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Keywords:

  • AEBSF and DFP;
  • aspartic protease;
  • cysteine protease;
  • endoprotease activity;
  • inhibitor specificity;
  • Iris x hollandica;
  • metalloprotease;
  • senescence

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    Visible senescence of the flag tepals in Iris x hollandica (cv. Blue Magic) was preceded by a large increase in endoprotease activity. Just before visible senescence about half of total endoprotease activity was apparently due to cysteine proteases, somewhat less than half to serine proteases, with a minor role of metalloproteases.
  • • 
    Treatment of isolated tepals with the purported serine protease inhibitors AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride] or DFP (diisopropyl-fluorophosphate) prevented the increase in endoprotease activity and considerably delayed or prevented the normal senescence symptoms.
  • • 
    The specific cysteine protease-specific E-64d reduced maximum endoprotease activity by 30%, but had no effect on the time to visible senescence. Zinc chloride and aprotinin reduced maximum endoprotease activity by c. 50 and 40%, respectively, and slightly delayed visible senescence. A proteasome inhibitor (Z-leu-leu-Nva-H) slightly delayed tepal senescence, which indicates that protein degradation in the proteasome may play a role in induction of the visible senescence symptoms.
  • • 
    It is concluded that visible senescence is preceded by large-scale protein degradation, which is apparently mainly due to cysteine- and serine protease activity, and that two (unspecific) inhibitors of serine proteases considerably delay the senescence symptoms.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The visible symptoms of plant senescence are preceded by large-scale degradation of macromolecules. A large increase in net breakdown was observed, for example, in petal RNA (Baumgartner et al., 1975), DNA (Winkenbach, 1970a,b), lipids (Bieleski & Reid, 1992) and proteins (Sultan & Farooq, 1996). In nonsenescent cells a considerable part of protein degradation occurs in proteasomes, located both in the cytosol and nucleus. Some protein degradation in these cells may also be carried out by (nonproteasome) proteases, thought to be located mainly in vacuoles (Genschik et al., 1998; Vierstra, 2003). During senescence the proteasome pathway seems to become up-regulated. Proteins are targeted for proteasome degradation after they become complexed with ubiquitin, and prior to visible senescence in daylily tepals an increase in protein ubiquitination was observed (Courtney et al., 1994).

Proteasome-independent protease activity also increases prior to visible senescence (Sultan & Farooq, 1996; Stephenson & Rubinstein, 1998). Nonproteasome proteases are often divided into exo- and endoproteases. Two exoproteases were present in daylily tepals; one being up-regulated prior to visible senescence (Mahagamasekera & Leung, 2001). The endoprotease group includes cysteine-, serine-, aspartic-, and metalloproteases, termed after the amino acid residues or metals required for the cleavage reactions (Vierstra, 2003). In a number of species, one or more cysteine protease genes were reportedly up-regulated prior to visible petal senescence (Jones et al., 1995; Guerrero et al., 1998; Eason et al., 2002; Wagstaff et al., 2002; van Doorn et al., 2003). Aspartic proteases have been observed in Arabidopsis petals (Chen et al., 2002) and were suggested to be involved in leaf senescence (Bhalerao et al., 2003). Serine proteases and metalloproteases have apparently not been described in petals, but seem to be involved in leaf senescence (Roulin & Feller, 1998; Roberts et al., 2003).

Apart from their role in protein degradation, proteases may be part of a signal transduction pathway. Numerous cysteine proteases (Stennicke & Salvesen, 1998) – called caspases – and some serine proteases (Smyth et al., 1996), are involved in signal transduction that leads to programmed cell death in animal cells. Caspases are defined by their active site motif. Caspases have not been found in the Arabidopsis genome but related families, called metacaspases and paracaspases have been identified, which may have a similar function (Lam, 2004). Programmed death of tracheary elements was delayed by a caspase inhibitor (Fukuda, 1997). Some reports suggest that meta-caspases, homologues of animal caspases, may be involved in programmed cell death in plants (Bozhkov et al., 2004). A cysteine protease seems also involved in the complex signalling pathway that regulates the degree of autophagy in cells (Mizushima et al., 2003). Programmed cell death in Iris tepals involves the increase in vacuole size, disappearance of cytoplasm and organelles, and vacuolar rupture, all typical for autophagy (van Doorn et al., 2003).

