Plant proteases, protein degradation, and oxidative stress: role of peroxisomes

Plant Physiol. Biochem. 40 (2002) 521–530 www.elsevier.com/locate/plaphy

Review

Plant proteases, protein degradation, and oxidative stress: role of peroxisomes José M. Palma *, Luisa M. Sandalio, F. Javier Corpas, María C. Romero-Puertas, Iva McCarthy, Luis A. del Río Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, CSIC, Apartado 419, 18080 Granada, Spain Received 4 December 2001; accepted 22 January 2002

Abstract Growth and development in all organisms occur as a result of an overall balance between synthesis and proteolysis. In plants, protein degradation is a crucial mechanism in some developmental stages such as germination, morphogenesis and cell biogenesis, senescence, and programmed cell death. In this work, the main proteases that take part in these processes are reviewed. Proteolysis is also an important component together with protein oxidation in oxidative stress situations induced by senescence and heavy metals. The presence of exo- and endoproteolytic activity in plant peroxisomes is analyzed, and the role of peroxisomal proteases in different physiological events that take place under oxidative stress situations is discussed. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Aminopeptidases; Endoproteases; Oxidative stress; Peroxisome; Programmed cell death; Senescence

Growth and development in most organisms occur as a result of the overall balance between protein synthesis and organelle biogenesis, and proteolysis and organelle turnover. In plants, protein degradation linked to different developmental stages, such as germination, differentiation and morphogenesis, senescence, and programmed cell death (PCD), has been reported [6,17,40,98]. On the other hand, there are an increasing number of references that report that, in certain circumstances, proteolysis is also associated to oxidative stress promoted by reactive oxygen species (ROS; O2–·, H2O2, ·OH) [86]. At cellular level, proteolytic processes commonly take place as a consequence of a regulated turnover of most cell components, including organelle biogenesis and autophagy [46]. During organelle biogenesis, translocation of preproteins to their respective target

compartments, i.e. mitochondria, chloroplasts, endoplasmic reticulum, etc., often requires the cleavage of a signal peptide. Once the polypeptide crosses the membrane, it is folded to form the final mature protein. In this event, chaperones are present on both sides of the membrane, thus facilitating the whole processing of proteins. This mechanism, which has been well studied in mitochondria and chloroplasts, is less understood in peroxisomes, where proteolysis is necessary for the import of some proteins like thiolase, acyl-CoA oxidase, and malate dehydrogenase [29,37,53]. Abnormal/misfolded and mistargeted polypeptides occurring by mistakes during the translation are also proteolytically degraded. These errors might be a consequence of mutations not only in those polypeptides but also in the set of enzymes/proteins involved in their synthesis, from transcription to the final processing in their target organelles [1,98].

Abbreviations: AP, aminopeptidase; CPE, chloroplast-processing enzyme; EP, endopeptidase; MDH, malate dehydrogenase; PCD, programmed cell death; PTS, peroxisomal targeting signal; ROS, reactive oxygen species; VPE, vacuolar-processing enzyme; XDH, xanthine dehydrogenase; XOD, xanthine oxidase * Corresponding author. E-mail address: [email protected] (J.M. Palma).

Protein degradation in plants is a complex process involving a multitude of proteolytic pathways that can be carried out in different cell compartments. The presence of proteolytic activity has been reported in several cell loci, such as vacuoles, chloroplasts, cell wall, microsomes, mitochondria, cytosol, and the Golgi apparatus [10,23]. However, little is known on the localization of proteases in

1. Introduction

© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 8 1 - 9 4 2 8 ( 0 2 ) 0 1 4 0 4 - 3

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peroxisomes. In this paper, the presence of exo- and endoproteolytic activity in plant peroxisomes and the proteolytic cleavage of plant proteins by peroxisomal endoproteases under oxidative stress conditions is reviewed, and the implications of the peroxisomal proteolytic metabolism in different physiological processes is discussed.

