The cleavage of proteins and peptides in plants is catalyzed by a series of peptide hydrolases differing in their subcellular localization, in their substrate specificities or in their regulatory properties (Barrett, 1986). The hydrolysis of peptide bonds is not restricted to the catabolism of mature proteins to free amino acids, but is also relevant for the modification and maturation of proteins. The removal of signal or transit peptides from larger precursors is linked to protein synthesis and intracellular sorting. The degradation of damaged or unassembled polypeptides is important for housekeeping in a cell. Furthermore, protein turnover is a prerequisite for the adaptation of the protein pattern (e.g., enzymes, translocator proteins) to changing conditions. All the previously mentioned processes occur in differentiating or mature cells and are not necessarily related to cell death. The proteolytic enzymes involved and the regulatory mechanisms may not be identical for protein maturation and for protein remobilization.

Different physiological situations may lead to the death of a cell (Greenberg, 1996; Noodén et al., 1997; Bleecker, 1998; Guarente et al., 1998). The degradation of macro-molecules in such cells and the export of low molecular weight compounds to sinks within the same plant represent a last contribution of these cells to other plant parts. Since proteins represent the predominant nitrogen fraction in leaves or seeds and these proteins are not transported as such in the phloem, the important role of proteolysis and its control for the redistribution of nitrogen is obvious. Under certain conditions, proteolysis may not only allow the export of nitrogen compounds after initiation of cell death, but it may also directly be involved in causing the death of a cell by degrading essential proteinaceous constituents.

Plant Cell Death Processes 107

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II. Selective Hydrolysis of Peptide Bonds

A. Protein Maturation and Removal of Peptides from Larger Precursors

The removal of peptides from larger precursors is specific for the protein and for the peptide bond within the protein. The cotranslational removal of the signal peptide in the rough endoplasmic reticulum (Chrispeels, 1991) and the post-translational cleavage of the transit peptide from proteins imported from the cytosol into plastids or mitochondria (Eriksson et al., 1996; Schatz and Dobberstein, 1996; Whelan and Glaser, 1997; Keegstra and Cline, 1999) are examples of a highly specific proteolytic process allowing the proper sorting and maturation of newly synthesized polypeptides. Processing enzymes in protein-storage vacuoles and in lytic vacuoles cleave larger precursor polypeptides also at well-defined positions (Okamoto etal., 1994; Hara-Nishimura etal., 1998). This type of protein cleavage is related to protein synthesis and not primarily to protein degradation or cell death.

B. Degradation of Damaged Proteins and of Unassembled Subunits

The degradation of damaged (Desimone et al., 1996; Stieger and Feller, 1997), mistargeted (Halperin and Adam, 1996) or not properly assembled (Schmidt and Mishkind, 1983) proteins is specific for the polypeptide, but may be unspecific for the peptide bonds within the polypeptide. After the initial cleavage, the polypeptide may be degraded completely to free amino acids. In general, fragments produced by a first cleavage of a mature protein are rapidly degraded to amino acids or small peptides and do not accumulate in the cell.

C. Protein Turnover and Adaptation of the Metabolism to Changing Conditions

Some proteins are quite stable in a plant cell, while other proteins are turned over rapidly (Mattoo et al., 1984). Protein turnover (synthesis and degradation) allows a modification of the protein pattern and as a consequence also an adaptation of the metabolism to altered conditions (Dungey and Davies, 1982). The susceptibility of proteins to proteolysis is an important intrinsic property and may be further influenced by the actual environment (Feller and Fischer, 1994). Proteins in the same compartment may differ considerably in their stability (Mitsuhashi and Feller, 1992).