The aim of the present work is to test two hypotheses: first that all four groups of endoproteases are involved in senescence-associated bulk protein degradation; and second that protease inhibitors delay the visible symptoms of senescence. This is apparently the first report to show that serine proteases seem responsible for a large part of bulk protein degradation prior to visible senescence and that serine-protease inhibitors can markedly delay the appearance of senescence symptoms.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant material

Iris×hollandica cv. Blue Magic flowers were obtained from a commercial grower. Flowers were harvested early in the morning, directly placed in water and transported in water to the laboratory, where they were stored at 2°C until use (the same day or the next morning). Stems were recut to 40 cm length and individually placed in vases with distilled water, at 20°C and 60%rh, with 12 h white light from fluorescent tubes, at 15 µmol m−2 s−1, and 12 h darkness. Iris flowers contain two whorls of three tepals each, of which the outer one (containing the flag tepals) was used in the present study. Their senescence progresses downward from the distal edge to the tepal base. Only the distal most 3 mm of tissue was used for measurements.

Other tests were carried out with flag tepals that were isolated on day 0 and placed in aqueous solutions of various inhibitors, at the same environmental conditions. Tissue sampling occurred by cutting 3 mm from the distal edges of the flag tepals, starting and ending at the two places where the tepal diameter is widest.

Protein levels and endoprotease assays

Flowers were held in water and the tepal edges were harvested at intervals. The material was ground to a fine powder in liquid nitrogen. The material was freeze-dried and analysed for protein levels using the assay according to Bradford (1976) and bovine serum albumin as a standard.

We used azoalbumin and resorufin-labelled casein as substrates for in vitro estimation of total endoprotease activity. In the albumin assay, one gram of freshly cut material from the distal tepal edges was ground to a fine powder in liquid nitrogen, and placed in 5 ml extraction medium (25 mm Hepes at pH 8.2, 3.5% NaCl and 2 mm DTT). The solution was desalted over a PD-10 column (Pharmacia) with 3.5% NaCl, and 2 mm DDT. CaCl2 20 mm was added to stabilise the proteins. The reaction medium consisted of 100 µl of the desalted extract, 50 µl of a 400 mm citrate/phosphate buffer at pH 5.5 (unless stated otherwise) and 50 µl 4% azoalbumin (final concentration 1%). Incubation occurred for 60 min at 25°C. The reaction was stopped by adding 600 µl trichloracetic acid 10%. Precipitation of the TCA-insoluble peptides occurred on ice for 20 min. After centrifugation, the absorption of the supernatant was measured at 440 nm. Protease activity was expressed in absorption units per mg protein. The data were corrected for absorption in controls without substrate.

The use of resorufin-labelled casein (Boehringer) in assaying endoprotease activity has been described previously (Fernandez et al., 1999). We used the same extraction and desalting steps as in the azoalbumin assay. The incubation medium consisted of 70 µl of desalted extract, 35 µl incubation buffer (400 mm citrate/phosphate buffer at pH 5.2, unless stated otherwise) and 35 µl of substrate (final concentration 0.1%). The reaction occurred at 37°C for 30 min, and was halted by adding 336 µl trichloroacetic acid 5%. Precipitation occurred at 37°C for 10 min. After centrifugation, 400 µl of the supernatant was mixed with 600 µl of 0.5 m Tris-HCl at pH 8.8. Absorption was measured at 574 nm. Protease activity is expressed in absorption units per mg protein. The data were corrected for absorption in controls without substrate.

Protease inhibitors and other chemicals

All inhibitors were obtained from Sigma (St Louis, MO, USA), unless otherwise noted. These inhibitors were applied in vitro, using the resorufin-labeled casein assay. Their effect on the time to the visible senescence symptoms was also tested. Isolated tepals were placed in aqueous solutions of these compounds, at a range of concentrations.

