. 15 mm; 6, radicle, hypocotyl and cotyledons ca. 20 mm; 7, radicle, hypocotyl and cotyledons ≥25 mm). Arrows on the left indicate major storage proteins. Sizes of the protein markers are indicated on the right (kDa).

surrounding the embryo (Figure 1A). After germination, the megagametophyte cells at the micropylar end of the seed and closest to the embryo died first (Figure 1B, a). Cell death progressed within this tissue at the later post-germinative stages, such that at a stage when the length of the seed’s radicle, hypocotyl and cotyledons was >25 mm (Figure 1B, c and d), most megagametophyte cells stained blue due to a lack of membrane integrity. Internucleosomal DNA cleavage of nuclear DNA, which is characteristic of PCD in animals and also occurs during some forms of plant PCD, occurred during the process of megagametophyte cell death. DNA laddering became evident at stages when the length of the seed’s radicle, hypocotyl and cotyledons was ≥15 mm (Figure 1C).

Megagametophyte cells accumulate storage proteins in protein bodies during seed maturation. The major storage proteins are insoluble proteins with molecular masses of ca. 42, 36 and 21 kDa; the soluble storage proteins are ca. 27 kDa and ca. 24 kDa (Figure 2) (Misra and Green, 1990). Most of these storage proteins were mobilized during the period between germination and when the length of radicle and hypocotyl was ca. 15 mm (Figure 2; stages 2–5). Protease activities of the megagametophyte were examined during and after seed germination (Figure 3). Two phases of increased protease activities were observed (Figure 3): the first phase corresponded with storage protein mobilization (compare Figure 2 to Figure 3); the second phase was coincident with the death of the megagametophyte cells as indicated by viability staining (compare Figure 1B to Figure 3). In-gel activity assays were conducted to assess qualitative changes in protease activities during the different post-germinative stages. As shown in Figure 4, the post-germinative proteases exhibited preferences for different substrates and different activities at pH 4.5 and pH 5.5. Some of the protease activities were detected exclusively at early post-germinative stages (e.g. the ca. 100 kDa protease in Figure 4A and the ca. 65 kDa protease in Figure 4C). Other protease activities were present prior to germination (i.e. in mature moist-chilled seeds) and persisted during the earlier post-germinative stages (e.g. the ca. 200 kDa protease

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Figure 4. In-gel activity assays with gelatin (A and B) or hemoglobin (C and D) as substrate. Megagametophyte protein extract (10 µl) was loaded in each lane. Stages 1–9 correspond to those shown in Figure 2. Sizes of the pre-stained protein markers are indicated on the left (kDa). White asterisks indicate protease activities associated with earlier post-germinative growth; square brackets on the right indicate protease activities associated with later post-germinative growth.

in Figure 4B, the ca. 97 kDa protease in Figure 4C and the ca. 45 kDa protease in Figure 4D). These proteases are likely involved in storage protein mobilization. Later post-germinative seedling growth was accompanied by the induction of different protease activities (e.g. those associated with bands ca. 45 kDa and 62– 83 kDa in Figure 4A and proteases of 30–38.5 kDa in Figure 4C). These proteases may be more related to the programmed death of megagametophyte cells. Since cysteine and serine proteases have been implicated in the programmed death of plant cells (see Introduction), we examined the protease activities in megagametophytes during late post-germinative stages (when the seed’s radicle, hypocotyl and cotyledons was >25 mm) by using zymograms and classspecific protease inhibitors. As shown in Figure 5A and B, five protease activities, with sizes ranging from 60 to 120 kDa, showed reduced activities in the presence of 1 mM PMSF (a serine protease inhibitor). These are likely serine proteases since their activities

Figure 5. Analysis of protease activities in the megagametophyte at a late post-germinative stage with specific inhibitors. A and C show activities in buffer without inhibitors as controls; B and D show activities in buffer containing 1 mM PMSF (B) or 50 µM E-64 (D). Arrows on the right indicate the inhibited protease activities. Sizes of the pre-stained protein markers are indicated on the left (kDa).

were not affected by pepstatin A (an aspartic protease inhibitor) or E-64 (a cysteine protease inhibitor) (data