D. Degradation of Mature Proteins in Relation to Nitrogen Remobilization

Mature proteins can be rapidly degraded during nitrogen mobilization from germinating seeds (Harvey and Oaks, 1974; Chrispeels and Boulter, 1975) or from cells in vegetative plant parts (Nooden et al., 1997) prior to cell death. The storage proteins in seeds are located in the protein bodies (Chrispeels and Boulter, 1975). A cysteine endopeptidase is de novo synthesized into the endoplasmic reticulum at the onset of germination, processed and then either transported in vesicles to the protein bodies (e.g., legumes) or secreted from the aleurone layer and the scutellar epithelium into the endosperm of cereals (Callis, 1995; Müntz et al., 1998). The proteins in photosynthesizing leaf cells differ considerably in their properties and in their subcellular compartmentation from storage proteins in seeds. In mesophyll cells of C3-plants, proteins (e.g., soluble enzymes, membrane proteins, regulatory proteins) are present mainly in the chloroplasts (Peoples and Dalling, 1988). During germination and during senescence, the final steps of protein remobilization coincide with the death of the cells, although the initial situation is quite different in storage tissues of seeds and in metabolically active leaf cells. The proteolytic systems involved and the regulation of proteolysis are not identical for germination and leaf senescence.

III. Proteolytic Activities in Plants A. Classification of Peptide Hydrolases

Exopeptidases remove an amino acid, a dipeptide or a tripeptide from the N-terminus (aminopeptidases) or from the C-terminus (carboxypeptidases) of a peptide or of a protein (Barrett, 1986). The exopeptidases in higher plants were reviewed in detail by Mikola and Mikola (1986). Since no clear function (especially no regulatory function) of exopeptidases in relation to cell death has been identified so far, these types of peptide hydrolases are not considered in more detail here.

Endopeptidases hydrolyze polypeptide chains to fragments of three or more amino acid residues in length. Based on the active center, various classes of endopeptidases can be distinguished (Barrett, 1986). Serine endopeptidases (EC 3.4.21), cysteine endopeptidases (EC 3.4.22), aspartate endopeptidases (EC 3.4.23) and metal endopeptidases (EC 3.4.24) represent such classes. The various types of endopeptidases may differ in their substrate specificities, in their subcellular localization, in their physiological function and in their inhibition properties.

Cysteine endopeptidases have been detected in a series of plant species (Ryan and Walker-Simmons, 1981; Callis, 1995). These enzymes are most active in the slightly acidic pH range and were found to be localized in the vacuole (Heck et al., 1981; Canut et al., 1985). Cysteine endopeptidases are key enzymes for the remobilization of storage proteins during germination (Chrispeels and Boulter, 1975; Callis, 1995). High activities of such enzymes were also detected in leaves (Feller et al., 1977; Peoples and Dalling, 1988). An increased level of transcription of genes encoding a cysteine proteinase during senescence has been reported by several groups (Buchanan-Wollaston, 1997; Valpuesta et al., 1995). Navarre and Wolpert (1999) suggested that a calcium-activated cysteine protease causes the cleavage of the large subunit of Rubisco in oats after treatment with victorin, a host-selective fungal toxin. Caspases, the well known cell death-related proteases in animals, belong also to the cysteine proteases (Ruoslahti and Reed, 1999; Wolf and Green, 1999). Caspases represent a special family of highly conserved cysteine endopeptidases, exist often as latent zymogens and are involved in inflammation and/or in regulatory cascades during apoptosis. It remains open whether this type of "cell killer" enzyme plays an important role in senescence of plant cells. The presence of caspase-like plant protease(s) in tobacco tissues infected with the tobacco mosaic virus (hypersensitive response) was reported recently (Del Pozo and Lam, 1998). The question, to what extent caspases or caspase-like proteases are important in plant cell death or certain types of plant cell death, remains to be answered (Chapter 1).

Far less information than for cysteine endopeptidases is available for plant serine endopeptidases. A serine-type protease has been found to mediate the degradation of a lightstress protein (Adamska etal., 1996). A marked increase of a vacuolar serine endopeptidase was detected in maize roots under sugar starvation (James et al, 1996). As judged from activity gel assays, a 60-kDa serine endopeptidase accumulated during in vitro tracheary element differentiation in Zinnia elegans (Ye and Varner, 1996). The activity of a 70-kDa serine endopeptidase was found to be low in young leaves and high in artificially senescing parsley leaves (Jiang et al., 1999). The latter reports suggest but cannot prove that serine endopeptidases play a role in plant cell death.

