JXB Advance Access originally published online on August 28, 2007
Journal of Experimental Botany 2007 58(11):2949-2958; doi:10.1093/jxb/erm137
RESEARCH PAPER |
Programmed cell death of the nucellus during Sechium edule Sw. seed development is associated with activation of caspase-like proteases
1Department of Biology University of Pisa, Via Ghini 5, I-56126 Pisa, Italy
2Department of Crop Plant Biology University of Pisa, Via Mariscoglio 34, I-56124, Pisa, Italy
* To whom correspondence should be addressed. E-mail: llombardi{at}biologia.unipi.it
Received 16 March 2007; Revised 24 May 2007 Accepted 25 May 2007
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Abstract |
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The nucellus is a maternal tissue that embeds and feeds the developing embryo and secondary endosperm. During seed development, the cells of the nucellus suffer a degenerative process soon after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been considered to be a form of developmentally programmed cell death (PCD). It was investigated whether or not this degenerative process is characterized by apoptotic hallmarks. Evidence showed that cell death is mostly localized in the border region of the tissue adjacent to the expanding endosperm. Cell death is accompanied by profound changes in the morphology of the nuclei and by a huge degradation of nuclear DNA. Moreover, an increase of activity of different classes of proteinases is reported, and the induction of caspase-like proteases sensitive to specific inhibitors was detected. Nucellar caspase-like proteases are characterized by an acid pH optimum suggesting a possible localization in the vacuole.
Key words: Cell death, DNA fragmentation, endosperm, nucellus, proteases, viability
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Introduction |
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In flowering plants, the double fertilization, which gives rise to a diploid zygote and to a triploid endosperm, occurs in the embryo sac embedded in maternal tissues, which have always been considered to play an important role in seed development (Russell, 1993). After a rapid expansion that allows growth of the whole seed, the nucellus undergoes a degenerative process very soon after fertilization (Russell, 1979). The major seed storage organ, the cellular endosperm, expands and accumulates reserves at the expense of the nucellus which is no longer present in mature seeds (Chen and Foolad, 1997; Xu and Chye, 1999; Dominguez et al., 2001; Hiratsuka et al., 2002).
Nucellar cell degeneration has been described from the ultrastructural and cytological point of view in several plant species including cotton (Jensen, 1975), barley (Norstog, 1974; Chen and Foolad, 1997), and Oenothera biennis (de Halac, 1980). In such systems cytoplasmic disorganization, nuclear condensation, and membrane blebbing have been reported. In Ricinus communis, nuclear DNA fragmentation in nucellar cells has been observed together with many ultrastructural changes like vesiculation of the cytosol, chromatin condensation, and vacuolar collapse (Greenwood et al., 2005). All these observations lead to the hypothesis that nucellar degeneration occurs by means of a developmentally regulated programmed cell death.
Cell death is an integral part of plant development and morphogenesis; PCD has been found to occur during many plant developmental processes like organ senescence, sex determination, dismantling of aleurone and suspensor, differentiation of tracheary elements, and aerenchyma formation in the cortex (Lam, 2004; Suarez et al., 2004). All of these are characterized by the presence of characteristic hallmarks like cytoplasmic shrinkage, chromatin condensation, cytochrome c leakage out of mitochondria, altered nuclear morphology, DNA fragmentation, and activation of proteases, similar to those found in animal apoptosis (Lam et al., 1999; Danon et al., 2000). Caspases (cysteinyl aspartate-specific proteinases) are fundamental components of animal PCD; once activated, they irreversibly trigger the executing phase of the cell death programme (Shi, 2002). Although caspase homologues were not found in plants, sequencing of the Arabidopsis genome revealed the presence of several metacaspase genes (Uren et al., 2000). The existence of true caspase-like activities has been reported during plant PCD (Rotari et al., 2005, and references within). Caspase 1, 3, and/or 6-like proteolytic activities have been correlated both to developmental and chemical-induced plant PCD and the latter can be abolished or delayed by the use of caspase inhibitors (Woltering et al., 2002; Sanmartìn et al., 2005).
As many of the reported experiments dealt with chemical- and pathogen-induced cell death, they are not sufficiently exhaustive in elucidating the correlation between the appearance of apoptotic hallmarks during PCD and death during plant development.
Developing seeds offer an extraordinary opportunity to study the delicate balance between cell differentiation and cell death during embryogenesis. In this work, the possibility has been addressed that degeneration of the nucellus of Sechium edule occurs by a process of PCD characterized by some apoptotic hallmarks.