2. Proteases and proteolysis in plants Among the functions assigned to proteolysis are: (a) the removal of abnormal/misfolded, modified, and mistargeted proteins; (b) the supply of amino acids needed to make new proteins; (c) contribution to the maturation of zymogens and peptide hormones by limited cleavages; (d) the control of metabolism and homeostasis by reducing the abundance of key enzymes and regulatory proteins; and (e) the cleavage of targeting signals from proteins prior to their final integration into organelles [98]. These molecular mechanisms form a part of more sophisticated global processes related to plant growth and development. Thus, the role of proteases in some developmental stages, such as germination, morphogenesis and cell biogenesis, senescence, and programmed cell death, is essential. Most proteases act either on the interior of peptide chains (endopeptidases, EP) or on their termini (exopeptidases). Exopeptidases have been differentiated according to their substrate specificity as aminopeptidases (AP), which are able to cleave peptides at the N-terminus, and carboxypeptidases (CP), which degrade peptides at the C-terminus [17,40]. Aminopeptidases are ubiquitous enzymes that have been identified in several tissues from a large number of plant species, although only a few of them have been purified and characterized so far. There are many references that correlate plant aminopeptidase activity with different stages of growth and development, seed germination, seed and fruit maturation, host–pathogen interactions, wounding processes [34,101], and leaf senescence [27]. A thorough review of plant aminopeptidases has been provided by Walling and Gu [101]. Carboxypeptidases are one of the major classes of proteases found in seeds, although their presence has been also reported in mature green and senescent leaves [27]. The seed carboxypeptidases are located in the protein bodies of dicotyledonous plants and in the starchy endosperm of germinating cereals [25,44]. As general features, seed carboxypeptidases have serine in their active site, a pH optimum within the 4–6 range, and low specificity. However, the behavior of these isozymes after germination is variable depending on the species [44]. Endopeptidases are classified according to their catalytic mechanism, which implies a specificity in the enzyme active sites. It has been suggested that the term endopeptidase should be used synonymously with proteinase. In plants, four classes of endopeptidases have been described: serineproteinases (EC 3.4.21), cysteine-proteinases (EC 3.4.22),

aspartic-proteinases (EC 3.4.23), and metallo-proteinases (EC 3.4.24). 2.1. Serine-proteinases Serine-proteinases (Ser-EP) are neutral proteinases harboring an active Ser in the catalytic site, which interacts with the proteic substrate. Ser-EPs have been found to be involved in a variety of physiological processes, such as senescence [24,40], programmed cell death [6], xylogenesis [104], tissue differentiation [32], the infection of plant cells [48], pathogenesis in virus-infected plants [96], and germination [92]. Many plant serine-proteinases have been purified and characterized so far. The most studied serineproteinases in plants are the chloroplastic ATP-dependent ClpP proteases [1,15], subtilisins (including cucumisin-like proteins), and kexin-type enzymes [6,85] (Table 1). ClpP proteases are constitutive, but some lightstimulation in their expression has been observed. The involvement of ClpP proteases in the degradation of mistargeted proteins has been suggested, although it appears that these enzymes might also participate in the protein import process by means of their putative chaperone-like activity [1]. However, the direct role of ClpP in both processes needs to be demonstrated. Subtilisins are a large multigene family with distinct expression patterns at the organ level [6,58]. Several other events, such as pathogenand development-specific regulation of plant subtilisins, reveal the requirement for these enzymes in a number of proteolytic processes throughout the plant’s life [6,96]. Subtilisin-like proteins have also been proved to participate in nodule formation by Frankia in both Betulaceae and Casuarinaceae [48]. Kexin-like proteins may process bioactive cell wall peptides to initiate signal transduction pathways in the extracellular matrix [85]. Therefore, kexin-type enzymes have been associated to plant defense responses and also to plant PCD. The participation of these proteolytic isozymes in germination has been also reported [6].