Inhibitors of metalloproteases were 1,10-phenantrolin, EDTA, and EGTA; inhibitors of cysteine and serine proteases were leupeptin, pepstatin, TPCK (N-alpha-tosyl-phenyl-alanine chloromethyl ketone), and TLCK (N-alpha-tosyl-lysine chloromethyl ketone). Inhibitors of cysteine proteases were diamide, iodoacetamide, N-ethylmaleimide, and zinc chloride. E-64 is a specific inhibitor of cysteine protease. We also used E-64d in testing with tepals, as it is able to pass membranes. Serine protease inhibitors tested were aprotinin, PMSF (phenylmethyl-sulfonyl fluoride), AEBSF [4-(2-aminoethyl)-benzene-sulfonyl fluoride], and DFP (diisopropylfluorophosphate). The effect of an analogue of AEBSF, called pABSF [4-(amidino)-benzenesulfonyl fluoride hydrochloride] was compared with that of AEBSF.

We also placed isolated flag tepals in aqueous solutions of acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-cmk), an inhibitor of ICE-type of caspases in animal cells, and acetyl-Asp-Glu-Val-Asp-chloromethyl-ketone (Ac-DEVD-cmk), an inhibitor of CPP32, which belongs to another caspase family (Enari et al., 1996).

Statistics

All experiments involved 10 replications (10 tepals or 10 flowers, individually placed in aqueous solutions). When using isolated tepals, all 10 replications originated from different flowers. All experiments were repeated at least once. Data were compared by analysis of variance, and F-test at P > 0.05, using the Genstat V program (Rothamsted, UK).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Senescence symptoms and protein levels

Blue Magic Iris flowers were harvested (day 0) when the tips of the blue tepals were just visible. When placed in water at room temperature the flower opened mainly on day 0–1. Opening was complete by the end of day 2. Visible senescence symptoms in the flag tepals were found on day 4. These symptoms were: discolouration of the distal edges, followed by slight inward rolling of the edges (stage 1), further inrolling of the whole flag region (stages 2–4), full discolouration to white (stage 5). The symptoms were also observed in intact plants, and in isolated tepals placed in water. Tepals that remained attached to cut stems showed these symptoms at about the same time as tepals isolated on day 0. Only in a few repeat experiments the isolated tepals showed visible senescence c. 0.5 d earlier than attached ones. We also observed that the effects of exogenous chemicals on isolated tepals were similar to those on tepals attached to cut flowers that stood in aqueous solution.

Cell death in Iris tepals starts from the distal edge and proceeds to the tepal base. To increase homogeneity of the samples we used only the distal most 3 mm of the flag tepals, in all measurements.

Visible senescence symptoms on day 4 were preceded by a decrease in protein level, both in tepals that were attached to cut flowers placed in water (Celikel & van Doorn, 1995) and in isolated tepals placed in water (Fig. 1).

image

Figure 1. Protein levels in the distal margin of flag tepals from Iris × hollandica, isolated on day 0 and placed in water (control) or in 5 mm AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride]. Data are means of four replications ± SD.

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Endoprotease activity

Using 0.1% resorufin-labelled casein as a substrate endoprotease activity showed a rapid increase from day 2. Activity was high by day 4, the time of the first visible senescence symptoms. The activity had further increased by day 5, when the tepal margins had extensively rolled in (Fig. 2). The pH dependence of the reaction was determined using extracts of tepals on day 4. From pH 5 upward, activity decreased until it was lowest at pH 8. At a pH lower than 5 the dependence could not be tested, due to precipitation. Similar results on endoprotease activity and pH dependence were obtained with azoalbumin as a substrate, which confirmed the results with resorufin-labelled casein. The latter was used in further tests, as the standard deviation was lower than in experiments with azoalbumin (results not shown).

image

Figure 2. Effect of 5 mm AEBSF on total endoprotease activity in the distal margin of flag tepals from Iris × hollandica, which were isolated on day 0 (flower about to open). AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride] was applied on day 0, 1, 2, and 3. Data are means of four replications ± SD.