In the present study, a description of the early events correlated to nucellar degeneration and characterized by profound changes in nuclear morphology and DNA fragmentation is reported. The presence of biochemical hallmarks characteristic of animal apoptosis suggests that the nucellus of Sechium edule degenerates after fertilization by means of a developmentally controlled programmed cell death. Moreover, the induction of a set of proteases is reported, among which caspase-like proteolytic activities appear to have a relevant role.
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Materials and methods |
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Plant material
Plants of Sechium edule Sw. (Cucurbitaceae) were grown in the field from June to September and fruits were harvested from the end of September. Seeds were cut longitudinally under a stereomicroscope and the endosperm and the embryo removed. Nucellar tissue was gently removed from the seed and, if not immediately used, stored at –80 °C. All material was kept in an ice bath during the whole procedure.
Viability staining
Viability of cells in intact nucellus was determined by staining with fluorescein diacetate (FDA, 2 µg ml–1 in 20 mM CaCl2, Sigma) for 15 min followed by N-(3-triethylammoniumpropyl)-4- 6-(4-(diethylamino) phenyl)-hexatrienyl) pyridinium dibromide (Synaptored, 20 µM in 20 mM CaCl2, Sigma) for 3 min (Fath et al., 2001). Nucella were observed under a Leica DMLB microscope and images were captured by a Leica DC 300F CCD camera. An argon and a krypton laser were used for visualization of the FDA (
ex 488 nm, em 502–540) and Synaptored (ex 515, em 625) signals, respectively.
Isolation and analysis of plant DNA
For the isolation of intact, high molecular weight DNA, the CTAB method was used (Ausubel et al., 2002). Briefly, frozen nucellar tissue was ground in N2 to a fine powder, and extracted with 2 vols of CTAB buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM TRIS–HCl pH 8, and 0.2% β-mercatptoethanol) for 45 min at 65 °C. The DNA was extracted with 1 vol. of chloroform:isoamyl alcohol (24:1 v/v) and the aqueous phase was precipitated with 1 vol. of isopropanol. After a centrifugation for 20 min at 14 000 rpm the supernatant was discarded and the pellet resuspended in TE buffer (10 mM TRIS–HCl pH 8, 1 mM EDTA) supplemented with RNase A (100 µg ml–1). DNA (10 µg) was subjected to electrophoresis on a 2% (w/v) agarose gel, stained with 0.5 µg ml–1 ethidium bromide for 30 min and observed on a UV light box. A 100 bp ladder was used as the standard.
In situ detection of DNA fragmentation (TUNEL assay)
Small blocks constituted by the seed teguments with adhering nucellus were fixed overnight at 4 °C in 4% (w/v) paraformaldehyde in phosphate buffer saline pH 7.4. After dehydration through an ethanol series samples were embedded in Paraplast Plus (Paraplast, Sherwood Medical Industries). Sections of 10 µm were cut and stretched on poly-lysine coated slides. The sections were then dewaxed in xylene and rehydrated before examination.
TUNEL assay was performed using an ‘In situ cell death detection kit’ (Promega) according to the manufacturer's instructions. To facilitate the entry of TdT enzyme into the tissue sections, the slides were treated with proteinase K (20 mg ml–1) for 20 min. The labelling reaction was performed at 37 °C in a humidified chamber in the dark for 1 h. A negative control was included in each experiment by omitting TdT from the reaction mixture. As a positive control, permeabilized sections were incubated with DNase I (10 U ml–1) for 10 min before TUNEL assay. Counter-stain was done with toluidine blue 0.05% (w/v) in NaCO3 1% pH 8.2. The yellow-green fluorescence of incorporated fluorescein-12-dUTP was observed using a Leica DMLB microscope equipped with a filter set for fluorescein and a Leica DC 300F CCD camera.
Experiments were repeated three times and each time five slides were labelled for both the P and the D regions. About 200 tissue sections were analysed.
Proteolytic activity assay using azocasein
Protein extracts were prepared by homogenization of the nucellar tissue in 1 vol. of ice-cold extraction buffer (TRIS–HCl 50 mM pH 7.4, EDTA 1 mM, DTT 1 mM, Triton X-100 0.1% v/v). Cell debris was pelleted by centrifugation for 10 min at 17 000 rpm at 4 °C, the supernatant collected and, if not immediately used, stored at –80 °C. Proteolytic activity was assayed in 500 µl reaction mixtures containing 225 µl acetate buffer 0.05 M pH 5, 250 µl azocasein 0.4% (w/v), 5 µl β-Me 0.25 M, and 20 µl extract. Reactions were incubated overnight at 32 °C, stopped by adding 125 µl TCA 50% p/v. Reaction mixtures were centrifuged 10 min at 17 000 rpm then absorbance at 330 nm was measured.