Table 1 Main endopeptidases (proteinases) reported in plants Type of proteinase

Protease name

References

ClpP Subtilisins Kexin-type proteins Cysteine-proteinases Caspase-like proteins Vacuolar-processing enzyme (VPE) Papain-like peptides Cathepsin-type proteases Asparaginyl endopeptidases Aspartic-proteinases Cardosins Cathepsin D-like proteins Metallo-proteinases FtSH Chloroplast-processing enzyme (CPE) Matrix-like enzymes

[1,15] [6,48,58] [6,85] [18,19,95] [36] [35,83,104] [3,39,93,97] [9,65] [12,71] [55,79] [1,51] [74] [52]

Serine-proteinases

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2.2. Cysteine-proteinases Cysteine-proteinases (Cys-EP) are the group of plant proteases that have been most thoroughly studied. The catalytic mechanism of these enzymes involves a cysteine group in the active site. In plants, many biological events have been reported where Cys-EPs take part. They include senescence [26,35,97], differentiation of mesophyll cells into tracheary elements and xylogenesis [28,104], pathogenlinked programmed cell death [3,6,19], mobilization of proteins during germination [25,83,92], and wounding [97]. The best-known cysteine-proteinases are caspase-like proteins, vacuolar-processing enzymes (VPEs), papain-like peptides, and cathepsin-type proteases (Table 1). Caspases play a central role in the execution of PCD in mammalian cells. These enzymes have the ability to cleave adjacent to an aspartic acid residue. It has been reported that caspaselike activity is also associated to plant apoptosis, and that caspase-specific inhibitors abolish bacteria-induced PCD [18,19]. Recently, the involvement of poly(ADP–ribose) polymerase—another PCD marker—and the activation of caspase-3-like protease have been reported in heat shockinduced apoptosis in tobacco suspension cells [95]. However, no caspase genes or caspase enzymes have been isolated from plants so far. VPEs are responsible for the maturation of various vacuolar proteins and are widely distributed in several plant organs. The analysis of the expression of the VPE system suggests that these enzymes play a role in the maturation of seed stored proteins and the activation of senescence-associated hydrolytic enzymes in the lytic vacuole, thus preparing cells for death [36]. The family of papain-like peptidases is the most thoroughly investigated of the cysteine-proteinases. These enzymes are synthesized as zymogens and have broad substrate specificity and the requirement of reducing agents for their full activity. In stems, the expression of a papain-like gene was reported to be associated with differentiating xylem [104]; a papain homolog cDNA clone whose expression is strongly upregulated in flower tepal senescence has also been isolated [35]. Very recently, it was reported that several papain-like proteins were present in the axes and cotyledons of dry seeds as well as during the germination and seedling growth of vetch (Vicia sativa L.) [83]. In this plant species, proteinase A is also present. Proteinase A is a papain-like protein that possibly participates in the mobilization of storage proteins, by their hydrolysis, during the late stages of germination [5]. Another intensively studied group of cysteineproteinases are cathepsin-type proteins. Cathepsins are lysosomal glycoproteins present in all mammalian cell types [8]. These proteases are active against a wide range of small peptides and large protein substrates, and show different catalytic mechanisms: cathepsin R is a serine-proteinase, cathepsins D and E are aspartic-proteinases, whereas cathepsins B, K, H, N, S, M, and T are cysteine-proteinases [8]. In plants, the purification of aleurain, a cysteine-protease

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with aminopeptidase activity and a 65% amino acid sequence identity, functionally homologous to cathepsin H, has been carried out [39]. Aleurain, which plays an important role in the mobilization of storage proteins in barley, has been located in lytic vacuoles [93]. Finally, a protein strikingly similar to cathepsin K has been detected in potato during early stages of the incompatible interaction with Phytophthora infestans [3]. The expression of cathepsinlike proteins has been found to be enhanced under natural senescence conditions and reduced by wounding [97]. Cysteine-proteinases also include asparaginyl endopeptidases, which are mainly involved in the mobilization of storage proteins during seed germination of several species [9,65]. In vetch, an asparaginyl endopeptidase that hydrolyzed bonds at the carboxyl side of Asn residues in the A and B chains of insulin was found to be unable to degrade the native 11S globulin from dry seeds. The enzyme only degraded this substrate after an initial hydrolysis by proteinase A. On the basis of this segmental hydrolytic action on the 11S globulin, the name proteinase B was proposed for the vetch asparaginyl endopeptidase [9]. 2.3. Aspartic-proteinases Aspartic-proteinases have a preference for peptide bonds flanked by hydrophobic amino acid residues, and are active at acidic pH [6,8]. Although these enzymes have been described in a wide range of plant species, most studies have been conducted in barley grains, where aspartic-proteinases are abundant. Less attention has been paid to asparticproteinases than to Ser-EPs and Cys-EPs, and, therefore, little is still known about their biological function [99]. Aspartic-proteinases are apparently implicated in the degradation of extracellular pathogenesis-related proteins, and the proteolysis of the globular storage proteins of cocoa seeds [99] and castor beans [38]. These enzymes are common in ungerminated seeds, but their activity has been proved to be essential during the germination and development of seeds [7,55]. An aspartic-proteinase has also been found to be induced in tomato leaves as a systemic wound response protein [82]. Phytepsins (plant pepsin-type enzymes) belong to the aspartic-proteinase group and are commonly inhibited by pepstatin. They include cardosins and cathepsin D-like proteins (Table 1). Cardosins are proteins extracted from the dried flowers of Cynara cardunculus that are used as milk-clotting enzymes for the production of sheep’s milk cheese in Portugal and western Spain [12,71]. Cathepsin D-like aspartic-proteinases have been found in ungerminated seeds, during germination, and also in all the living tissues of barley grain, including shoots and roots [55,79]. 2.4. Metallo-proteinases Plant metallo-proteinases are less known than the other groups of proteases. Most enzymes contain Zn, although