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In vitro effects of protease inhibitors

The effect of protease inhibitors on total endoprotease activity was tested in vitro, using extracts from tepals that had been isolated on day 0 and held in water for 4 d. Resorufin-labelled casein was used as a substrate, at pH 5.5. Pepstatin, an inhibitor of aspartic proteases, had no effect at 0.1–5.0 mm (Table 1). Inhibitors of metalloproteases such as 1,10-phenanthroline had a relatively small effect on total activity (Table 1). Compounds that are known to inhibit both serine and cysteine proteases, such as TLCK and TPCK, decreased total activity by 44–60%. Chemicals that reportedly are serine protease inhibitors decreased total endoprotease activity by 35–45% (Table 1). Some inhibitors of cysteine proteases decreased total activity by 50–66%. Some of these chemicals may not be very specific, and this may result in overestimation of cysteine protease activity. Howerer, the largest reduction of activity was obtained with E-64, purportedly a specific cysteine protease inhibitor (Table 1).

Table 1.  Effect of purported protease inhibitors on endoprotease activity and visible senescence
ChemicalIn-vitro inhibition (day 4,% of control)*Inhibition after application to isolated tepals (day 4,% of control)*Delay of senescence (h)
  • *

    Data in column 1 and 2 are percentage inhibition compared to controls without inhibitor treatment (means ± SD, n = 3).

  • First column: in vitro endoprotease activity after addition of various protease inhibitors to extracts of Iris × hollandica tepals, prepared on day 4 after the onset of opening. Second column: tepal endoprotease activity, after inclusion of inhibitors in the aqueous solution in which Iris petals were stood. Inhibitors were included in the water on day 0, extracts were made on day 4. Third column: delay of visible senescence in isolated tepals, following inclusion of inhibitors in the aqueous solution on day 0. For each inhibitor a concentration range has been tested. The concentration which had the largest effect is here given. nd, Not determined.

Metalloprotease inhibitors
 1,10-Phenanthroline (10 mm)25 ± 3  0 0
 EDTA (10 mm)19 ± 4  0 0
 EGTA (10 mm)17 ± 3  0 0
Aspartic protease inhibitor
 Pepstatin A (5 mm) 0  0 0
Inhibitors of cysteine and serine proteases
 Leupeptin (0.01 mm)60 ± 4  0 0
 TLCK (0.1 mm)59 ± 6  0 0
 TPCK (0.1 mm)44 ± 6  0 0
Cysteine protease inhibitors
 E-64 (0.01 mm)66 ± 5 ndnd
 E-64d (0.1 mm)53 ± 4 32 ± 4 0
 Iodoacetamide (1 mm)56 ± 5  0 0
 N-ethylmaleimide (10 mm)50 ± 5  0 0
 Zinc chloride (2 mm)51 ± 6 55 ± 5 8 ± 2
Serine protease inhibitors
 Aprotinin (5 mm)42 ± 4 43 ± 512 ± 2
 PMSF (5 mm)45 ± 3  0 0
 AEBSF (5 mm)41 ± 410048-indefinitely
 pABSF (10 mm)nd nd 0
 DFP (10 mm)35 ± 610048-indefinitely

Effect of protease inhibitors applied to isolated Iris tepals, on endoprotease activity, protein level, and visible senescence

Tepals were isolated on day 0 and placed with their proximal ends in aqueous solutions of protease inhibitors (Table 1). Three compounds that inhibit metalloproteases (EDTA, EGTA and 1–10 phenanthroline) had no effect on endoprotease activity and did not delay the time to visible senescence. Some inhibitors of both cysteine- and serine proteases (leupeptin, TLCK, and TPCK) were also ineffective with regard to visible senescence and endoprotease activity. With the exception of zinc chloride, various inhibitors of cysteine proteases also had no effect on visible senescence. Zinc chloride delayed senescence by c. 8 h and reduced endoprotease activity on day 4 to about half (Table 1, see Fig. 3 for activity on other days). E-64d, the membrane-permeable form of E-64, had no effect on senescence, although it inhibited endoprotease activity on day 4 by c. 30% (Table 1; Fig. 3 shows activity on other days). Aprotinin delayed senescence by about 12 h and reduced protease activity on day 4 by > 40% (Table 1).