In the experiments with inhibitors, the latter were added to the above reaction mixtures and incubated for 15 min at 30 °C prior to the addition of azocasein. The inhibitors used were: 5 µM trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), 10 µM pepstatin, 10 µM leupeptin, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 1 mM EDTA. As some inhibitors were dissolved in DMSO and ethanol, controls of protease activity in the presence of these chemicals were made to check their effects on activity.
To determine the optimum pH for enzymatic activity, the assays were performed in acetate buffer 0.05 M pH 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 or Na-phosphate buffer 0.05 M pH 6.0, 6.5, 7.0, 7.5.
In vitro caspase-like protease activity assay
Protein extracts were prepared by homogenization of nucellar tissue in 0.8 vol. of ice-cold extraction buffer (50 mM HEPES-KOH pH 7.0, 10% [w/v] sucrose, 0,1% [w/v] CHAPS, 5 mM DTT, 1 mM EDTA). Cell debris was pelleted by centrifugation for 10 min at 17 000 rpm at 4 °C, the supernatant collected and immediately used or stored at –80 °C. Proteolytic activity was measured in 500 µl reaction mixtures containing 50 µg protein and 75 µM of substrates specific for individual mammalian caspase. The following colorimetric substrates were used: Ac-YVAD-pNA, Ac-DEVD-pNA, and Ac-VEID-pNA (all from Sigma) dissolved in DMSO. To determine the optimum pH for the caspase-like activities, the assays were performed in 50 mM acetate buffer pH 3.5, 4.0, 4.5, 5.0 5.5 or in 50 mM HEPES-KOH buffer pH 6.0, 6.5, 7.0. Reactions were incubated for 5 h at 32 °C then absorbance at 405 nm was taken against a blank containing buffer and substrate alone. All assays were performed in duplicate. In the experiments with inhibitors, the latter were added to the reaction mixtures and incubated for 15 min at 30 °C prior to addition of the substrates. The inhibitors used were: 5 µM E-64, 10 µM pepstatin, 1 mM PMSF, 100 µM Ac-YVAD-CHO, 100 µM Ac-DEVD-CHO, and 100 µM Ac-VEID-CHO, all from Sigma.
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Results |
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Seed development and viability of the nucellus
The large Sechium edule seed represents an interesting model system to study nucellar degeneration. The nucellar tissue is a transparent, glossy and easy to isolate tissue made by a few layers of cells adhering to the integuments. During seed development, the extent of nucellar degeneration coincides with the progression of endosperm growth and the time-course of this process can easily be appreciated even with naked eye (Fig. 1A). Cell death enables regression of the nucellus border which leaves space for the rapidly growing endosperm. The two tissues are barely in contact so the dead nucellar cells are not squashed back by the endosperm but rather undergo autolysis.
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S. edule seeds were classified into four different stages based on endosperm growth and, consequently, on the degree of nucellus degeneration: stage A (endosperm is rather visible), stage B (its length is one-third of the seed), stage C (it occupies one-half of the seed), and stage D (it has almost filled the cavity of the seed) (Fig. 1C).
The viability of intact nucellus was examined using a double-staining procedure with the fluorescent dyes FDA and Synaptored. FDA detects living cells, as they are capable of metabolizing FDA yielding fluorescein molecules; by contrast, Synaptored is only able to enter dead, damaged cells without an intact membrane. As shown in Fig. 2, orange-red dying cells are mostly localized in the border region of the tissue proximal to the expanding face of the developing endosperm, while the remaining portion of the tissue is alive. This marginal band of dead cells is very narrow and the width of the dying portion does not vary from the very early to the late stage of seed development. Based on the distribution of the dying cells, nucellar tissue was conventionally divided into a proximal region (P) representing the narrow band characterized by cell death, and a distal region (D) where all the cells are alive. A sub-proximal region (SP), adjacent to the P region was also taken into account for further experiments (Fig. 1B).