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cobalt or manganese can also be present to activate the water molecule for the hydrolysis of peptide bonds [8]. Among the most studied plant metallo-proteinases are the chloroplastic ATP-dependent FtSH [51], possibly implicated in the removal of unassembled and incompletely synthesized polypeptides [1], and the chloroplast-processing enzyme (CPE; Table 1) [74]. A metallo-proteinase has been also localized almost exclusively in the apoplast of soybean leaves and was very abundant during the late stages of leaf expansion [31]. More recently, the purification and characterization of a metallo-proteinase from alfalfa senescent leaves [61] and the cloning of an Arabidopsis thaliana cDNA homologous to the matrix metallo-proteinase [52] have been reported. Matrix metallo-proteinases include a number of animal endopeptidases involved in the degradation of extracellular matrix proteins. Results obtained with animal matrix metallo-proteinases suggest that the plant enzymes may be important regulators of growth and development, and could even participate in plant PCD [6]. 2.5. The ubiquitin–26S proteasome system The main mechanism by which proteins from the cytosol and nucleus in eukaryotes are degraded in a nonlysosomal way implies the proteasome [10,41]. This system consists of several subunits that are folded as a 700-kDa cylinder through which unfolded polypeptides are channeled and cleaved internally to produce peptides 4–10 residues in length. The so-called 20S proteasome, according to its sedimentation coefficient, associates with another multisubunit protein, the 19S cap, thus forming the ATP-dependent 2100-kDa, 26S proteasome [6,98]. The proteasome does not belong to any of the classes of proteinases described above, although lately several authors have included it in a new class of proteases, the threonine endopeptidases (EC 3.4.25) [6,72]. Three main types of proteolytic specificities are known for the proteasome: trypsin-like, chymotrypsin-like, and peptidylglutamyl peptide-hydrolyzing [6]. Previous to their degradation by the proteasome, proteins are tagged in a polyubiquitin manner. Those proteins that are fated for degradation in the cytosol and the nucleus are polyubiquitinated. The proteins thus labeled are degraded by the 26S proteasome [41,98]. Besides, the ubiquitin pathway is also important for developmental regulation by selectively removing various cell-cycle effectors, transcription factors, and cell receptors such as phytochrome A [98]. Also, it has been reported that the small polypeptide ubiquitin participates in a variety of fundamental cellular events, such as cell differentiation, stress response, and PCD [100], and in pollen metabolism [2,87]. For a detailed review of this topic, readers are recommended to [41,98,100]. 2.6. Signal peptidases Another type of proteases, which is not usually included in the groups mentioned in the previous sections, is that

Fig. 1. Model proposed for the degradation of oxidized proteins in animal cells. Oxidized proteins are mainly cleaved by the 20(26)S proteasome, which prevents aggregate formation and further tissue damage. Proteolysis can also help in removing potentially toxic protein fragments and provide amino acids for new protein synthesis (modified from Grune et al. [33]). Thick arrows indicate the predominant pathways.

formed by the signal peptidases. These enzymes play an essential role in the processing of immature polypeptides to their final fate either in mitochondria, endoplasmic reticulum or the different chloroplastic sites [4]. All signal peptidases are integral membrane proteins with the catalytic site facing the external side or the membrane. They are ideally positioned to cleave membrane-bound preproteins already translocated to their target organelle, but still attached to the membrane by their signal peptide [16].