image

Figure 3. Total endoprotease activity in the distal margin of flag tepals from Iris × hollandica, isolated on day 0 and placed in water on day 0 (control), and after placement in aqueous solutions of E-64d at 10 mm, DFP (diisopropyl-fluorophosphate) at 10 mm, and ZnCl2 at 2 mm. Data are means of four replications ± SD.

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Inclusion of 5 mm AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride] in the solution in which flag tepals were stood largely prevented the decrease in protein level (Fig. 1). Inward rolling of the tepals was also considerably delayed or even completely prevented after treatment with 1–5 mm AEBSF. At 1 mm, tepal inrolling was virtually absent, although some tepals started to slightly roll inward by day 5. The blue colour had then also faded, in some localised patches. The tepals wilted from day 6 and had desiccated by day 7. These symptoms were different from those observed during senescence, both in tepals attached to the flowering stem and in isolated tepals. At 5 mm the normal senescence symptoms such as inward rolling were not observed. Instead the tepals showed wilting from day 6, followed by desiccation. Application of 5 mm AEBSF on day 0 prevented the increase in protease activity (Fig. 2). Application of 5 mm AEBSF on day 1 was almost as effective, with regard to inhibition of total protease activity, as treatment on day 0, but application on day 2 or 3 was considerably less effective (Fig. 2). The time to inward rolling of the tepals was delayed for at least 40 h (or indefinitely) by application on day 1, which is similar to treatment on day 0. If given on day 2 or 3, AEBSF did not affect the time to tepal inrolling. The AEBSF analogue pABSF [4-(amidino)-benzenesulfonyl fluoride hydrochloride]; Diatchuk et al. (1997), applied at 1–10 mm, had no effect on the time to visible senescence (Table 1).

DFP (diisopropylfluorophosphate) had similar effects on the time to visible tepal senescence as AEBSF (Table 1). DFP delayed senescence from 5 mm, and had a maximum effect at 10 mm. At 10 mm it reduced total endoprotease activity to almost zero, at least until day 6 (Fig. 3).

Effect of granzyme inhibitors on tepal senescence

In some animal systems, programmed cell death is regulated by granzymes, which are serine proteases. In a few cell lines, cell death was delayed by IGA [(7-(phenylureido)-4-chloro-3-(2-isothioureidoethoxy) isocoumarin], a specific granzyme A inhibitor (Anel et al., 1997). Granzyme B-induced cell death reportedly occurs in several animal systems, and can be inhibited by Z-Ala-Ala-Asp-chloromethylketone (Z-AAD-cmk), an active-site-directed compound, and by 3,4-dichloroiso-coumarin (DIC; Hudig et al., 1991). These two compounds did not delay visible tepal senescence in Iris (Table 2).

Table 2.  Effect of inhibitors (of granzymes, CPP32, ICE caspase, and proteasome activity) on the time to visible senescence in flag tepals on cut Iris flowers, or in flag tepals isolated from Iris flowers just before floral opening
ChemicalTime to visible tepal senescence in cut flowers (d)Time to visible tepal senescence in isolated tepals (d)
  • **

    Statistically different from the controls.

  • The time to senescence started in the closed bud stage, just before flower opening. Inhibitors were included in the aqueous solution on day 0.