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Cellular morphology
Compared with cells of the integuments, nucellar cells are bigger and irregularly shaped. They contain a very large vacuole and no chloroplasts (Fig. 3A). Alive and metabolically active cells of the D region contain nuclei with a regular envelope and a visible nucleolus. The big and rounded nuclei are displaced in the periphery towards the cell membrane (Fig. 3B–E). Strikingly, the dying cells located in the P region of the nucellus show morphologically altered nuclei lacking the nucleolus (Fig. 3G–M). In the two or three cellular layers in contact with the inner integuments, the nuclei never show nucleoli but still appear approximately round shaped (Fig. 3M). Small clusters of condensed chromatin undergo marginalization, being stuck on the inner side of the nuclear envelope. In the upper layers of the nucellus, towards the inner cavity of the seed, nuclei progressively lose their round shape and become deeply lobed and stretched (Fig. 3H–L). Condensation of chromatin appears to be completed and small clusters of chromatin are distributed along the outstretched nucleus.
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DNA fragmentation during nucellus degeneration
To determine whether or not DNA fragmentation occurs in the nuclei of the dying nucellus, transverse sections of nucellar tissue adhering to integuments were subjected to TUNEL assay. In the control D region, no TUNEL-positive nuclei are present (Fig. 3F), while nuclear DNA degradation becomes evident in the P region, as demonstrated by the presence of green-fluorescent TUNEL-positive nuclei (Fig. 3N–Q). In detail, fluorescein labelling reveals DNA degradation in morphologically altered nuclei still evident by toluidine blue staining. Appropriate control treatments were conducted for every set of slides. If the DNase I treatment preceded the TUNEL assay, all nuclei in the whole section were fluorescent (positive control, not shown). On the other hand, when TdT enzyme was omitted from the labelling mixture no fluorescence was observed (negative control, not shown).
Conventional agarose gel electrophoresis of genomic DNA confirms that extensive DNA degradation characterizes the P region as indicated by a conspicuous uniform smear. DNA from the distal portion of the tissue appears as a unique undegraded band with high molecular weight (Fig. 4). DNA degradation characterizes all stages of nucellus re-absorption. This result confirms the observation of nuclear DNA degradation by the TUNEL assay. A clear laddering pattern, given by multiples of internucleosomal units of 180 bp, has never been observed. Consistent results were obtained in several independent DNA extractions and electrophoresis.
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Characterization of proteolytic activities
Among the basic components of PCD is the activation of different classes of proteinases which act both as signals and executioners of the cell death programme.
In order to analyse the importance of proteolysis during the process of nucellar death, protease activities in the P and SP portions of the tissue was evaluated in a wide range of pH values and compared with the proteolytic activity of the control D region. In the P region the activity was higher at every pH value analysed and, in particular, it showed two peaks at pH 5 and 7, where activity is more than 20% higher than in the control region. No increase of proteolysis over the control could be detected in the SP region (Fig. 5A).
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To characterize proteinase activity further, class-specific inhibitors were used in the azocasein assay. As shown in Fig. 5B, none of the inhibitors used strongly compromised activity at pH 5; they all had only a partial effect causing a 10–30% reduction of activity with no evident differences for the two regions analysed.
Quite different results were obtained at pH 7 where the inhibitors used were more effective and caused a differential reduction of activity between the P and D regions (Fig. 5B). Proteolytic activity in the P region was not sensitive to leupeptin while it could be reduced by E-64 (25%), pepstatin (55%), PMSF (50%), and even more by EDTA (82%). In the D region pepstatin, PMSF, and EDTA were the only effective inhibitors, but the inhibition was weaker than in the P region. This is consistent with the activation of some cysteine, serine, and metallic proteases upon induction of death in the nucellar tissue.
Caspase-like proteases (CLP) induction
The increase of proteolytic activity detected by azocasein assay during the induction of the cell death programme in the nucellus is due to the activation of different classes of proteases, as shown by using class-specific inhibitors. In order to determine whether or not nucellus cell-free extracts contain any caspase-like proteolytic activity (CLP), synthetic, colorimetric, four amino acid substrates were used for three distinct members of the animal caspase family. The substrates tested were Ac-YVAD-pNA (for caspase-1), Ac-DEVD-pNA (for caspase-3), and Ac-VEID-pNA (for caspase-6).
All the three different substrates were efficiently cleaved by extracts obtained from nucellar tissue (Fig. 6A–C). YVADase and DEVDase specific activities were strongly induced in the P and SP regions, as they were 40–45% higher than in the control region D. Instead, cleavage of the Ac-VEID-pNA substrate did not appear to be specifically correlated to cell death as less than 20% induction of activity, compared with the D region, is reported here.