3. Protein degradation and oxidative stress in plants In addition to the involvement of proteases in the biological processes described above, protein degradation also occurs under conditions that induce oxidative stress. In fact, since early works obtained in Professor Kelvin Davies’ laboratory, a number of reports have shown that cells exhibit increased rates of proteolysis following exposure to oxidative stress-inducing agents [33,67]. The working hypothesis for these reports implies that intracellular proteins are oxidatively modified by free radicals and/or related oxidants, and these modified proteins are selectively recognized and preferentially degraded by intracellular proteolytic enzymes [33] (Fig. 1). This model has been proved to be valid in all eukaryotic organisms, including plants [86]. The protein modification promoted by oxidative stress is characterized by the production of carbonyl groups in the molecule [50,73]. Basal levels of carbonyl groups are detected in plants, as a result of their generation as byproducts of normal physiological processes. However, increases in carbonyl contents have been observed in maize seedlings after chilling-induced oxidative stress [45,69], in senescing nodules from pea and bean [56], in isolated chloroplasts exposed to oxygen radical-generating systems [88], in thylakoid proteins from water-stressed leaves of wheat [94], and in leaves of pea plants grown under toxic Cd concentrations [76]. In the case of Cd, it was found that

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this metal induced oxidative stress in pea plants [81], and some peptides, such as Rubisco, glutathione reductase, manganese superoxide dismutase, and catalase, were specifically oxidized by treatment with Cd. It was also found that Cd enhanced the proteolytic activity, and by using specific antibodies, it was demonstrated that the oxidized proteins were more efficiently digested by proteases [76]. Lascano and colleagues [49] also reported that exposing chloroplastic glutathione reductase to an ·OH generating system brought about the breakdown of the enzyme by a sulfhydryl- and metal-containing protease. Works by other authors have associated the expression of different proteases and/or their proteolytic activity to conditions that usually induce oxidative stress, i.e. anoxia [91], drought and temperature stress [90], and the pathogen-promoting hypersensitive response [103]. However, the specific combination oxidative stress–proteolysis was not directly addressed. Different evidence obtained during the last years substantiated the idea that PCD and senescence [70] are the physiological archetypes where controlled proteolysis is linked to the oxidative stress generated by ROS. PCD is one of the biological processes most thoroughly studied nowadays. It is generally accepted that ROS trigger PCD and antioxidants inhibit mechanisms leading to apoptosis [43,86]. During apoptosis, protein degradation also takes place in a modulated way [6,28]. In recent years, several proteases (Cys-EPs and Ser-EPs) have been shown to be involved in plant PCD, mainly in virus-, bacteria-, and fungi-promoted pathogenesis [3,19,47], and organogenesis [32,102]. Plant senescence is a natural process mainly characterized by an intensive loss of proteins and chlorophyll, as well as severe increases in lipid peroxidation and membrane permeability due to a notable enhancement in the metabolism of activated oxygen [20]. Senescence in plants has also been reported to be accompanied by an increase in the proteolytic activity measured either using exogenous substrates or following the degradation of endogenous proteins [22,24,42]. In fact, senescence has been postulated to be a genetically regulated process, controlled by internal and external signals, in which proteases play a key role [11,64]. Moreover, the ability of cells to switch from one developmental state to another or to adapt to new environmental conditions often requires the rapid dismantlement of existing regulatory networks through proteolysis [98]. At the subcellular level, most of the information available on the combined action of oxidative stress plus proteolysis has been obtained from studies on chloroplasts. Thus, Ishida and colleagues [42] reported that, in wheat chloroplasts, the large subunit (LSU) of Rubisco is broken down by ROS into 37- and 16-kDa polypeptides. Similar results were also found in pea plants [77]. In this plant species, the degradation of chloroplastic phosphoglycolate phosphatase, glutamine synthetase, and other enzymes took place as well [89]. These studies also revealed that the degradation of the stroma proteins was light dependent,

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although some light-independent degradation may also occur [78]. The fact that proteins that become nonfunctional due to interactions with oxygen species are further degraded by proteolysis is particularly significant in the thylakoid membranes. In these membranes, enzymes operate in a highly oxidizing environment and are susceptible to structure and function impairments [51]. Within the thylakoid membrane, photosystem II (PSII) is the most susceptible component to oxidative damage. The degradation of the PSII D1 protein has been found to be carried out by the metallo-peptidase FtSH in a reaction that requires GTP [51].