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Figure 7. Western blot analysis of the white spruce Cys-EP-related protein in megagametophyte protein extracts using antibodies directed against (A) KDEL and (B) Cys-EP. Proteins were extracted from megagametophytes of seeds at two late post-germinative stages (1, radicle, hypocotyl and cotyledons ca. 25 mm [putative pro-form and mature Cys-EP present]; 2, radicle, hypocotyl and cotyledons ≥30 mm [primarily mature Cys-EP present]).

not shown). Six proteases, with apparent molecular masses in the range of 20–48 kDa, are likely cysteine proteases; their activities were inhibited by E-64 (Figure 5C and D), but not by PMSF (Figure 5B), pepstatin A, or EDTA (data not shown) and were enhanced by 2mercaptoethanol (data not shown). These six protease activities were induced or became more abundant after storage proteins were mobilized, suggesting they may be involved in the process of megagametophyte cell death. A Cys-EP-related protein is induced in megagametophyte cells after germination Figure 6. A. Northern blot analysis showing the steady-state levels of transcripts encoding the white spruce Cys-EP-related protein in megagametophytes before and after germination. Total RNA was extracted from megagametophytes at different stages (1, before germination; 2, radicle 2–5 mm; 3, radicle and hypocotyl ∼10 mm; 4, radicle, hypocotyl and cotyledons ∼15 mm; 5, radicle, hypocotyl and cotyledons ∼20 mm; 6, radicle, hypocotyl and cotyledons ∼25 mm; 7, radicle, hypocotyl and cotyledons ∼30 mm; 8, radicle, hypocotyl and cotyledons ∼35 mm). RNA (10 µg) was loaded in each lane. The blot was hybridized with the Cys-EP cDNA probe (top); a DNA probe encoding 18S rRNA was used to standardize equal loading (bottom). When the seed’s radicle, hypocotyl and cotyledons were ca. 35 mm, both Cys-EP mRNA and 18S rRNA exhibited degradation. The size of the Cys-EP-related transcripts is ca. 1.3 kb. B. Western blot analysis of Cys-EP-related protein in white spruce megagametophyte protein extracts with antibody directed against castor bean Cys-EP. Proteins were extracted from megagametophytes of seeds at different stages (as indicated in A). The expected sizes of the proform of Cys-EP (45 kDa) and the mature form of Cys-EP (35 kDa) appear as larger bands: 48 kDa and 38 kDa, respectively, under our gel conditions. C. In-gel protease activity assay of the protein (ca. 38 kDa) corresponding to the putative mature form of Cys-EP. The 38 kDa protein was electro-eluted from the SDS-PAGE gel and analyzed by the in-gel activity assay. Gel slices were incubated in 50 mM NaOAc buffer (pH 5.5) (a), and in the same buffer containing 50 µM E-64 (b).

Cys-EP, a cysteine protease, has been implicated in PCD of castor bean endosperm cells following seed germination (Schmid et al., 1999). To investigate whether the cysteine protease activities of white spuce megagametophyte cells are related to this CysEP, northern and western blot analyses were performed. Transcripts encoding the Cys-EP-related protease were not present in megagametophytes of mature seeds upon moist chilling, nor were they present during germination and early post-germinative growth (radicle length ca. 2–5 mm) (Figure 6A). Transcripts (ca. 1.3 kb) became detectable when the seed’s radicle and hypocotyl were ca. 10 mm and became very abundant at later post-germinative stages (ca. 25– 30 mm) (Figure 6A). Thereafter, the steady-state level of Cys-EP transcripts declined, likely as a result of enhanced turnover of total RNA. At post-germinative stages more advanced than those shown in Figure 6A, the amount of extractable RNA was extremely low, presumably as a result of cell death.