Metallo-endopeptidases contain zinc and were detected in plants by several groups (Ragster and Chrispeels, 1979; Barrett, 1986; McGeehan et al., 1992; Bushnell et al, 1993). In soybean leaves, a fraction of a monomeric metallo-proteinase with a molecular mass of only 15 to 20 kDa is partially present in the extracellular space, where the specific activity is 50-fold higher than in crude leaf extracts (Graham et al., 1991). The level of this enzyme increases with leaf age. Metallo-endopeptidases were also detected in the chloro-plasts (Abad et al., 1989; Musgrove et al., 1989; Bushnell et al, 1993; Roulin and Feller, 1998). In intact chloroplasts isolated from pea leaves, the degradation of stromal proteins in darkness is strongly inhibited by EDTA (Fig. 7-1). This activity in intact chloroplasts is stimulated by divalent cations (Roulin and Feller, 1998). Considering these results, a key role of metallo-endopeptidase(s) in the degradation of stromal proteins in the intact chloroplasts appears likely.

Aspartic endopeptidases are characterized by a low pH optimum (below pH 5). This type of endopeptidase was also detected in plants, but little is known about their functions (Callis, 1995).

B. Complex Proteolytic Systems

Proteolytic activities can be integrated in larger polypeptide complexes (Lupas et al., 1997). The proteolytic cores of the cytosolic proteasome (20S proteasome) and of the plastidial Clp system (ClpP) form a barrel-shaped structure with the proteolytic sides oriented to the central pore. Only polypeptides entering the central pore can be degraded by the intact structures (Lupas et al., 1997). The proteolytic core may be able to degrade unfolded polypeptides in an ATP-independent manner, while the additional ATPase moieties of the two systems may serve as a funnel and may be required for unfolding proteins present in a stabilized three-dimensional structure. Complete unfolding and the absence of disulfide bonds are prerequisites for the degradation of polypeptides by the 20S proteasome. Possible interactions between complex proteolytic systems and substrate proteins are schematically shown in Fig. 7-2.

A series of reports concern the plant proteasome (Vierstra, 1993; Belknap and Garbarino, 1996; Bahrami and Gray, 1999). This complex proteolytic system is ubiquitous in eukary-otic cells (located in the cytosol and in the nucleus) and is present under two forms (the 20S proteasome and the 26S proteasome). The 20S proteasome represents the barrel-shaped pro-teolytic core with a narrow pore in the center and contains the Ntn hydrolases (Lupas et al., 1997). The assembly of the 20S proteasome is quite well known (Chen and Hochstrasser, 1996), but the disassembly of this complex is not yet elucidated. The 26S proteasome is a very complex structure and contains 14 different subunits in the proteolytic core (20S proteasome) and about 20 different subunits in the two 19S caps (Lupas et al., 1997). The 19S caps have ATPase and ubiquitin-binding activities and are attached to the end of a 20S core. The precise functions are not yet known for the various subunits of this self-compartmentalizing protease (Baumeister et al., 1998). The proteasome is involved in the

Proteasome 20s And 19s

Figure 7-1. Influence of EDTA on proteolysis in intact pea (Pisum sativum) chloroplasts incubated at 25°Cin the light (45 |imol m-2 s-1) or in darkness. Intact chloroplasts were isolated on Percoll steps and incubated in a medium containing 1 mM DTT, 1 mM MgCl2 and 2 mM EDTA (A) or in a modified medium lacking EDTA (B). Following incubation and re-isolation of the intact organelles, changes in the levels of the large (LS) and small (SS) subunit of Rubisco and of the light-harvesting chlorophyll a/b-binding protein (LHCII) were visualized by coomassie brilliant blue-staining (CBB) of the gels. The degradation of LS, phosphoribulokinase (PRK), glutamine synthetase (GS) and ferredoxin-dependent glutamate synthase (GOGAT) was detected by immunoblotting with specific antibodies. Equal quantities of chlorophyll (1 |g) were loaded on each lane. [From Roulin and Feller (1998), with permission.]