4. Proteases and plant peroxisomes In plants, the presence of proteolytic activity has been reported in several cell compartments, such as vacuoles, chloroplasts, the cell wall, microsomes, mitochondria, cytosol, and the Golgi apparatus [10,23]. However, the demonstration of the presence of proteolytic activity in plant peroxisomes is recent, and the information in this field is still scarce. Peroxisomes are subcellular respiratory organelles, containing, as basic enzymatic constituents, catalase and H2O2-producing flavin oxidases [21]. These organelles have an essentially oxidative type of metabolism, and it has become increasingly clear that they carry out vital functions in plant cells and play an important role in the generation of signal molecules [13,21]. Several metabolic processes support the idea that some proteolytic activity must exist in peroxisomes. The conversion of glyoxysomes into leaf peroxisomes during the germination of seeds, as well as the opposite process converting leaf peroxisomes into glyoxysomes, implies the degradation of the organelle’s pre-existing proteins [62]. During the translocation of some polypeptides into the peroxisome, a proteolytic cleavage of the N-terminus takes place [53,60,66]. This partial proteolysis has been observed in in vitro import assays with some peroxisomal proteins, such as thiolase, MDH, and acyl-CoA oxidase, whose molecular mass inside the organelle is lower than that of the translation product [29,37,53]. The cleaved peptidic sequence corresponds to the peroxisomal targeting signal located at the N-terminus (PTS2), and the process is accomplished in a way similar to that reported for mitochondrial and chloroplastic proteins [4]. On the other hand, it has been reported that plant peroxisomes contain the xanthine oxidoreductase system [21]. Xanthine oxidoreductase is an FAD-, molybdenum-, iron-, and sulfur-containing hydroxylase that has been found as two interconvertible forms (D and O). The conversion of form D (xanthine dehydrogenase, XDH) into form O (xanthine oxidase, XOD) is carried out by either reversible or irreversible pathways. In the irreversible step, XDH is converted into XOD by a proteolytic cleavage [24,63]. In peroxisomes from senescent pea leaves, a much higher increase of XOD activity is observed compared to XDH activity [68]. This

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Table 2 Proteases characterized in plant peroxisomes. Peroxisomes were purified from young (15-d-old) and senescent (50-d-old) leaves, and from leaves of pea plants grown with 50 µM CdCl2. +, Present; –, absent; n.d., not determined Isozyme Exopeptidases Leu-AP Endopeptidases EP1 EP2 EP3 EP4 EP5 EP6 EP7

Young plants

Senescent plants Plants grown with 50 µM CdCl2

+

n.d.

