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Purification and Characterization of Serine Proteases That Exhibit Caspase-Like Activity and Are Associated with Programmed Cell Death in Avena sativa

Department of Botany and Plant Pathology and Center for Gene Research and Biotechnology, Oregon State University, Corvallis, Oregon, 97331-2902

¹ To whom correspondence should be addressed. E-mail wolpertt{at}bcc.orst.edu; fax 541-737-3573.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Victoria blight of Avena sativa (oat) is caused by the fungus Cochliobolus victoriae, which is pathogenic because of the production of the toxin victorin. The victorin-induced response in sensitive A. sativa has been characterized as a form of programmed cell death (PCD) and displays morphological and biochemical features similar to apoptosis, including chromatin condensation, DNA laddering, cell shrinkage, altered mitochondrial function, and ordered, substrate-specific proteolytic events. Victorin-induced proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is shown to be prevented by caspase-specific and general protease inhibitors. Evidence is presented for a signaling cascade leading to Rubisco proteolysis that involves multiple proteases. Furthermore, two proteases that are apparently involved in the Rubisco proteolytic cascade were purified and characterized. These proteases exhibit caspase specificity and display amino acid sequences homologous to plant subtilisin-like Ser proteases. The proteases are constitutively present in an active form and are relocalized to the extracellular fluid after induction of PCD by either victorin or heat shock. The role of the enzymes as processive proteases involved in a signal cascade during the PCD response is discussed.

	INTRODUCTION

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Victoria blight of Avena sativa (oat) is caused by the necrotrophic fungus, Cochliobolus victoriae (Meehan and Murphy, 1946), which is pathogenic because of the production of the host-specific toxin, victorin (Meehan and Murphy, 1947). Isolates of C. victoriae that produce victorin are pathogenic on susceptible A. sativa (Meehan and Murphy, 1947), whereas mutants or outcrosses that do not produce the toxin are nonpathogenic. Host susceptibility and victorin sensitivity are conferred by a dominant allele at the Vb locus (Litzenberger, 1949). Homozygous recessive genotypes (vb/vb) are victorin insensitive and resistant to the fungus. The interaction between victorin and the Vb gene product induces a response in A. sativa that displays characteristics of programmed cell death (PCD) (Navarre and Wolpert, 1999; Tada et al., 2001; Yao et al., 2001; Curtis and Wolpert, 2002, 2004).

PCD is a genetically controlled, organized form of cellular suicide that functions in eliminating unnecessary or aged cells. It is essential for cellular maturation and morphogenesis and is required to maintain cellular homeostasis in multicellular organisms. In addition, improper regulation of PCD has been implicated in a wide variety of animal diseases (Polverini and Nör, 1999; Wang and Wang, 1999).

PCD also has been associated with several processes in plants, including senescence (Bleecker and Patterson, 1997; Miller et al., 1999; Schmid et al., 2001), stress (Katsuhara, 1997; Solomon et al., 1999), development (Runeberg-Roos and Saarma, 1998; Groover and Jones, 1999; Schmid et al., 1999), and the hypersensitive response (HR) to pathogens (Dangl et al., 1996; Mittler et al., 1997; Pontier et al., 1998; Mackey et al., 2002; Abramovitch et al., 2003). Currently, very little is known about the fundamental processes that control and regulate PCD in plants.

Apoptosis, the most characterized form of PCD, has been extensively studied in animal systems and can be distinguished by unique characteristics. Cells undergoing apoptosis display morphological changes, including cell shrinkage, chromatin condensation, and apoptotic body formation. Biochemically, apoptotic cells exhibit DNA fragmentation (also referred to as DNA laddering) and activation of a family of Cys proteases called caspases (cysteine aspartases) (reviewed in Vaux and Korsmeyer, 1999; Hengartner, 2000). The name caspase is derived from their active site residue (which is a Cys) and substrate specificity (caspases cleave only after aspartate residues, aspase).

The victorin-induced PCD response in sensitive A. sativa demonstrates similar morphological and biochemical traits to animal apoptosis, including cell shrinkage and collapse (Yao et al., 2001; Curtis and Wolpert, 2004), chromatin condensation (Yao et al., 2001), DNA laddering (Navarre and Wolpert, 1999; Tada et al., 2001), mitochondrial depolarization and permeability transition (Curtis and Wolpert, 2002, 2004), and ordered, substrate-specific proteolytic events (Navarre and Wolpert, 1999). Furthermore, victorin-induced PCD in A. sativa is easily initiated, proceeds in a rapid and synchronous manner, and appears to encompass at least all leaf mesophyll cells. Therefore, victorin treatment of A. sativa provides an appropriate system in which to study the mechanism and progression of plant PCD.

The proteolytic cleavage of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has been identified as a specific PCD-induced event in victorin-treated A. sativa cells (Navarre and Wolpert, 1999). Rubisco cleavage occurs after the first 14 amino acids (apparently after glutamate-14) and is prevented by Cys protease inhibitors (Navarre and Wolpert, 1999). Rubisco degradation has been identified as a characteristic of senescence (Weidhase et al., 1987; Ferreira and Davies, 1989), also a form of PCD, and has been shown to occur in A. sativa chloroplast after oxidative stress (Casano and Trippi, 1992), a treatment shown to induce PCD (Amor et al., 1998; Solomon et al., 1999). Additionally, chloroplast-localized proteases have been reported that appear to recognize Rubisco as a substrate (Bushnell et al., 1993; Casano et al., 1994). These data indicate that a specific proteolytic process is required to degrade Rubisco and that this process is common to several types of PCD.