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To test whether the cleavage of the three substrates was executed by caspase-like activities or by other proteases, the caspase assays were performed in the presence of different inhibitors. As showed in Fig. 6D–F, the cysteine protease inhibitor E-64, the aspartic protease inhibitor pepstatin, and the serine protease inhibitor PMSF had only a weak effect on the cleavage of the three substrates by nucellus undergoing cell death. By contrast, when the specific inhibitors Ac-YVAD-CHO (for caspase-1), Ac-DEVD-CHO (for caspase-3), and Ac-VEID-CHO (for caspase-6) were included in the respective assays, the proteolytic activity was suppressed significantly. The caspase-1 like activity showed by the control region D was sensitive to E-64 and pepstatin, and the caspase-3 like activity was also sensitive to PMSF, suggesting the contribution of different proteases to the activity detected in the region not involved in cell death.
Biochemical studies on animal caspases (Stennicke and Salvesen, 1997) indicated for caspase activity a high sensitivity towards pH changes. Therefore, to study the effect of pH on CLP activities in nucellus cell-free extracts proteolytic activity was compared in assay buffer containing acetic acid (pH 3.5, 4, 4.5, 5, 5.5) or HEPES (pH 5.5, 6, 6.5, 7). As shown in Fig. 6, the optimal pH for the cleavage of the three synthetic substrates was 4–4.5 and there was no detectable activity at neutral pH.
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Discussion |
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Nucellus degeneration in flowering plants appears to be a default process in ovule ontogeny, although some variations in the temporal and spatial pattern of such degradation may be observed in different species. Nucellar cells die right after fertilization to supply the young embryo and the expanding endosperm with nutrients.
In Sechium edule seed, the nucellus starts to degenerate when the endosperm begins to develop after double fertilization. Autolysis of the nucellus gradually leaves a cavity inside the ovule that is filled by both the growing endosperm and embryo. Cell death in the nucellus occurs in a very thin and narrow band of cells proximal to the expanding face of the endosperm, suggesting the involvement of a death signal coming from the endosperm itself. The near, downstream alive cells may respond later to the same hormonal stimuli or simply be induced to undergo PCD by the neighbouring dying cells.
The largeness of the region characterized by cell death never changes during the whole process of nucellus disappearance, similar to that observed in the nucellus of Ricinus by Greenwood et al. (2005). These authors report that only cells lying two or three cell layers proximal to the expanding endosperm show sympthoms of PCD.
The nuclei of degenerating nucellar cells undergo profound morphological changes, which differ depending on the position of the cells inside the tissue. In the layer of cells closer to the integuments, round-shaped nuclei show the presence of chromatin aggregates, peripherally stuck on the nuclear envelope. The nucleolus is never visible in these nuclei, suggesting that this is the first nuclear structure to dismantle. In the nucellar cell layer far from the integuments and closer to the internal cavity of the ovule, nuclei become stretched, deeply lobed, and condensed. Similar nuclear modifications also occur in dying nucellar cells of other plant species, like Oenothera biennis (de Halac, 1980) and barley (Norstog, 1974). Furthermore, they are similar to nuclear shape alterations and chromatin condensation in other forms of programmed cell death, both in animals and plants.
When assayed by in situ TUNEL the stretched and convoluted nuclei appear to be all positive, indicating that a DNA fragmentation process is taking place. Moreover, DNA extracted from dying nucellar cells show a diffuse pattern of degradation when electrophoresed. Nuclear DNA fragmentation is considered one of the typical hallmarks of PCD and it has been reported also to occur during nucellus degeneration in wheat (Dominguez et al., 2001), barley (Linnestad et al., 1998), and Ricinus, where it was also visualized by in situ TUNEL assay. A typical DNA laddering that characterizes apoptotic cell death in animals and in some plant systems is not reported here. DNA laddering is usually difficult to demonstrate in extracts from tissues in which a variable percentage of the cells is undergoing the fragmentation process (Wang et al., 1996; Bethke et al., 1999; Ruey-Hua and Chen, 2002; Gunawardena et al., 2004). The proximal region of the nucellus is characterized by the simultaneous presence of cells at different stages of PCD: living cells, dead cells, and cells that are just committed to die. Indeed, a DNA ladder is usually observed in systems characterized by cells beginning the death programme in a synchronous way, being at any time in the same phase of the process. This is the case of cultured cells induced in PCD by external triggers such as microbial toxins (Kusaka et al., 2004), mannose (Stein and Hansen, 1999), menadione (Sun et al., 1999), cold (Koukalová et al., 1997), and salt stress (Katsuhara, 1997).