Figure 7-1. Influence of EDTA on proteolysis in intact pea (Pisum sativum) chloroplasts incubated at 25°Cin the light (45 |imol m-2 s-1) or in darkness. Intact chloroplasts were isolated on Percoll steps and incubated in a medium containing 1 mM DTT, 1 mM MgCl2 and 2 mM EDTA (A) or in a modified medium lacking EDTA (B). Following incubation and re-isolation of the intact organelles, changes in the levels of the large (LS) and small (SS) subunit of Rubisco and of the light-harvesting chlorophyll a/b-binding protein (LHCII) were visualized by coomassie brilliant blue-staining (CBB) of the gels. The degradation of LS, phosphoribulokinase (PRK), glutamine synthetase (GS) and ferredoxin-dependent glutamate synthase (GOGAT) was detected by immunoblotting with specific antibodies. Equal quantities of chlorophyll (1 |g) were loaded on each lane. [From Roulin and Feller (1998), with permission.]

ubiquitin-dependent degradation of proteins (Vierstra, 1993). The N-terminus, the presence of a lysine residue for the covalent attachment of ubiquitin (isopeptide bond) and the three-dimensional structure of the protein must be considered as important factors influencing the susceptibility of a protein for degradation in this pathway (Vierstra, 1993; Byrd et al., 1998). The ubiquitin-dependent proteolysis in the cytosol is most likely important for the

flBflB FjATPase

• i J.

Proteolytic complex

Native Modified protein protein

Free amino acids, peptides

A 3


b gg ¿j

% *


c 22^



Figure 7-2. Scheme representing possible interactions between complex proteolytic systems and substrate proteins. (A) The ATPase moiety may serve as a funnel and feed the substrate protein into the proteolytic moiety. (B) Native proteins may be stable in the absence of a functional ATPase moiety. (C) Modified (unfolded) proteins may directly serve as substrates for the proteolytic moiety. (D) The separation of the subunits forming the proteolytic complex may expose catalytic sites of the peptide hydrolase(s) and allow a rather unspecific attack of proteins.

turnover of short-lived proteins, for the degradation of abnormal proteins, for the regulation of the cell cycle and for stress responses (Vierstra, 1993; Belknap and Garbarino, 1996). On the other hand, it appears unlikely that a major portion of leaf proteins is degraded during senescence via the ubiquitin/proteasome pathway (Bahrami and Gray, 1999).

During the past few years, more information has become available concerning Clp (Shanklin etal., 1995; Schaller and Ryan, 1995; Crafts-Brandner etal., 1996; Ostersetzer and Adam, 1996; Desimone etal, 1997; Sokolenko etal., 1998; Nakabayashi etal, 1999). This complex proteolytic system is located in the plastids and consists of two types of subunits (ClpP and ClpC). ClpP, a 23-kDa polypeptide, is encoded in the plastome and contributes the proteolytic moiety to the complex (Shanklin et al., 1995). ClpC (a polypeptide of about 90 kDa equivalent to ClpA in E. coli) is nuclear-encoded, synthesized as a larger precursor in the cytosol, imported into the plastids and cut to its final size by the removal of the transit peptide. ClpC bears the ATPase activity. The mRNAs (Crafts-Brandner et al, 1996; Ostersetzer and Adam, 1996) and the proteins (Shanklin et al., 1995) for ClpP and ClpC are present throughout the life cycle of a leaf and are not expressed in a senescence-specific manner. However, Nakabayashi et al. (1999) reported an increased transcription of some clp genes during dark-induced and natural senescence. Nuclear genes encoding ClpP (nclpP) have been detected recently beside the clpP gene on the plastome (Sokolenko et al., 1998; Nakabayashi et al., 1999). The exploration of the various components of the Clp system, of the genes encoding them and of their physiological functions remains a challenge for future research.