+

57

– + – + + – –

+ + + + + + +

– – + + + – +

220 88 76 64 50 46 34

suggests that the conversion of XDH into XOD seems to occur inside peroxisomes, as has been observed in mitochondria [80]. In spite of this evidence, very little attention has been paid to the study of proteases in peroxisomes. In the early 1990s, endopeptidase activity and a leucine-aminopeptidase (Leu-AP) were detected in the soluble fraction of peroxisomes from pea leaves [14]. The peroxisomal Leu-AP was characterized as a serine-protease and had a maximal activity at pH 7.5, a molecular mass of 56.8 kDa, and a pI of 5.3. In a recent work, the presence of an additional Leu-AP in pea leaf peroxisomes has been reported, and the activity of the two Leu-APs increased in plants subjected to metal stress by cadmium [57]. Gietl and colleagues [30] found in castor bean a cysteine endoprotease, which was initially localized in glyoxysomes. Nevertheless, further studies showed that this enzyme was located in ricinosomes, a new type of organelle, whose name is due to their presence in the endosperm of castor bean (Ricinus communis L.) [84]. Ricinosomes have been characterized by ultrastructural and cytochemical studies. These organelles are slightly smaller than glyoxysomes, and seem to develop from the endoplasmic reticulum [84]. The presence of endoprotease activity in peroxisomes has been investigated in pea leaves. In assays with peroxisomes from young leaves in the presence of azocasein, a specific substrate for endoproteases, a low EP activity of approximately 167 nKat mg–1 protein was measured. By contrast, an up to sixfold higher endoproteolytic activity was observed in senescent plants [23]. Through analysis by SDS –PAGE in gelatin-containing gels, three EP isozymes were detected in peroxisomes from young leaves, which were designated EP2, EP4, and EP5. However, four additional isoenzymes (EP1, EP3, EP6, and EP7) were detected in senescent plants [23]. The endopeptidases were characterized by using different class-specific inhibitors (Table 2). All proteases were neutral, and the serine-proteinases (EP1, EP3, and EP4) represented about 70% of the total EP activity of peroxisomes from senescent leaves. Besides, these serine-proteinases showed a notable thermal stability, not being inhibited by incubation at 50 °C [23]. In a recent work, the endoproteolytic activity of leaf peroxisomes was

Molecular mass (kDa)

Type of isozyme

References

Serine-proteinase

[14,57]

Serine-proteinase Cysteine-proteinase Serine-proteinase Serine-proteinase ? Cysteine-proteinase Metallo-proteinase

[23] [23] [23,57] [23,57] [23,57] [23] [23,57]

found to be increased by growing pea plants with Cd. This enhancement was a consequence of the overall increase in the activity of the EP isozymes detected in SDS/gelatincontaining gels. In these experiments, the isozymes EP3, EP4, EP5, and EP7 were also present in peroxisomes from Cd-treated plants (Table 2) [57].

5. Role of peroxisomal proteases in oxidative stress situations The higher proteolytic activity found in peroxisomes from senescent leaves suggested a role for peroxisomal EPs at that developmental stage. Senescence in plants is associated to several metabolic events similar to those that occur at the subcellular level during oxidative stress situations. In particular, plant peroxisomes were postulated to play an activated oxygen-mediated function during natural senescence [68]. The relevance of peroxisomes in senescence has also been shown in hepatocytes [105]. In order to know how peroxisomal endopeptidases participate in the processes occurring during senescence, the degradation of peroxisomal and nonperoxisomal proteins by the endoproteases of purified peroxisomes from senescent pea leaves was investigated [24]. It was found that most peroxisomal proteins were endoproteolytically degraded, and this was specifically demonstrated in the case of catalase, glycolate oxidase, and glucose-6-phosphate dehydrogenase. Peroxisomal proteases were also able to cleave nonperoxisomal proteins like Rubisco and urease [24]. These results indicate that proteases from plant peroxisomes might play an important role in the turnover of peroxisomal proteins during senescence. This means that the organelle’s own proteolytic machinery could participate in the mechanism of conversion of leaf peroxisomes into glyoxysomes, which is known to occur in senescent tissues [24]. Also, peroxisomal EPs might be involved in the turnover of proteins located in other cell compartments during advanced stages of senescence, when deterioration of membranes and leakage of the organelles’ soluble fraction takes place (Fig. 2). In experiments carried out in vitro, it was demonstrated that bacterial XDH was converted into XOD in the presence

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Fig. 2. Possible cellular functions of peroxisomal proteases. The leucine aminopeptidase (Leu-AP) might be involved in the cleavage of the N-terminus of polypeptides harboring a PTS2, such as acyl-CoA oxidase, thiolase, and malate dehydrogenase. The conversion of xanthine dehydrogenase (XDH) into xanthine oxidase (XOD), taken from the model proposed by Nishino [63], can be carried out by peroxisomal endoproteases (EPs). The degradation of peroxisomal proteins (catalase, glycolate oxidase, glucose-6-phosphate dehydrogenase, and, probably, other proteins) is accomplished by the organellar EPs. Under senescence conditions, when deterioration of membranes takes place, EPs might be also involved in the degradation of nonperoxisomal proteins.

