oxidative stress lnduces partia1 degradation of the large ...lnstitut für biologie ii, universitat...

Plant Physiol. (1996) 11 1 : 789-796

Oxidative Stress lnduces Partia1 Degradation of the Large Subunit of Ribulose-l,5-Bisphosphate

Carboxylase/Oxygenase in lsolated Chloroplasts of Barley

Marcelo Desimone, Axel Henke, and Edgar Wagner*

lnstitut für Biologie II, Universitat Freiburg, Schanzlestrasse 1, D-79104 Freiburg, Germany

The effects of oxidative stress on the degradation of ribulose-1,5- bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) were studied in isolated chloroplasts from barley (Hordeum vulgare L. cv Angora). Active oxygen (AO) was generated by varying the light intensity, the oxygen concentration, or the addition of herbicides or ADP-FeCI,-ascorbate to the medium. Oxidative treatments stimulated association of Rubisco with the insoluble fraction of chloroplasts and partia1 proteolysis of the large subunit (LSU). The most prominent degradation product of the LSU of Rubisco showed an apparent molecular mass of 36 kD. The data suggest that an increase in the amount of AO photogenerated by O, reduction at photosystem I triggers Rubisco degradation. A possible relationship between AO-mediated denaturation of Rubisco and proteolysis of the LSU is discussed.

Rubisco degradation plays an important regulatory role in at least two physiological processes. First, during foliar senescence there is a pronounced redistribution of nutri- ents from leaves to reproductive structures. The vast ma- jority of such metabolites come from foliar protein degradation. Rubisco is the most abundant protein in leaves and therefore represents a source of amino acids for reproductive organs (Peoples et al., 1980; Makino et al., 1984; Fer- reira and Teixeira, 1992). Second, environmental stress factors can cause reversible and irreversible inactivation of Rubisco (Brüggemann et al., 1994; Eckardt and Pell, 1995). Irreversibly inactivated Rubisco has to be degraded and replaced by new synthesized copies to fully reestablish the photosynthetic function.

One common event in plants during these two physiological conditions is the development of oxidative processes mediated by AO species. AO can be formed in chloroplasts either by direct transfer of excitation energy from chlorophyll to oxygen, producing singlet oxygen, or by single electron reduction, resulting in 0,- and its de- rivatives H,O, and OH. (for review, see Asada and Taka- hashi, 1987). During foliar senescence or under unfavorable environmental conditions, the concentration of AO can rise to toxic levels, causing cellular injuries such as lipid peroxidation, inactivationl denaturation of enzymes, and

* Corresponding author; e-mail wagnerQsun2.ruf.uni- freiburgde; fax 49 -761-203-2840.

789

DNA damage. The direct effect of AO on isolated proteins has been well characterized (Stadtman, 1993). Considering albumin as a model protein, Davies (1987), Davies and Delsignore (1987), and Davies et al. (1987a, 1987b) found that AO treatment can increase the hydrophobicity of the protein, modify certain amino acid residues, and produce intra- and intermolecular cross-linking and protein fragmentation. In addition, an increase in the susceptibility to proteolysis of AO-modified proteins has been reported (Stadtman, 1990). In chloroplasts, oxidative stress cause inhibition of the enzymes of the Calvin cycle (Kaiser, 1979), inactivation and rapid degradation of the D1 protein (Kyle, 1987; Aro et al., 1990), aggregation of thylakoid proteins (Roberts et al., 1991), and modifications of Rubisco structure (Mehta et al., 1992; García-Ferris and Moreno, 1994).

Proteolytic activities in chloroplasts able to degrade Rubisco in vivo or in vitro have been reported by severa1 authors (Nettleton et al., 1985; Musgrove et al., 1989; Buschnell et al., 1993; Otto and Feierabend, 1994). How- ever, the physiological relevance and the regulation of these proteases are still not well understood (Huffaker, 1990). In particular, it is not clear if elevated amounts of AO trigger Rubisco degradation by a putative interna1 proteolytic system of chloroplasts. In this context, two method- ological problems deserve to be mentioned. First, Rubisco degradation has been measured either as a decrease in the intensity of the 55-kD band corresponding to the LSU in SDS-PAGE or as the appearance of bands with lower molecular masses that react with Rubisco antibodies on western blot analysis. However, loss or appearance of typical bands under oxidative stress do not necessarily indicate proteolysis, because proteins exposed to AO can form highly insoluble aggregates or undergo nonenzymatic fragmentation (Stadtman, 1993). Second, the use of isolated chloroplasts for degradation experiments offers the possi- bility that clearer conclusions about the subcellular com- partmentation can be obtained. However, Miyadai et al. (1990) point out that proteolytic activities previously reported in mechanically isolated chloroplasts could be caused by contamination with vacuolar proteases.