Proteolytic alteration of key cellular proteins is a fundamental characteristic of animal apoptosis and is executed by the highly specialized caspases. Multiple cellular targets exist for caspases, all of which are directly or indirectly involved in the ordered disassembly of the cell. Two types of caspases exist, initiator and effector caspases. Initiator caspases are activated by autoproteolysis, after which they are able to proteolytically activate effector caspases. Effector caspases target cellular proteins, such as poly(ADP-ribose) polymerase (PARP), which is proteolytically inactivated, resulting in a signature 89-kD fragment (reviewed in Solary et al., 1998). In addition, CAD, a caspase-activated DNase, is indirectly activated when an effector caspase cleaves ICAD (inhibitor of CAD), allowing CAD to disassociate and process DNA into nucleosomal fragments (reviewed in Nagata, 2000). Caspase proteolysis typifies the ordered processive nature of apoptosis because caspases function with exquisite control, cleaving at limited site(s) found within specifically targeted key proteins, differing from the degradative role of other proteolytic pathways required during apoptosis (e.g., the proteasome-ubiquitin pathway of protein degradation) (Solary et al., 1998).

Current research has demonstrated that caspase-specific inhibitors can prevent different forms of plant PCD, suggesting that caspase-like enzymes may be involved in these responses (reviewed in Woltering et al., 2002). For example, Tobacco mosaic virus–induced HR in Nicotiana tabacum leaves was prevented by treatment with short peptide inhibitors that preferentially inhibit caspase-1 and caspase-3 proteases (del Pozo and Lam, 1998). Other forms of PCD in N. tabacum, including those induced by bacteria (Richael et al., 2001) and treatment with a fungal elicitor (Elbaz et al., 2002), also were prevented by caspase inhibitors. Similar results were obtained with caspase inhibitors in preventing or reducing cell death after treatment with camptothecin (De Jong et al., 2000), staurosporine (Elbaz et al., 2002), and isopentenyladenosine (Mlejnek and Procházka, 2002). In addition, heat shock–induced PCD in N. tabacum suspension cells stimulated cleavage of PARP and activation of a caspase-3–like protease (Tian et al., 2000), whereas N. tabacum protoplast treated with menadione displayed PARP cleavage (Sun et al., 1999). However, to date, no protease that recognizes caspase substrates or is inhibited by caspase inhibitors has been identified in plants.

Studies have described proteases associated with several types of plant PCD, including senescence (Delorme et al., 2000; Schmid et al., 2001; Eason et al., 2002), oxidative stress (Solomon et al., 1999), seed development (Schmid et al., 1998, 1999; Wan et al., 2002), tracheary element development (Runeberg-Roos and Saarma, 1998; Groover and Jones, 1999), and the HR (Vera and Conejero, 1988; D'Silva et al., 1998; Krüger et al., 2002). However, characterization of these proteases reveals that they are degradative, not processive enzymes; thus, they are unlike caspases or other proteases involved in signaling pathways. A degradative nature suggests that their role in PCD may be associated with the terminal decomposition of the dying cell but not in the initiation or progression of the PCD signal.

This research describes the purification and characterization of two proteases that possess caspase-like specificity but contain an active-site Ser residue. Therefore, because of their aspartate specificity (aspase) and active-site Ser residue, they have been termed saspases. The saspases, purified from A. sativa, contain amino acid sequence similarities to subtilisin-like Ser proteases from plants and likely function in a PCD-induced signaling cascade involving other proteases that leads to the proteolytic processing of Rubisco. They also are constitutively present in the cell, not being transcriptionally or translationally activated during the response, but released into the extracellular fluid (ECF) upon induction of PCD. Heat shock–induced PCD also is further characterized and displays similar biochemical features as victorin-induced PCD, including DNA laddering, Rubisco proteolysis, and release of the saspases into the ECF. These results and the implications of numerous subtilisin-like Ser proteases involved in plant disease, development, and death are discussed.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Evidence for a Protease Cascade Leading to Rubisco Proteolysis
Rubisco proteolysis is a characteristic of victorin-induced PCD and has been shown to be inhibited by two general protease inhibitors, E-64 (a Cys protease inhibitor) and leupeptin (a Cys and Ser protease inhibitor) (Navarre and Wolpert, 1999). Two caspase and a granzyme B inhibitor were tested for their effects on Rubisco proteolysis. These and a large variety of other short peptide inhibitors (and corresponding substrates) were designed based on the caspase recognition sites in known substrates. These inhibitors are typically preferential for a given caspase but can often inhibit a wide variety of caspases (Kidd, 1998). One inhibitor initially evaluated, Ac-VAD-CMK, is considered a pan-caspase inhibitor because it can inhibit most caspases (Thornberry et al., 1997; Kidd, 1998; Ekert et al., 1999), whereas another inhibitor, Ac-DEVD-CMK, is more specific for caspase-3 (Thornberry et al., 1997; Ekert et al., 1999). In addition, we tested Z-AAD-CMK, which inhibits granzyme B (Ekert et al., 1999), a Ser protease that specifically cleaves peptide bonds after aspartate residues (the recognition sequence for the three inhibitors is in single-letter amino acid code). Preincubation of peeled A. sativa leaves with the caspase and granzyme B inhibitors prevented victorin-induced Rubisco proteolysis (Figure 1). The effectiveness of these inhibitors was similar to that of E-64 and leupeptin.