of peroxisomes from senescent leaves [24]. These data suggest that peroxisomal EPs could also participate in regulatory mechanisms that do not necessarily imply full degradation of proteins (Fig. 2). On the other hand, it was reported that 50 µM CdCl2 produced an enhancement of the hydrogen peroxide concentration in peroxisomes and also an increase in the activity of some antioxidative enzymes of the ascorbate–glutathione cycle and NADP-dependent dehydrogenases located in these cell organelles [75]. Furthermore, in western blotting assays using an antibody against dinitrophenylhydrazone (DNPH), which recognizes oxidatively modified proteins, a higher level of oxidized proteins was found in peroxisomes from Cd-treated plants [76]. Under the same experimental conditions, Cd induced senescence symptoms in leaf peroxisomes, with an enhancement of the endogenous proteolytic activity and the activity of the glyoxylate cycle enzymes malate synthase and isocitrate lyase [57]. Taken together, these results indicate that the peroxisomal proteases could participate in the metabolic changes produced by Cd, by degrading the oxidized proteins, and possibly also in the transition of leaf peroxisomes into glyoxysomes, which is known to occur during senescence [20,62].

6. Future prospects At present, one of the most studied aspects of peroxisome biology is the biogenesis of these organelles. Peroxisomal

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proteins are synthesized in free polyribosomes, with a targeting signal that drives proteins to their organelle. Much is known about the different targeting signals of peroxisomal proteins (PTS) and the required cofactors for the translocation inside the organelle [53,60,66]. Two main PTSs have been reported so far. PTS1 resides in the C-terminus with a tripeptide analogous to A/C/G/S/TH/K/L/N/R-I/L/M/Y [59]. PTS2, as indicated above, is in the N-terminus and is usually cleaved upon the translocation of polypeptides through the membrane. This process possibly implies the involvement of an aminopeptidase. The occurrence of a Leu-AP in peroxisomes suggests that it might be involved in the processing of imported precursor polypeptides in peroxisomes (Fig. 2). The purification of the peroxisomal Leu-AP, and its further characterization and assays with its specific substrates, will help in clarifying the mechanisms of processing of polypeptides till their mature form during the translocation to the peroxisome. On the other hand, the study of the combined mechanism of oxidation plus proteolytic degradation of proteins under certain conditions is gaining relevance in plant biology. Senescence and metal toxicity by cadmium are representative of an oxidative stress taking place together with an enhanced proteolytic activity [76,81]. In these experimental conditions, a parallel behavior was observed in the metabolism of leaf peroxisomes. The molecular and biochemical characterization of peroxisomal proteases will allow the comparison of their properties with those available in databanks. It will provide information on their catalytic mechanism and their putative substrates. Studies on the role of peroxisomal proteases in oxidative stress situations can be accomplished by the incubation of enzymes with different cell components. This is particularly relevant in the light of recent results in Arabidopsis plants that demonstrated that oxidative stress induced PEX genes involved in the peroxisome biogenesis [54]. The control of proteolysis will be also of interest in terms of increasing plant productivity. This control could be established by using specific inhibitors. Plants are known to contain important levels of protease inhibitors that regulate enzyme activity. This strategy of manipulating senescence could overlap other strategies such as that of breeding or genetic engineering to improve crop yields by keeping leaves photosynthetically active for longer. Today, much attention is being devoted to the investigation of PCD in plants, a physiological stage characterized by a notable oxidative stress and the activation of several proteolytic pathways. At the subcellular level, there is still little information on the events that occur in PCD processes in plants. Taking into account that senescence is a type of PCD, the cellular and molecular study of the possible involvement of peroxisomal proteases in apoptosis could provide deeper insights into this important physiological process.

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Acknowledgements The authors apologize to many colleagues whose work could not be cited directly because of space limitations. This work has been supported by contract HPRN-CT-200000094 from the European Union and grant PB98-0493-01 from the DGESIC (Ministry of Education and Culture), and the Junta de Andalucía (Research Group CVI 0192). M.C.R.-P. and I.M. acknowledge their Ph.D. fellowships from the Junta de Andalucía and Fundación Ramón Areces, respectively.

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