737 The putative pro-form of Cys-EP (48 kDa) first became detectable in the megagametophyte when the seed’s radicle and hypocotyl were ca. 15 mm, and at a later post-germinative stage (ca. 20 mm), the putative mature form of Cys-EP (38 kDa) became detectable (Figure 6B). At the stage when the seedling was >35 mm, the Cys-EP-related protein was cleaved into smaller peptides (Figure 6B). The 38 kDa protein corresponding to the putative mature form of Cys-EP was electroeluted from the SDS-PAGE gel and its activity assessed by an in-gel protease activity assay. The protein exhibited protease activity which was inhibited by E-64 (a cysteine protease inhibitor), suggesting that it is a cysteine protease (Figure 6C). A key feature of the Cys-EP of castor bean is that the proform has a KDEL sequence (Schmid et al., 1999, 2001). Western blot analysis confirmed that the Cys-EP-related protein of white spruce megagametophyte also has a KDEL sequence in its putative proform and the KDEL sequence is cleaved in the putative mature form (Figure 7). In castor bean endosperm cells, the proform of the Cys-EP is accumulated in ‘ricinosomes’; the mature form is released from these organelles at a late post-germinative stage, upon ‘ricinosome’ disruption (Schmid et al., 1999, 2001). Immunocytochemical studies showed that the Cys-EP-related protein of white spruce megagametophyte cells was not detectable at an early stage after germination (Figure 8a). When the length of the seed’s radicle and hypocotyl was ca. 10 mm, the protein was detected in megagametophyte cells close to the cotyledons (Figure 8c). Later, in megagametophytes from seeds in which the radicle, hypocotyl and cotyledons were ca. 20 mm (Figure 8e), the protein was abundant in all parts of the megagametophyte tissue. At this stage, the fluorescent-labeled discrete bodies at the periphery of cells located within the central parts of the megagametophyte tissue suggest the biogenesis of ricinosomelike organelles containing the Cys-EP-related protein in the megagametophyte cells. The diffuse labeling in the outer and inner layers of the megagametophyte (i.e. those located closest and farthest away from the cotyledons) may indicate the release of the protease from the ‘disintegrated’ ricinosome-like organelles. Caspase-like protease activities increase during PCD of megagametophyte cells Caspase-like protease (CLP) activities were barely detectable in the megagametophyte before seed ger-

mination. After germination, they gradually increased reaching a maximum in the megagametophyte when the length of the seed’s radicle, hypocotyl and cotyledons was ca. 25 mm (Figure 9, stage 7). This is a time when the megagametophyte cells are dying as indicated by viability staining and TUNEL assays (He and Kermode, 2003). The CLP activities were inhibited by the specific inhibitor Ac-DEVD-CHO (an inhibitor more specific for caspase-3) and cysteine protease inhibitors, E-64 and leupeptin, but not by aprotinin (a serine protease inhibitor) or by pepstatin A (an aspartic protease inhibitor) (Figure 10A). The CLP activities were also inhibited to a lesser extent by AcYVAD-CHO (an inhibitor more specific for caspase-1) (Figure 10A); however, there were no detectable activities when AC-YVAD-AMC (a synthetic peptide substrate for caspase-1) was used as a substrate (data not shown). The optimal pH of the CLP activities was 5.0, and there were little detectable activities when the pH was neutral (Figure 10B). These results may indicate that the CLP activities are not localized in the cytoplasm; alternatively, they may function when the cytoplasm becomes acidified before or after tonoplast rupture. Taken together, our results suggest that the CLP activities detected in the post-germinative megagametophyte are distinct from caspases, but exhibit some similarities to CLP activities induced during other forms of plant PCD (reviewed in Thornberry and Lazebnik, 1998; Lam and del Pozo, 2000). Caspase-3 inhibitor treatment attenuates PCD of megagametophyte cells To investigate whether the CLP activities are associated with PCD of megagametophytes, germinated seeds with radicle lengths of ca. 2 mm were treated over an 8-day period with water or 100 µM AcDEVD-CMK (a cell permeable caspase-3 inhibitor). As indicated in Figure 11A, the peak of CLP activities in inhibitor-treated megagametophytes was delayed 1 day as compared to that in the water-treated controls. Viability staining of 4-day inhibitor-treated and 4-day water-treated megagametophytes indicated that the inhibitor attenuated the extent of programmed death of the inner cell layers of the megagametophyte (Figure 11A inset). Internucleosomal DNA cleavage accompanies the PCD of megagametophyte cells; thus, we also examined the degradation of nuclear DNA within the inhibitor-treated and water-treated megagametophytes. In the presence of the inhibitor, advanced