View larger version (39K): [in this window] [in a new window]   Figure 1. Inhibition of Rubisco Proteolysis with Protease Inhibitors. Leaf segments with epidermis removed were pretreated with 200 µM of the indicated inhibitor for 2 h then with 20 ng/mL of victorin for 4 h. Coomassie blue–stained 12% SDS-PAGE of total protein extract. Only the region containing Rubisco is shown.

View larger version (28K): [in this window] [in a new window]   Figure 2. Characterization of Two Caspase-Like Proteolytic Activities. (A) Activity profile of fractions from a HIC column. The protease in activity A was assayed by incubating 50 µL of each fraction with 50 µL of 20 mM Mops, pH 7, with 20 µM substrate for 2 h. Activity B was assayed as described in Methods. RFU, relative fluorescence units. (B) Substrate profile of activity A. Activity A was assayed as described above for proteolytic activity with indicated substrates. RFU, relative fluorescence units. (C) Substrate profile of activity B. RFU, relative fluorescence units. (D) Inhibitor profile of activities A and B. Samples were pretreated with inhibitor (200 µM) for 2 h and then assayed for hydrolytic activity with Z-DEVD-AFC (activity A) or with Z-VAD-AFC (activity B). Expressed as percent inhibited of noninhibited control.

View larger version (37K): [in this window] [in a new window]   Figure 3. The Caspase-Like Protease Is an 84-kD Ser Protease. (A) Reversibility of inhibition of the protease in activity B. Three distinct inhibitors all containing the VAD recognition motif were incubated at a concentration of 200 µM with activity B for 2 h. Samples were washed twice in buffer then reconstituted to original volume. After initial incubation and after each wash, aliquots were removed and assayed for Z-VAD-AFC hydrolytic activity. Irreversible inhibition of the protease was achieved only with the CMK-based inhibitor. (B) Biotin blot of the protease in activity B. Each lane contains a 30-µL sample of activity B from the HIC column. Samples were preincubated 2 h with a nonbiotinylated inhibitor (lanes 2 and 3) then incubated 2 h with biotin-YVAD-CMK (lanes 1 to 3). The caspase-like protease shows specific binding to biotin-YVAD-CMK because binding is competitive with Z-VAD-CMK, an inhibitor, but not Z-DEVD-CMK, which does not inhibit activity. Only the portion of the blot containing the 84-kD protein is shown.

Extracellular Localization of the Saspase after Victorin Treatment
Infiltration of victorin into unpeeled leaves caused the release of the saspase into the ECF. The use of leaves with the epidermis removed provides a very sensitive and rapid assay of victorin activity (Figure 1). However, because this assay cannot be used for the collection of the ECF, a different assay was developed. In this new assay, victorin was directly infiltrated into whole leaf segments, thus allowing the ECF to be removed by centrifugation. However, this leaf infiltration assay is less sensitive. Infiltration of victorin at 1 µg/mL resulted in the first observation of Rubisco proteolysis and DNA laddering (discussed below) at 8 h after infiltration. Observation of levels of Rubisco proteolysis and DNA laddering comparable to that seen after 4-h incubation of peeled A. sativa leaf segments in 20 ng/mL of victorin were not seen until 15 h after infiltration (data not shown). Thus, the relative rates of Rubisco proteolysis and DNA laddering and, presumably, release of the saspases into the ECF are different in the two assays.

ECF was collected from victorin-infiltrated leaves at 0 to 6 h after infiltration with victorin, and then saspase activity was measured by Z-VAD-AFC hydrolysis (Figure 4A). Protease activity was first detected at 1 h and was maximal at 5 h. The same ECF samples were incubated with 200 µM biotin-YVAD-CMK for 2 h, separated by SDS-PAGE, and blotted for biotin visualization. The appearance of the 84-kD saspase band in the ECF (Figure 4B) coincided with hydrolytic activity. Four-centimeter leaf sections were used for extraction of the ECF, and typically, eight leaf sections were used per treatment/experiment. For the first 5 h (the time when maximum saspase activity was recovered), the quantity of ECF recovered in untreated leaves ranged from 6.5 to 8 µL/leaf segment and averaged 7.5 µL (n = 56), whereas toxin-treated leaf segments ranged from 6.5 to 10.75 µL/leaf segment and averaged 9 µL (n = 52). Slightly more ECF was consistently recovered from toxin-treated leaf segments at all time points likely because of the fact that toxin causes substantial leakage of electrolytes (e.g., Wheeler and Black, 1962; Sammadar and Scheffer, 1968), which would increase the water potential of treated cells.