Control4.1 ± 0.24.0 ± 0.1
Granzyme inhibitors
 Z-Ala-Ala-Asp-chloromethylketone4.0 ± 0.14.1 ± 0.2
 3,4-Dichloroiso-coumarin (DIC)4.1 ± 0.14.0 ± 0.3
CPP32 caspase inhibitor
 Acetyl-Asp-Val-Asp-chloromethylketone3.9 ± 0.34.0 ± 0.2
ICE caspase inhibitors
 Acetyl-Tyr-Val-Ala-Asp-chloromethylketone3.9 ± 0.24.1 ± 0.1
 Boc-Asp (OBzl) chloromethylketone4.1 ± 0.14.0 ± 0.3
 Z-Asp-2,6-dichlorobenzoyloxymethylketon4.0 ± 0.23.9 ± 0.2
Proteasome inhibitor
 Z-leu-leu-Nva-H4.6 ± 0.1**4.6 ± 0.1**

Effect of inhibitors of caspases and proteasome activity

In animal systems, several caspases are involved in transduction of the cell death signal. Cut Iris flowers or flag tepals, isolated on day 0, were placed in aqueous solutions containing caspase inhibitors. The tetrapeptide acetyl-Asp-Glu-Val-Asp -chloromethylketone (Ac-DEVD-cmk), a specific inhibitor of CPP32 caspase (Enari et al., 1996), did not delay visible senescence in Iris tepals (Table 2). Ac-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-cmk; ICE inhibitor II), a specific inhibitor of ICE caspases (Enari et al., 1996), Boc-Asp (OBzl) chloromethylketone (Corasaniti et al., 2001) and Z-Asp-2,6-dichlorobenzoyloxymethylketone (ICE inhibitor III; Harrison-Shostack et al., 1997) also did not affect the time to visible senescence (Table 2). By contrast, the proteasome inhibitor Z-leu-leu-Nva-H slightly delayed the onset of senescence (Table 2).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Rise in protease activity prior to visible senescence

A large increase in protease activity occurred in Iris tepals, starting from day 2 of vase life. The pH optimum of the in vitro reactions with the two tested substrates was consistent with the idea that most of the protease activity tested was located in an acidic compartment such as the vacuole. The effect of inhibitors, used in vitro, suggested that more than half of total protease activity was due to cysteine proteases. This mainly followed from the effect of E-64, which is purportedly specific. Inhibitors of serine proteases also reduced activity to c. 40%, indicating that serine proteases account for a considerable part of total activity. As inhibitors of metalloproteases only had a small effect, metalloproteases may play a minor role. The present results seem the first evidence for the presence of serine- and metalloprotease activity associated with senescence of petals.

Effects of proteasome and protease inhibitors on the time to visible senescence

A proteasome inhibitor delayed visible senescence by c. 12 h. Although this effect was small it is noteworthy, as it may indicate a role of the proteasome in the development of the senescence symptoms in tepals. Others have also found evidence for a role of proteasomes in senescence. During the differentiation of tracheary elements, proteasome inhibitors delayed programmed death during its commitment phase. These inhibitors had no effect during the lytic phase (Woffenden et al., 1998; Fukuda, 2000).

A few protease inhibitors tested delayed visible senescence: zinc chloride, aprotinin, AEBSF, and DFP. None of these inhibitors seems specific. Zinc ions inhibit cysteine proteases (Weis et al., 1995), but also some metalloproteases (Zuo & Woo, 1998), and a number of other enzymes such as nucleases (Gerhold et al., 1993). Zinc ions may also delay senescence by inhibiting homologues of caspases in animal cells (Enari et al., 1996). Aprotinin slightly delayed senescence and is known to inhibit serine proteases (Uchikoba et al., 1995), although it is not clear how specific this effect is. Both AEBSF and DFP are serine protease inhibitors and resulted in a considerable delay of visible senescence. DFP also inhibits deacylation reactions (Guther et al., 2001). In animal systems it inhibits esterases such as acetylcholinesterase (Testylier et al., 1999), and lecithin-cholesterol acyltransferase (LCAT), which hydrolyses oxidized polar phospholipids generated during lipoprotein oxidation (Goyal et al., 1997). DFP may therefore also have an effect through lipid metabolism. AEBSF also affects enzymes other than serine proteases. It slightly inhibited in vitro activity of papain, a cysteine protease (10–20% decrease; C. Pak, unpublished results). Furthermore, AEBSF has been reported to be an in vitro inhibitor of plant phospholipase D (Andrews et al., 2000). Its effect on the time to visible senescence (and on protease activity) may therefore be due to inhibition of lipid degradation. It seems that in Iris tepals large-scale lipid degradation precedes large-scale protein degradation (van Doorn et al., 2003). In neuron cells, AEBSF inhibited NADPH oxidase and attenuated the increase in oxygen free radicals (Hwang et al., 2002). In the same system, AEBSF delayed cell death by inhibiting caspase activity, but it remained unclear if this was due to inhibition at the caspase protein level (Rideout et al., 2001). It follows that both DFP and AEBSF can interact with several enzymes. Hence their mode of action in Iris tepal senescence, although suggesting inhibition of serine proteases is at present not clear. Whatever enzyme is inhibited by AEBSF, the reaction apparently depends on the presence of two contiguous CH2 residues between the amino residue and the benzene ring. Replacement of the two CH2 residues with one such residue and an extra lateral amino residue, in pABSF, resulted in loss of action (Table 1). When the activity of these two compounds on NADP oxidase was tested, both had a large inhibitory effect (Diatchuk et al., 1997).