738

Figure 8. Immunocytochemical analysis of the white spruce Cys-EP-related protein in megagametophytes after seed germination. Sections are of post-germinative megagametophytes at different stages (a and b, radicle ca. 2 mm; c and d, radicle and hypocotyl ca. 10 mm; e and f, radicle, hypocotyl and cotyledons ca. 20 mm.). Sections a, c and e were labeled with Cys-EP antibody and the Cys-EP protein was detected with an FITC-conjugated secondary antibody. Sections b, d and f are negative controls which were incubated in pre-immune serum. Arrows indicate the inner side of the megagametophyte, close to the cotyledons. Bar represents 50 µm.

DNA degradation was delayed by 1 day (5 days vs. 6 days), as was DNA laddering (6 days vs. 7 days) (Figure 11B).

Discussion Temporal separation of protein reserve mobilization from cell death Megagametophyte cells of white spruce seeds accumulate storage proteins during seed maturity and these proteins are mobilized upon germination. After reserve mobilization is completed, megagametophyte

739

Figure 9. Analysis of caspase-like protease activities in protein extracts of megagametophytes (1, before germination; 2, radicle ca. 2 mm; 3, radicle ca. 5 mm; 4, radicle ca. 10 mm; 5, radicle and hypocotyl ca. 15 mm; 6, radicle, hypocotyl and cotyledons ca. 20 mm; 7, radicle, hypocotyl and cotyledons ca. 25 mm; 8, radicle, hypocotyl and cotyledons ca. 30 mm; 9, radicle, hypocotyl and cotyledons ≥35 mm). Data are based on the mean of three replicates ± SD.

cells undergo PCD (He and Kermode, 2003). Numerous proteases are involved in degradation of the storage proteins and in the cell death process. Two phases of increased protease activities occurred in the megagametophyte: the first phase corresponded to the stage of storage protein degradation; the second phase coincided with cell death. Some of the proteases active at earlier stages (as indicated by in-gel activity assays) were different from those active at later stages. Five serine protease and six cysteine protease activities (ca. 60–120 kDa and 20–48 kDa, respectively) were identified in the post-germinative megagametophytes at the later stages. These protease activities were either induced or became more abundant when megagametophyte cells were dying, suggesting that they play a role in cell death. Subcellular localization of the specific proteases induced during early and later post-germinative stages may help distinguish their respective roles in storage protein mobilization versus cell death. Potential involvement of a KDEL-containing cysteine protease in the programmed death of white spruce megagametophyte cells

Figure 10. Characteristics of the caspase-like protease (CLP) activities in white spruce megagametophytes. A. Inhibition of CLP activities by different protease inhibitors. CLP activities were measured in the presence of aprotinin, pepstatin, E-64, leupeptin, Ac-DEVD-CHO and Ac-YVAD-CHO, all at a concentration of 20 µM. B. Optimal pH of the CLP activities in megagametophytes at a late post-germinative stage (when the length of the radicle, hypocotyl and cotyledons was ca. 25 mm). Data are based on the mean of three replicates ± SD.

Cysteine proteases have been associated with different forms of plant PCD (reviewed in Beers et al., 2000). One of the cysteine proteases in the late postgerminative white spruce megagametophyte is related to Cys-EP, a cysteine protease associated with PCD of the endosperm of castor bean seeds. This cysteine protease was synthesized de novo after germination. The transcripts and Cys-EP-related protein were detected at a stage when the majority of storage proteins had been degraded. The putative proform of the megagametophyte Cys-EP-related protein has a KDEL sequence, while the putative mature form does not. Immunolocalization studies indicated that the Cys-EP-related protein was localized to punctate bodies that may be ricinosome-like organelles in white spruce megagametophyte cells, similar to those of endosperm cells of the castor bean seed (Schmid et al., 1999). In seed storage tissues, the degradation of storage proteins also involves cysteine proteases, but these proteases should be co-localized with storage proteins to protein storage vacuoles (as occurs in barley aleurone cells) (Bethke et al., 1996), or at the least, their accumulation should be coordinated with storage protein degradation. Although we cannot rule out the possibility that the Cys-EP-related protein of white spruce plays a role in protein reserve mobilization, the