View larger version (81K): [in this window] [in a new window]   Figure 4. Release of the Saspase into the ECF after Victorin Treatment. (A) and (B) Leaves were infiltrated with 1 µg/mL of victorin (vic) or water (no victorin control), and the ECF was removed at the indicated time. (A) Z-VAD-AFC hydrolytic activity in the ECF. Activity expressed as relative fluorescence units. (B) Biotin blot of the ECF. Twenty microliters of each sample was incubated with 200 µM biotin-YVAD-CMK for 2 h before electrophoresis and blotting. (C) and (D) Leaves were preinfiltrated with 400 µM biotin-YVAD-CMK, preincubated 2 h, then infiltrated with 1 µg/mL of victorin mixed with 100 µM biotin-YVAD-CMK. (C) Biotin blot of the ECF. (D) Biotin blot of total protein extract. Note that the saspase is uniformly labeled in all samples including the water only/no victorin control (last lane).

Heat Shock–Induced PCD Is Similar to Victorin-Induced PCD
Heat shock has been successfully used to induce PCD in Cucumis sativus (cucumber) (Balk et al., 1999) and N. tabacum (Tian et al., 2000). We wanted to ascertain weather or not heat shock could induce characteristics similar to victorin-induced PCD in A. sativa. Victorin-sensitive and -insensitive leaves were infiltrated with water (Figures 5A to 5D, lanes 1 and 4), 1000 ng/mL of victorin (Figures 5A to 5D, lanes 2 and 5), or heat shock (45°C for 1 h) (Figures 5A to 5D, lanes 3 and 6). The ECF was collected 4 h after start of the treatment, and protein and DNA samples were collected after 15 h. Heat shock induces the typical characteristics of victorin-induced PCD in victorin-sensitive and -insensitive A. sativa, including Rubisco proteolysis (Figure 5A, lanes 3 and 6), DNA laddering (Figure 5B, lanes 3 and 6), and release of the 84-kD saspase into the ECF (Figures 5C and 5D, lanes 3 and 6). The water controls for both sensitive (Figure 5, lane 1) and insensitive (Figure 5, lane 4) leaves did not display any of the characteristics associated with PCD, nor did victorin-treated insensitive leaves (Figure 5, lane 5). Heat shock–induced Rubisco proteolysis, like that induced by victorin, was prevented by the caspase inhibitors Ac-VAD-CMK and Ac-DEVD-CMK, the granzyme B inhibitor Ac-AAD-CMK, and the general Cys protease inhibitors E-64 and leupeptin (Figure 6).

View larger version (91K): [in this window] [in a new window]   Figure 5. Heat Shock–Induced PCD in Victorin-Sensitive and -Insensitive A. sativa. Victorin-sensitive and -insensitive A. sativa leaves were infiltrated with water (lanes 1, 3, 4, and 6) or 1 µg/mL of victorin (lanes 2 and 5). Water-infiltrated leaves in lanes 3 and 6 were heat shocked for 60 min at 45°C and then incubated at 25°C for indicated times below. (A) Coomassie blue–stained SDS-polyacrylamide gel of total protein extracted 15 h after treatment. Rubisco cleavage is seen in sensitive leaves treated with victorin and sensitive and insensitive leaves that were heat shocked. Only the region containing Rubisco is shown. (B) 1.5% agarose gel of DNA extracted 15 h after treatment. (C) Biotin blot of ECF collected 4 h after treatment. Samples were incubated with 200 µM biotin-YVAD-CMK for 2 h before electrophoresis. (D) Z-VAD-AFC hydrolytic activity in the ECF. Activity expressed as relative fluorescence units.

View larger version (44K): [in this window] [in a new window]   Figure 6. Inhibition of Heat Shock–Induced Rubisco Proteolysis. A. sativa leaves were preincubated with 400 µM of indicated inhibitor for 2 h and then heat shocked for 60 min at 45°C (sample in lane 1 did not undergo heat shock). Proteins were isolated 15 h after heat shock. Only the region containing Rubisco is shown.

View larger version (15K): [in this window] [in a new window]   Figure 7. Anion Exchange Chromatography Separated Two Distinct Peaks of Saspase Activity. Silver stained gel of active fractions (20 µL loaded/lane) eluted off the anion exchange column. Z-VAD-AFC hydrolytic activity of each sample is indicated below the gel, expressed as relative fluorescence units. Fractions 42 to 52 (SAS-1) and 54 to 64 (SAS-2) were pooled for individual purification of SAS-1 and SAS-2 by size exclusion chromatography.

View larger version (44K): [in this window] [in a new window]   Figure 8. Electrophoresis of SAS-1 and SAS-2 after Purification by Size Exclusion Chromatography. Silver stained gel of purified SAS-1 and SAS-2; 15 µL of active fraction was loaded per lane.

View larger version (25K): [in this window] [in a new window]   Figure 9. Molecular Mass of both SAS-1 and SAS-2 Is 74.5 kD as Indicated by Mass Spectrometry. Spectrograph of purified SAS-1 (top spectrograph) and SAS-2 (bottom spectrograph) were individually analyzed by MOLDI-TOF mass spectrometry. First peak is the doubly charged ion, and the second peak is the singly charged ion.