Abbreviations: AO, active oxygen; Chl, chlorophyll; DTE, di- thioerythritol; LSU, large subunit; MDA, malondialdehyde; MV, methylviologen; OH , hydroxyl radical; 02-, superoxide anion; SOD, superoxide dismutase; SSU, small subunit.

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790 Desimone et al. Plant Physiol. Vol. 11 1 , 1996

The principal goal of the present work was to clarify whether a photogenerated increase in AO can induce degradation of Rubisco inside chloroplasts. Therefore, we subjected isolated chloroplasts to standard treatments such as variation of light intensity or oxygen concentration, addition of herbicides, specific scavengers, an AO-generating system, or combinations of the above.

MATERIALS A N D METHODS

Seeds of barley (Hordeum vulgare L. cv Angora; Raiffeisen-Zentralgenossenschaft, Bad Krozingen, Ger- many) were sown in vermiculite and grown under alter- nating light conditions (12 h of dark, 12 h of white light) at constant temperature (25°C) and a RH of 70%. White light of an intensity of 80 W m-' was provided by Xenon arc lamps of 10 kW (Osram, Munich, Germany).

lsolation of Chloroplasts

Primary leaves were harvested after 7 d and homoge- nized in a grinding medium containing 50 mM Tris-HC1 (pH 7.8), 330 mM sorbitol, 1 mM EDTA, 1 mM DTE, 1% (w/v) protease-free BSA, and 10 pg mL-' leupeptin with a blender (Waring). After filtration through a nylon mesh (70 pm), the suspension was centrifuged at 2,OOOg for 60 s. The resulting pellets were resuspended in washing medium containing 50 mM Tris-HC1 (pH 7.8), 330 mM sorbitol, 1 mM EDTA, and 1 mM DTE and centrifuged as above. This washing step was repeated twice. The pellets were subsequently resuspended in 3 mL of washing medium and filtered again through a nylon mesh (20 pm). The filtrate was mixed with a solution containing 50% (v/v) Perco11 (Pharmacia), 3% (w/v) PEG 6000, 1% (w/v) Ficoll, and the same ingredients as the washing medium. The mixture was centrifuged at 30,OOOg for 15 min and the lower green band was recovered. This fraction was diluted with washing medium ( 1 : l O ) and centrifuged at 2,000s for 2 min. The chloroplasts were resuspended in a washing medium containing 5 mM MgC1, and 0.5 mM MnCl, to a final Chl concentration of 100 pg mL-'. A11 procedures were carried out between O and 4°C.

The integrity of chloroplasts was estimated by the ferri- cyanide photoreduction assay and phase-contrast microscopy (Walker et al., 1987). The isolation yielded, on aver- age, a recovery of 90% intact chloroplasts as verified by both methods. No contamination by other subcellular compartments was detected by microscopy or by the measurement of the marker enzymes Cyt c oxidase, catalase, acid phosphatase, and PEP carboxylase (data not shown).

Oxidative Treatments

For light treatments, equal volumes of isolated, intact chloroplasts were placed in glass vials, diluted with resuspension medium to a final concentration of 50 pg Chl mL-l, and exposed to light (5, 100, and 500 W m-') generated by a slide projector with a 300-W tungsten lamp at room temperature (22-24°C). Variation of oxygen concentration in the reaction medium was achieved by continuous

passage of air, 100% oxygen, or 100% nitrogen through the vials. Additional effectors (100 p~ MV or 100 p~ DCMU) were added together with resuspension medium before light exposure. After 1 h of incubation under 500 W m-', the apparent intactness of the chloroplasts was roughly 90% of the initial value, as measured by phase-contrast microscopy. Studies with antioxidants were carried out using broken chloroplasts prepared by hypoosmotic shock in 50 mM Tris-HC1 (pH 7.8) and 5 mM MgC1,. Aliquots of this preparation were mixed with either 100 units mL-l SOD (EC 1.15.1.1, Boehringer Mannheim), 100 units mL-' catalase'(EC 1.11.1.6, Sigma), 100 mM mannitol, or 10 mM EDTA. MV (100 p ~ ) was added to a11 probes before illumination (100 W m-'). Experiments with ADP-FeC1,- ascorbate were performed using intact or broken chloroplasts (both 50 pg Chl mL-') or isolated Rubisco (1 mg mL-'). ADP-FeCl, (1, 10, 100, or 1000 p~ final concentrations) and, subsequently, sodium ascorbate (1 mM final concentration) were added and incubated at 25°C. The reactions were stopped by addition of 10 mM EDTA at the beginning or after 1 h.