C. ATP-Dependency of Proteolysis

No ATP is required for the hydrolytic cleavage of an accessible peptide bond. Nevertheless, several ATP-dependent proteolytic systems have been identified in plant cells. Such proteolytic activities are present in various subcellular compartments (Vierstra, 1993; Shanklin et al., 1995; Whelan and Glaser, 1997). In some cases the ATPase and the peptide hydrolase activities are present on different subunits (e.g., proteasome, Clp), while in other cases the two activite sites are present in the same polypeptide (e.g., in the mitochondrial protease Lon) as summarized by Lupas et al. (1997). Since ATP is not directly necessary for the cleavage of an accessible peptide bond, the ATP hydrolysis may be required for changes in the conformation of substrate proteins making them susceptible to proteolysis by exposing hydrolyzable peptide bonds to the active center of a peptide hydrolase. The ATPase moiety of proteolytic systems may be responsible for the substrate specificity by unfolding only those polypeptides with certain properties (e.g., destabilizing sequences, damage, partial denaturation).

D. Compartmentation

The vacuole contains various endopeptidases and carboxypeptidases (Heck et al., 1981; Canut et al., 1985; Barrett, 1986). The vacuole is frequently considered as the lytic compartment in plant cells. Although vacuoles contain a set of peptide hydrolases capable of hydrolyzing a series of leaf proteins including plastidial proteins, the role of this compartment in the remobilization of proteins prior to cell death is not yet clear. Several lines of evidence indicate that at least the initial steps in the degradation of plastidial proteins during senescence can occur inside the chloroplast (Feller and Fischer, 1994 and references therein). A transfer of peptides generated by plastidial protein degradation from the chloroplasts to the vacuole for final hydrolysis or the release of vacuolar enzymes into other cell compartments after membrane rupture could lead to an involvement of these peptide hydrolases in the catabolism of extravacuolar proteins and also to the remobilization of foliar proteins during senescence or other types of plant cell death (Guiamet et al., 1999). A defensive function of vacuolar peptide hydrolases was also considered. Since protein bodies represent a special type of protein-storing vacuoles, proteolysis in these vacuoles during germination is very important for the mobilization of nitrogen and the function of peptide hydrolases is obvious.

Peptide hydrolases have also been identified in the extracellular space (Graham et al., 1991; Groover and Jones, 1999). It was suggested that a 40-kDa extracellular protease plays an important role in the differentiation of tracheary elements (Groover and Jones, 1999), and that this protease is important at least for certain types of programmed cell death in plants.

The endoplasmic reticulum (Chrispeels, 1991), the mitochondria (Eriksson et al., 1996; Schatz and Dobberstein, 1996), the peroxysomes (Distefano et al., 1997) and the plastids (Reinbothe et al., 1995; Shanklin, 1995; Roulin and Feller, 1998) have their own set of proteolytic activities. The proteolytic enzymes in the various compartments may be involved in the processing of precursor proteins, in the removal of damaged or not properly assembled proteins, in protein turnover and finally also in the net protein remobilization prior to cell death.

Proteolytic activities in chloroplasts are of special interest for three reasons: (1) Most of the proteins present in a mesophyll cell of a C3-plant are in the chloroplasts (Peoples and Dalling, 1988). (2) Chloroplast functions and also chloroplast proteins are lost in an early phase of senescence, while other cell compartments are not yet affected (Makino et al., 1983; Gepstein, 1988; Feller and Fischer, 1994). (3) Intact chloroplasts are able to degrade abundant proteins present inside these organelles (Mitsuhashi et al., 1992; Desimone et al., 1996). Light is a major factor influencing protein catabolism in isolated pea chloroplasts (Fig. 7-1). Furthermore, different degradation products of the large subunit of Rubisco accumulated in the presence and absence of light. Only proteolysis in darkness was strongly inhibited by EDTA (most likely by inhibiting a metallo-endopeptidase). Possible mechanisms for the effect of light on the degradation of stromal proteins are summarized in Fig. 7-3. Several soluble and insoluble peptide hydrolases have been detected in chloroplasts (Musgrove et al., 1989; Bushnell et al, 1993; Shanklin et al., 1995; Lindahl et al., 1996; Ostersetzer et al, 1996). At least two types of proteases in the chloroplasts depend on ATP: the Clp system in the stroma with ClpP (protease moiety) and ClpC (ATPase moiety) and a membrane-bound homologue of the bacterial FtsH protease (Shanklin et al., 1995; Lindahl et al., 1996). The functions of these proteolytic enzymes in relation to cell death have not yet been identified.

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