View larger version (35K): [in this window] [in a new window]   Figure 10. Peptide Maps of SAS-1 and SAS-2. Partial tryptic digest of SAS-1 and SAS-2 analyzed by MALDI-TOF mass spectrometry. Major peptide peaks with molecular mass differences between SAS-1 and SAS-2 are indicated by an arrow. m/z, mass-to-charge ratio.

View larger version (24K): [in this window] [in a new window]   Figure 11. Effects of pH on Hydrolytic Activity of SAS-1 and SAS-2. Partially purified SAS-1 and SAS-2 (post-anion exchange chromatography) were assayed for hydrolytic activity at different pHs with the substrate Z-VAD-AFC. RFU, relative fluorescence units.

A protein gel blot of purified SAS-1 and SAS-2 probed with SAS-2 antiserum is shown in Figure 12. After addition of SDS-loading buffer to each sample (200 ng purified enzyme), the samples were either boiled 5 min (Figure 12, lanes 2 and 4) or not boiled (Figure 12, lanes 3 and 5) before loading on the gel. Nonboiled samples of both enzymes underwent autoproteolysis and accumulated lower molecular mass bands. The SAS-2 sample contained four distinct proteolytic bands—a, b, c, and d— whereas the SAS-1 sample only contained three bands—a, c, and d. Bands c and d are more visible when overexposed (data not shown).

View larger version (83K): [in this window] [in a new window]   Figure 12. Autoproteolysis of SAS-1 and SAS-2. Protein gel blot of purified SAS-1 and SAS-2 (200 ng each lane) boiled and not boiled before electrophoresis. Nonboiled enzymes underwent autoproteolysis with the appearance of lower molecular weight bands (a, b, c, and d).

	DISCUSSION

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The saspase proteases were originally implicated based on the effects of inhibitors that prevented victorin and heat shock–induced Rubisco proteolysis, a characteristic of PCD in A. sativa. Other inhibitors that prevent this response (Ac-DEVD-CMK, E-64, and leupeptin) do not inhibit the activity of either saspase, indicating the involvement of additional proteases. The identification of another protease with specificity for DEVD derived substrates and inhibitors, but not E-64 or leupeptin, suggested that at least two other proteases function within a protease cascade, with either or both of the saspase enzymes, leading to the proteolysis of Rubisco.

This evidence suggests that the saspases are components of a PCD-induced protease cascade that ultimately leads to the activation of a protease that is targeted to the chloroplast to cleave Rubisco. Although cleavage of Rubisco is inhibited by Ac-VAD-CMK and Z-AAD-CMK, both of which are recognized by the saspases, it is not likely that either saspase directly cleaves Rubisco in vivo. Four lines of evidence suggest that an alternative protease cleaves Rubisco: (1) E-64, leupeptin, and Ac-DEVD-CMK prevent Rubisco cleavage but do not affect SAS-1 or SAS-2 activity or their release into the ECF; (2) the cleavage site in Rubisco (Navarre and Wolpert, 1999) does not conform to the substrate specificity for the saspases; (3) purified SAS-1 and SAS-2 do not cleave Rubisco in vitro (data not shown); and (4) the saspases appear to function extracellularly.

A Gene Family
The caspase-like activity was purified into two distinct proteases, SAS-1 and SAS-2. The two proteases are nearly indistinguishable, in that their substrate and inhibitor profiles are identical, they have the same pH and salt requirements, they are predicted to have equivalent molecular masses determined by either electrophoretic migration or mass spectrometry, they possess similar peptide maps, they have an identical NH₂-terminal sequence (except the first amino acid of SAS-1), and they both bind heparin and are autoproteolytic. Although they recognize the same substrates and inhibitors, SAS-2 does have a higher hydrolysis rate for several caspase substrates and is slightly more sensitive to some caspase inhibitors. Furthermore, the difference in mass between two of the several major peaks in their peptide maps suggest a differential posttranslational modification (such as a methyl group) or a single amino acid substitution. The comparisons between SAS-1 and SAS-2 indicate that they are different yet highly similar proteases, most likely from two separate genes. But the possibility exists that they are differentiated by unique posttranslational modifications to the same gene product.

Gene families of subtilisin-like Ser proteases have been identified in several plant species. Lycopersicon esculentum (tomato) has had 10 genes for subtilisin-like Ser proteases thus far cloned and characterized (Vera and Conejero, 1988; Tornero et al., 1997; Jordá et al., 1999, 2000; Meichtry et al., 1999; Janzik et al., 2000; Riggs et al., 2001). Whereas Arabidopsis (Arabidopsis thaliana) has had only four genes characterized (Neuteboom et al., 1999; Berger and Altmann, 2000; Tanaka et al., 2001; Hamilton et al., 2003), at least 10 more genes have been annotated as being subtilisin-like Ser proteases. Rice has had only one subtilisin-like Ser protease gene characterized (Yamagata et al., 2000), but seven have been annotated from the genome, two of which have high sequence similarity to SAS-1 and SAS-2. Thus, it is likely that two or more subtilisin-like Ser proteases are present in A. sativa, and quite possibly a more divergent protease with DEVD specificity functions along with SAS-1 and SAS-2 within the protease cascade leading to Rubisco proteolysis. Because of the structural and immunological similarities between the saspases and the difficulty in purifying large amounts from the ECF, we have not been able to distinguish which saspase, if not both, is released into the ECF. However, initial studies have indicated that after induction of PCD and removal of the ECF from victorin-treated leaves, total saspase protein remaining in the whole leaf is dramatically reduced, suggesting that both SAS-1 and SAS-2 are released into the ECF. Further experiments need to be performed to confirm these observations.