Proteolytic enzymes are known to have a principal role in the execution of programmed cell death, hydrolysing targets involved in the organization and maintenance of cell structure and homeostatic pathways. In plants, several proteolytic activities have been associated with developmentally regulated cell death (Beers et al., 2000). An increase in total proteolytic activity correlated with cell death is reported here: a study of inhibitors showed that this increase is due to metallic, serine, aspartic, and cysteine proteases. It is worth noting that a gene encoding an aspartic proteinase called ‘nucellin’ is expressed in degenerating nucellar cells of barley (Chen and Foolad, 1997) and a rice homologue of nucellin is strongly expressed in the nucellus just after fertilization (Bi et al., 2005). Interestingly, wheat nucellus degeneration is also associated with expression of a serine-protease and a cysteine protease, whose transcripts also accumulate in the aleurone layer (Dominguez and Cejudo, 1998). A gene encoding a cysteine protease similar to the vacuolar processing enzyme and called ‘nucellain’ is expressed in association with nucellar degeneration in barley (Linnestad et al., 1998) and specific expression of another cysteine protease has been reported in the nucellus of brinjal (Solanum melongena) (Xu and Chye, 1999). Recently, the expression of a papain-type cysteine protease has been correlated to nucellar cell death in Ricinus (Greenwood et al., 2005).
The most representative cysteine proteases implicated in PCD are the caspases. Although caspase homologues were not found in plants, the existence of caspase-like activities during plant developmental PCD has been reported by using synthetic substrates specific for animal caspases (Rotari et al., 2005, and references within). The induction of CLP activities in the developmentally regulated PCD of nucellus is reported for the first time. Caspase-1-like and caspase-3-like activities are strongly induced in the P and SP regions of the nucellus and they are almost totally inhibited by their specific inhibitors. Caspase-6-like protease is not significantly induced in the SP region while it appears correlated to cell death in the P region. These data suggest that caspase-1, 3, and 6-like proteases are involved in executing nucellar cell death.
CLP activities detected in Sechium degenerating nucellus are active at acid pH and there is only barely detectable activity at neutral pH. This is in contrast with what was observed for animal cytoplasmic caspases (Stennicke and Salvesen, 1997), and for most of the CLP detected in plant PCD systems which are active at neutral or basic pH. Nevertheless, many authors have recently reported plant caspase-1, 3, and 6-like preference for acid pH (He and Kermode, 2003; Danon et al., 2004; Rotari et al., 2005). This pH requirement may suggest that Sechium CLPs are not localized in the cytoplasm but they may be vacuolar. Vacuoles are a reservoir for many hydrolytic enzymes which, upon rupture of the tonoplast, are released and lead to the recycling of cellular contents by autophagy and autolysis (Klionsky and Emr, 2000). Vacuolar collapse has been implicated in various types of plant developmental PCD as tracheary element differentiation, senescence, and suspensor degeneration (Gahan, 1982; Jones, 2001; Obara et al., 2001; Filonova et al., 2002). Recently, Hatsugai et al. (2004) showed that the vacuolar processing enzyme has caspase-1 like activity and is required for cell death in tobacco infected by mosaic virus. The barley VPE homologue nucellain is involved in nucellus cell death as are other VPE homologues in other species. Moreover, the cysteine protease associated with cell death in the nucellus of Ricinus is localized in the ricinosomes, ER-derived vescicles that act as ‘suicide bombs’ in the cytoplasm of the degenerating nucellus (Greenwood et al., 2005). Taken together, all these observations lead to the hypothesis that CLP activities detected in degenerating nucellus may be involved in the degradation of cellular contents inside acidic vesicles, perhaps through a series of events culminating in autophagic cell death (Patel et al., 2006).
From these results it is concluded that nucellar degeneration occurs by mean of a programmed cell death which displays the typical hallmarks of PCD. It implicates the degradation of nuclear DNA and the activation of many proteolytic enzymes which may be part of an autophagic mechanism of cell death.
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Abbreviations |
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CTAB, cetyl trimethyl ammonium bromide; DTT, dithiothreitol; HEPES. 4-(2-hydroxyethyl-1-piperazine-ethanesulphonic acid, ; TCA, trichloroacetic acid; TUNEL, terminal dUTP nick-end labelling; β-ME, β-mercaptoethanol; CLP, caspase-like protease.
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