740

Figure 11. Effects of a caspase-3 inhibitor on CLP activities and programmed cell death of white spruce megagametophyte cells. A. CLP activities in megagametophyte protein extracts from caspase-3 inhibitor-treated (100 µM) or water-treated seeds. Data are based on the mean of three replicates ± SD. The inset in A shows viability staining of cross sections of megagametophytes (after 4 days of treatment): a, water-treated; b, caspase-3 inhibitor-treated. Blue staining indicates dead cells. B.Agarose gel electrophoresis of megagametophyte DNA to show fragmentation. DNA was isolated from megagametophytes taken from seeds after 3–7 days of treatment. M: DNA markers.

temporal and spatial characteristics of its accumulation are not consistent with such a role. Rather, its major function may be in the programmed death of white spruce megagametophyte cells. Cysteine proteases with a carboxyterminal KDEL are unique to plant cells and have been detected in ER-derived precursor protease vesicles (PPVs) (Chrispeels and Herman, 2000). Some of the cysteine proteases involved in storage reserve mobilization including vicilin peptidohydrolase of mung bean (Vigna radiata) seeds and SH-EP of black gram (Vigna mungo) seeds are accumulated in cytoplasmic ‘foci’ or ‘KDEL-tailed cysteine protease-accumulating vesicles (KVs)’ (Chrispeels et al., 1976; Baumgartner et al., 1978; Okamoto and Minamakawa, 1998; Toyooka et al., 2000). However, in castor bean (Ricinus communis) endosperm cells, the Cys-EP whose proform

accumulates in ricinosomes is proposed to play a key role in PCD, in part because there is a distinct temporal separation of storage protein mobilization and the biogenesis of ricinosomes, in which the latter coincides with the death process (Schmid et al., 1999, 2001; Gietl and Schmid, 2001). As emphasized above, our results also support a role for the Cys-EP related protease in PCD of white spruce megagametophyte cells. KDEL-terminated cysteine proteases have been widely found in senescing tissues (Valpuesta et al., 1995; Nadeau et al., 1996; Cercos et al., 1999; Gietl and Schmid, 2001), and it will be interesting to investigate how they are integrated into the cell death program.

741 Caspase-like protease activities may be involved in PCD of white spruce megagametophyte cells Apoptosis of animal cells generally involves a complex activation cascade in which inactive caspase precursors serve as substrates for other caspases. All caspases are synthesized as inactive precursors containing an N-terminal pro-domain and a large and small subunit; to generate the active protein, the prodomain is cleaved off and either a heterodimer or a heterotetramer is assembled. The specific pathways of caspase activation are very complex and depend on both the nature of the initial death trigger (e.g. toxic chemicals, irradiation, withdrawal of growth factors, cell surface receptor-interacting factors) and on the cell type undergoing apoptosis (Cohen, 1997). Caspase-like protease (CLP) activities have been implicated in the programmed death of plant cells (see review by Lam and del Pozo, 2000). We examined CLP activities in megagametophytes of white spruce seeds upon germination. CLP activities were readily detectable in post-germinative megagametophytes using Ac-DEVD-AMC, a fluorogenic substrate specific for caspase-3 of animal cells, but no proteolytic cleavage was detectable using Ac-YVAD-AMC as a substrate (a substrate specific for caspase-1). The CLP activities were inhibited by the caspase-3specific inhibitor, Ac-DEVD-CHO, and to a lesser extent by the caspase-1-specific inhibitor, Ac-YVADCHO. Cysteine protease inhibitors E-64 or leupeptin effectively inhibited the CLP activities, but PMSF or aprotinin (serine protease inhibitors), and pepstatin A (an aspartic protease inhibitor) had no effect. The optimal pH of the CLP was 5.0. The CLP activities of plant cells then, are distinct from caspases involved in PCD of other eukaryotic cells. For example, cysteine protease inhibitors generally do not inhibit caspases, caspases cleave their substrates optimally at neutral pHs, and Ac-YVAD-CHO is a relatively ineffective inhibitor of caspase-3 (Nicholson, 1996; Thornberry and Lazenik, 1998). However, the CLP activities in our system exhibit some similarities to CLPs detected during the programmed death of certain plant cells. For example, substrate cleavage activity of CLPs associated with the hypersensitive response is inhibited by cysteine protease inhibitors (D’Silva et al., 1998) and by caspase-specific inhibitors, Ac-YVAD-CHO and Ac-DEVD-CHO (del Pozo and Lam, 1998). Lowering the pH increases cleavage activity of some CLPs (Korthout et al., 2000). It is interesting that during PCD associated with the hypersensitive response,