Processive Protease and Family Relations
The substrate specificity of SAS-1 and SAS-2 is distinct from all other known subtilisin-like Ser proteases. Among the substrates tested, only certain caspase substrates and the granzyme B substrate were hydrolyzed and to varying degrees, indicating that although an aspartate residue is required in the P1 position, specificity extends beyond this one residue. No other identified protease from plants has been reported to be specific for caspase substrates or to cleave exclusively after an aspartate residue. Plant subtilisin-like Ser proteases differ widely in their substrate specificity. Cucumisin (Yamagata et al., 1994), macuralisin (Rudenskaya et al., 1995), taraxalism (Rudenskaya et al., 1998), hordolisin (Terp et al., 2000), and Ser endopeptidase-1 (Fontanini and Jones, 2002) have been reported as having a broad substrate range and are thought to be degradative proteases, whereas Auxin Induced in Root3 (Neuteboom et al., 1999), Abnormal Leaf Shape1 (Tanaka et al., 2001), Stomatal Density and Distribution1 (SDD1) (Berger and Altmann, 2000), Tomato subtilase1 (LeSBT1) (Janzik et al., 2000), 50-kD Systemin Binding Protein (Schaller and Ryan, 1994), and Ara12 (Hamilton et al., 2003) are described as possessing greater substrate specificity and are thought to function as processive proteases. Until more is known about the biochemical and physiological properties of plant subtilisin-like Ser proteases, the biological significance of the difference between a degradative or processive function may prove challenging to determine. Because SAS-1 and SAS-2 do not generally degrade proteins or non-caspase-specific substrates (e.g., casein), require an aspartate residue in the P1 position of the substrate, and are likely involved in a signaling cascade, it appears evident that they function as processive proteases.

Plant subtilisin-like Ser proteases are members of the Pyrolysin family of subtilisin-like Ser proteases (Siezen and Leunissen, 1997) and are predicted, based on DNA sequence of the cloned proteases, to be expressed as pre-pro-protein precursors. Another family of subtilisin-like Ser proteases, the Kexin family, also is expressed as pre-pro-protein precursors. Kex2 from Saccharomyces cerevisiae, the prototype member of this family, has been well-characterized (Mizuno et al., 1988). In Kex2, the pre-domain is a signal peptide that directs the enzyme into the secretory system of the endoplasmic reticulum (ER) and is cleaved during import (Van de Ven et al., 1991). The pro-domain is autocatalytically cleaved while in the ER, which results in the formation of the active, mature form of the protease (Gluschankof and Fuller, 1994). The pre- and pro-domains of plant subtilisin-like Ser proteases are thought to each function in a similar manner to those of the Kex2 enzyme because several plant subtilisin-like Ser proteases have been localized extracellularly (Vera and Conejero, 1988; Yamagata et al., 1994; Taylor et al., 1997), and the NH₂ terminus of mature, active enzymes corresponds to the predicted mature NH₂ terminus absent the pre- and pro-domains (Terp et al., 2000; Popovi et al., 2002).

The saspases from A. sativa are extracellularly localized during the PCD response, and their NH₂-terminal sequence is similar to the mature, processed NH₂ terminus of most plant subtilisin-like Ser proteases, characteristics which correspond in function to the pre-pro-protein model of subtilisin-like Ser proteases. Furthermore, because pro-domains are autocatalytically processed and the pro-domain cleavage site indicates, to some degree, the cleavage specificity for the enzyme, it might be expected that in the saspases, the amino acid preceeding the cleavage site would be an aspartate. Interestingly, the protein with the most similarity to the sequenced saspase peptides, the putative protease annotated from the rice genome, does have an aspartate residue in the P1 position of the predicted pro-domain cleavage site, suggesting that the putative rice protease may possess similar cleavage specificity to the saspases from A. sativa.

Heparin and Autoproteolysis
In the early stages of characterization and before purification and partial sequence analysis, it was clear that the saspases were high molecular mass, extracellular Ser proteases apparently involved in a protease cascade. That depiction strongly resembled a Drosophila melanogaster protease called gastrulation defective (GD), which is a 72-kD extracellular Ser protease involved in a protease cascade leading to dorsal-ventral signaling (Konrad et al., 1998; Han et al., 2000; DeLotto, 2001; Dissing et al., 2001; LeMosy et al., 2001). After purification and partial sequencing, it became clear that GD and SAS-1 and SAS-2 are structurally unrelated, although they may share some similar biological characteristics. Two other characteristics were later identified as being in common between the saspases and GD, namely heparin binding and autoproteolysis.