The results may indicate that serine proteases are not only involved in bulk endoprotease degradation, but also are part of the processes that result in the large increase in total endoprotease activity. Following treatment with AEBSF (5 mm) and DFP (10 mm), starting on day 0, no rise in total protease activity was observed, at least until day 6. More than half of total protease activity on day 4 is apparently due to cysteine proteases, but AEBSF had only a small effect, in vitro, on cysteine proteases (C. Pak, unpublished; see this section). This means that AEBSF apparently inhibits the onset of the rise in total endoprotease activity. This is by contrast with application of AEBSF on day 2 or 3, which resulted in inhibition of only c. 50% of total protease activity. AEBSF may then only inhibit the serine protease proteins that have already been formed.

Although the data suggest that AEBSF regulates the onset of the increase in both serine- and cysteine proteases, it is unclear how it may do so. Very little is known about the regulation of the increase in total endoprotease activity. The effect of AEBSF may involve a regulatory serine protease. Apoptosis in several animal systems is regulated by granzymes (which are serine-proteases). However, we found that the granzyme inhibitors Z-AAD-cmk and DIC had no effect on the time to visible senescence in Iris. This we do not take as evidence against the idea that a serine protease – or even a granzyme homologue – is involved in the large increase in total endoprotease activity. We do not know how much of the granzyme inhibitors reached their potential targets, nor do we know how specific these inhibitors are.

The effect of AEBSF was by contrast to that of E-64d. If E-64d was included in the water on day 0 it inhibited total protease activity by c. 30%, which may coincide with direct inhibition of most cysteine proteases. E-64d therefore did not seem to regulate an early step in the onset of increased endoprotease activity.

The aim of the present work was to test two hypotheses: first that all four groups of endoproteases are involved in senescence-associated bulk protein degradation; and second that protease inhibitors delay the visible senescence symptoms. The first hypothesis was not fully substantiated as no positive evidence was found for aspartic protease activity. This conclusion is mitigated by the fact that we tested only one such inhibitor (pepstatin; other general aspartic protease inhibitors are apparently not available), but is reinforced by the finding that pepstatin is highly specific for aspartic proteases (Roberts & Taylor, 2003). The second hypothesis was substantiated, but only clearly so for purported serine protease inhibitors.

It is concluded that visible senescence of Iris tepals is preceded by a sharp increase in endoprotease activity. The onset of this increase showed a positive correlation with the onset of the visible senescence symptoms. In vivo data indicate that cysteine- and serine proteases, and probably metalloproteases, are involved close to the maximum of endoprotease activity. A number of purported protease inhibitors substantially reduced the increase in endoprotease activity and delayed the time to visible senescence. The timing of the rise in endoprotease activity therefore was correlated with the timing of the visible senescence symptoms.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The work was supported by grants from the Department of Agriculture and the Department of Economic Affairs of The Netherlands. The contribution of Y. Zhong in some experiments is gratefully acknowledged.

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  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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