only caspase-1-like activities are detected; in contrast, during leaf senescence, caspase-3 like activities are detected (Lam and del Pozo, 2000). As these forms of PCD are vastly different, the different CLP activities may reflect differences in the underlying mechanism or signal transduction pathways. In animal systems, there are several apoptotic effectors causing nuclear changes in apoptotic cells, such as caspase-activated DNase (CAD), apoptosis-inducing factor (AIF), cathepsin B and LDNase II (Zamzami and Kroemer, 1999). The wellcharacterized apoptotic DNase, caspase-activated DNase (CAD), is normally bound to its inhibitor (ICAD) and exists as an inactive complex. Upon receipt of apoptotic stimuli, activated caspase-3 cleaves ICAD to release CAD. CAD then enters the nucleus to degrade chromosomal DNA into internucleosomal fragments (Enari et al., 1998; Sakahira et al., 1998). Internucleosomal DNA laddering has been widely reported in some types of plant PCD (Ryerson and Heath, 1996; Young et al., 1997; Wang et al., 1996a, b, 1998, 1999; Orzaez and Granell, 1997; Young and Gallie, 1999, 2000a, b; Stein and Hansen, 1999; Xu and Hanson, 2000), but these reports did not determine CLP activities. DNA laddering occurs during PCD of post-germinative white spruce megagametophytes. The cell permeable caspase-3 inhibitor, Ac-DEVDCMK, when applied to germinated seeds, delayed the peak of CLP activities in megagmetophyte cells by 1 d and attenuated the extent of megagametophyte cell death as revealed by viability staining. Moreover, the extent of chromosomal DNA degradation and appearance of DNA laddering were delayed by 1 day. These results suggest that CLP activities are associated with megagametophyte cell death and internucleosomal DNA fragmentation. Whether there is a CAD-like DNase in megagametophyte cells will require further investigation. Post-germinative megagametophyte cells of white spruce seeds will provide a good model to investigate the functional link of CLP activities and nuclear DNA internucleosomal cleavage. There is presently no direct evidence for a caspasetriggered cascade in the control of plant PCD. More recently, using iterative database searches, Uren et al. (2000) defined that paracaspases (found in metazoans and Dictostelium) and metacaspases (found in plant, fungi, and protozoa) are two novel families of caspase-related proteins based on domain structure and sequence similarity. The plant metacaspases can be divided into two subclasses: type I plant metacaspases contain a prodomain that has a proline-rich

742 region and a zinc-finger motif typical of plant proteins that function in the hypersensitive response death pathway; type II metacaspases do not contain an obvious prodomain but have a conserved insertion of ca. 180 amino acids between the regions corresponding to caspase p20 and p10 subunits (Uren et al., 2000). The search of Arabidopsis genome sequences indicates that there is no similarity to proteins involved in regulating apoptosis in animal cells (e.g. classical caspases, bcl2/ced9 and baculovirus p35), but there are eight homologues of a newly defined metacaspase family (Arabidopsis Genome Initiative, 2000). Indirect evidence that caspase-like protease activities and mitochondria are involved in some types of plant PCD is mounting (see review by Lam and del Poza, 2000); moreover, the isolation of a PCDinduced homologue of human protein PIRIN in tomato provides some clues about the role of putative NFκB-associated pathways in plant defense mechanisms (Orzaez et al., 2001). There may indeed be a functionally conserved mechanism in the control of plant PCD. The cloning of the plant caspase-like proteases will help to elucidate their precise role in PCD.

Acknowledgements We are very grateful to Christine Gietl (Technische Universität München) for the Cys-EP antiserum and cDNA. This research was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant awarded to A.R.K.

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