Heparin, a sulfated glycosaminoglycan, is involved in several biological activities in animals, including binding antithrombin III. Such binding accelerates the rate of antithrombin III–mediated inhibition of Ser proteases involved in the blood coagulation cascade (reviewed in Petitou et al., 1988; Capila and Linhardt, 2002). Heparan sulfate, a heparin derivative, is ubiquitously distributed on the surfaces of animal cells and in the extracellular matrix where it mediates various physiological and pathological processes (reviewed in Capila and Linhardt, 2002). Though the biological significance of GD binding heparin is not known, Dissing et al. (2001) suggest that the pipe gene product, a heparan sulfate 2-O-sulfotransferase, might regulate activity of GD through binding of a yet unknown sulfated proteoglycan. Possibly, the binding of SAS-1 and SAS-2 to a heparin-like molecule in plants may have a regulatory role, similar to Ser proteases involved in the blood coagulation cascade or the putative role of heparin-like molecules in regulating GD.

Autoproteolysis by GD, SAS-1, and SAS-2 is an intriguing yet confounding characteristic. GD has been shown to undergo autoproteolysis only in the presence of the Snake protease and into two distinct bands of 44 and 46 kD (Dissing et al., 2001). Neither proteolytic activity nor a biological function has been identified for the products of proteolysis, though it was suggested that the unprocessed form of the protease was the zymogen and the proteolytic products might function as the active component of the cascade (Dissing et al., 2001). SAS-1 and SAS-2 autoproteolysis has only been observed in gel where it is likely dependent on concentration and the state of denaturation. Both proteases are stable at room temperature in biological buffers for several days (data not shown). Autoproteolysis by SAS-1 and SAS-2 is not unique among plant subtilisin-like Ser proteases because two other proteases have been shown to undergo a form of self-catalyzed proteolysis. LeSBT1, a 73-kD protease from L. esculentum, is autoprocessed into a 68-kD protease at pH 5.0 and 6, whereas it completely autodegrades at pH 4.0 (Janzik et al., 2000), and hordolisin from Hordeum vulgare (barley) was autoprocessed from 74 to 62 kD during storage at –20°C (Terp et al., 2000).

Regulation and Localization
A unique characteristic of the saspases is that they appear to be constitutively present in A. sativa cells, and release of active saspase into the ECF but not de novo synthesis of the enzyme is correlated with the induction of PCD. This is in contrast with other proteases associated with plant PCD (Delorme et al., 2000; Eason et al., 2002; Wan et al., 2002) or the HR (Vera and Conejero, 1988; Krüger et al., 2002), which are typically regulated at the level of expression. P-69B and P-69C, members of a family of L. esculentum subtilisin-like Ser proteases, are two pathogenesis-related (PR) proteins that show increased expression levels during HR (Jordá et al., 1999). The pathogen-induced P-69 enzymes also are secreted into the extracellular space (Tornero et al., 1996a). However, unlike saspases, these proteins are not present in the cell before induction of the HR (Tornero et al., 1997).

Release of the saspase(s) into the ECF occurs before the onset of other markers of PCD, indicating that saspase release could be causal to but is not a consequence of PCD. The saspase(s) starts to appear in the ECF at 1 h after victorin treatment, at least 7 h before other signs of PCD, such as Rubisco proteolysis and DNA laddering, start to occur. This indicates that saspase appearance in the ECF is not because of cell lysis but is a regulated event. This conclusion is further supported by time-course studies (Curtis and Wolpert, 2004) that have established that the plasma membrane and tonoplast of victorin-treated A. sativa cells remain structurally intact after the point at which Rubisco proteolysis, DNA laddering, and even cell collapse occur.

The question arises as to where the saspases are localized before release into the ECF. They could be retained in the ER where they await processing and secretion. The Kexin family of subtilisin-like Ser proteases is known to autocatalytically remove their pro-domain in the ER before continuing in the secretory pathway (Gluschankof and Fuller, 1994; Hill et al., 1995). A mutation in the Kex2 protease that inhibits removal of the pro-domain stimulates accumulation of proKex2 in the ER (Gluschankof and Fuller, 1994). The pro-domain is thought to contain an ER retention sequence. If the saspases are stored in the ER while awaiting processing and secretion, then an inactive pro-saspase form should be identifiable. However, analysis of proteins labeled in vivo with biotin-YVAD-CMK and extracted under denaturing conditions (phenol extracted) from victorin-treated and untreated tissue revealed similar concentrations of only the mature processed proteases (Figure 4D). Soluble protein extracts from victorin-treated and untreated tissue also contained comparable amounts of saspase activity (data not shown). These data suggest that the saspases are likely sequestered as active proteases.

Another possible saspase-retention area is the cell exterior, where it could associate with the extracellular matrix or a membrane protein. Heparin or heparin-like molecules are typically extracellular, either membrane bound or soluble (Petitou et al., 1988; Stringer et al., 2002), and because the saspases are ultimately extracellular, it is possible that a heparin-like molecule plays a physiologically significant role, either before release of the saspases or sometime during their biological function. Whatever their storage location, the fact that the saspases appear to be constitutively present in an active form suggests that they can be rapidly released and that a quick response is important for PCD.

PCD and Saspase Function
Though no physiological substrate(s) of the saspases has been identified, data suggest that they activate, either directly or indirectly, other proteases. Because the saspases recognize two of the inhibitors (and their complementary substrates) that prevent Rubisco proteolysis and because victorin treatment affects localization of the saspases, it appears likely that they are involved in the signal cascade leading to Rubisco proteolysis. The saspases also likely function early in the signal cascade because their release into the ECF is not prevented by any of the protease inhibitors that prevent Rubisco proteolysis, indicating that these inhibitors function either downstream or after release of the saspases. Furthermore, because multiple proteases appear to be involved in this cascade, one or both of the saspases are likely involved in activation of these other proteases. Are these proteases the direct substrate, or do the saspases interact with an extracellular protein, possibly membrane associated, which facilitates the activation of intracellular proteases? Several reports have implicated the interaction of extracellular proteases with Leu-rich repeat (LRR) proteins (LRP) during the HR and developmental processes. The pathogen-induced, subtilisin-like Ser protease P-69 from L. esculentum is able to proteolytically process an extracellular LRP (Tornero et al., 1996b). The LRP, predicted to be soluble in the extracellular matrix, also has increased expression levels during pathogen attack, but the function of the processed or unprocessed form is yet to be determined. Krüger et al. (2002) describe an extracellular Cys protease, Required for Cladosporium fulvum Resistance3 (Rcr-3), that is required for Cf-2–dependent HR. Cf-2 is a resistance gene in L. esculentum that encodes a transmembrane LRP with an extracellular LRR domain. Not only is Rcr-3 required for Cf-2–dependent HR, it also is necessary for suppression of Cf-2–dependent autonecrosis. Two other extracellular proteases, SDD1, a subtilisin-like Ser protease (Berger and Altmann, 2000), and Brassinosteroid-Insensitive1, a Ser carboxypeptidase (Li et al., 2001), have been described as necessary for stomata development and brassinosteroid signaling, respectively. Both proteases function upstream of LRR receptor–like proteins that are required for continuation of their respective signaling pathways (Li et al., 2001; Serna and Fenoll, 2002). Whether or not the saspases interact with another protease, an LRR receptor–like protein, or a ligand for a membrane receptor is currently not known. Identification of the physiological substrate for the saspases will be an important step in furthering the understanding of the processes of PCD in A. sativa.

Recently, Woltering et al. (2002) asked the question, "Do plant caspases exist?" This research addresses that question by providing insight into enzymes that possess caspase-like activity, though not structurally related to caspases. Numerous studies have indicated the involvement of caspase-like proteases in the regulation of plant PCD. Caspase-specific inhibitors prevent PCD in response to bacteria (del Pozo and Lam, 1998; Richael et al., 2001), fungal elicitors (Elbaz et al., 2002), and various forms of stress (Sun et al., 1999; Tian et al., 2000; Elbaz et al., 2002; Mlejnek and Procházka, 2002; Woltering et al., 2002). Although these reports indicate that there are proteases in plants that are inhibited by caspase-specific inhibitors and implicated in PCD, no enzyme had yet been identified that pertains to these observations. Saspases, plant enzymes possessing caspase-like specificity and associated with the PCD response, are subtilisin-like Ser proteases, and partial amino acid sequence analysis revealed homology to all plant subtilisin-like Ser proteases. The unusual requirement for aspartate residues in the P1 position of the substrate, the specificity of the enzyme among aspartate-containing substrates, the lack of general proteolytic activity, and the possible involvement in a signal cascade suggest that the saspases function as processive proteases. Release of one or both of the saspases into the ECF occurs during victorin and heat shock–induced PCD, two treatments that share other characteristics of PCD, namely Rubisco proteolysis and DNA laddering. The saspases appear to be involved in a PCD-induced signaling cascade based on inhibitor studies and relocalization during the PCD response, but unfortunately no direct evidence of their involvement has been identified. Initial experiments to induce PCD in A. sativa leaves by infiltration of purified saspase have provided interesting but inconsistent results. The ability to purify large amounts of enzyme (needed for these types of experiments) without first cloning the gene has been the largest impediment. The identification of the putative protease from rice, which possesses the highest amino acid sequence similarity, should facilitate cloning of the saspase genes from A. sativa. It also will be of interest to see if this putative protease from rice has caspase-like activity and if it is involved in rice PCD responses.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Experimental Materials
A. sativa seedlings of the line X469 (victorin sensitive) and X424 (victorin insensitive) were grown in a growth chamber for 6 to 7 d under a 16-h photoperiod at 24°C. All protease substrates and caspase inhibitors were purchased from Calbiochem (La Jolla, CA). The remaining chemicals and protease inhibitors were purchased from Sigma (St. Louis, MO).

Treatment of A. sativa Tissue
A. sativa leaves were treated with victorin by one of three methods. A. sativa leaves, with their epidermis removed, were floated on 20 mM 3-(N-morpholino)-propanesulfonic acid (Mops), pH 6.5, supplemented with 20 ng/mL of victorin. For leaf infiltration assays, intact A. sativa leaves were infiltrated with 1 µg/mL of victorin in water and then cut into 4-cm leaf segments. The third method, which was used to purify large quantities of protein, treated leaf slices