Measurement of Chl, Protein, and M D A

Chl was determined by the method of Lichtenthaler (1987); total protein was determined according to Bradford (1976) using BSA as standard; and MDA production as a marker for lipid peroxidation was determined by the method of Heath and Packer (1968).

Proteolysis of Rubisco

Proteolysis of Rubisco was assessed by following the appearance of degradation products of the LSU. After oxidative treatments, equal amounts of chloroplast suspension were mixed (2:l) with sample buffer containing 200 mM Tris-HC1 (pH 6.8), 4% (w/v) SDS, 14% (v/v) 2- mercaptoethanol, 20% (v/v) glycerol, and 0.001% (w /v) bromphenol blue, and boiled for 2 min. The denatured proteins were resolved by SDS-PAGE, and Rubisco and its degradation products were detected by immunoblotting.

Cel Electrophoresis and lmmunoblotting

SDS-PAGE was carried out using two systems: (a) mini- gels, as described by Laemmli (1970), with a homogeneous acrylamide concentration of 12.5%, and (b) precast Excelgel SDS, gradient 8 to 18% (Pharmacia). The separated proteins were transferred to PVDF membranes (Millipore) with a semidry electroblotter (Bio-Rad) according to the instruc- tion manual. Rubisco was detected using a rabbit anti- serum against purified protein from barley and a goat anti-rabbit IgG antibody conjugated to alkaline phosphatase (Schleicher & Schuell).

Quantification of Rubisco Associated with Soluble or Membrane Fractions

After treatment, chloroplasts were exposed to hypoosmotic shock and centrifuged at 14,5008 for 10 min. The supernatant and pellet were carefully separated and boiled



Degradation of Rubisco under Oxidative Stress 79 1

for 2 min in the presence of sample buffer. After electrophoresis and immunodetection, each lane was scanned three times with a laser densitometer (Ultroscan 2202, LKB, Bromma, Sweden) and integrated (3390A integrator, Hewlett-Packard). The lane for the soluble fraction and the membrane fraction were integrated and the sum of both lanes was taken as 100%.

Purification of a Degradation Product of LSU

Isolated intact chloroplasts were stimulated to degrade Rubisco by incubation with MV (100 PM) in light (100 W m-,). After 1 h the chloroplasts were broken and the soluble proteins were obtained by centrifugation and separated in SDS-PAGE (12.5% acrylamide). A Coomassie blue-stained band corresponding to the 36-kD polypeptide, which reacted with anti-Rubisco antibody in comparative immunoblotting, was carefully cut out from gels and elec- troeluted. The eluate was concentrated (Centricon 3, Ami- con, Witten, Germany), resolved in SDS-PAGE in gradient (8-18%), and transferred to PVDF membranes. Three polypeptides with molecular masses near 36 kD were detected in Coomassie blue stain, but only the most abundant of them reacted in anti-Rubisco immunoblotting. This band was cut out and the N-terminal sequence was determined. Additional analysis in two-dimensional electrophoresis showed that the preparation was composed mainly of the immunoreactive polypeptide.

Rubisco Purification

Isolated chloroplasts were broken by resuspension in distilled water. After 5 min extracts were equilibrated in chromatography buffer consisting of 50 mM Tris-HCI (pH 7.8), 1 mM EDTA, 1 mM DTE, and 5 mM MgCl,. Membranes and soluble fractions were separated by centrifugation at 40,OOOg for 15 min. Soluble proteins were applied to a column filled with Q-Sepharose Fast Flow (1 X 30 cm) and equilibrated with chromatography buffer. Bound proteins were eluted with a linear gradient of NaC1. Between 225 and 275 mM NaCl, an A,,, peak appeared that was con- firmed by SDS-PAGE to be mainly Rubisco. The corresponding fractions were pooled and concentrated by precipitation with (NH,),SO, (50% saturation). The pellet was resuspended in chromatography buffer containing 150 mM NaCl and loaded onto a column (1.4 X 90 cm) filled with Sephacryl S-300. Equilibration and elution of proteins were carried out using the same buffer. Fractions containing Rubisco were pooled and concentrated by precipitation with (NH,),SO, (5oy0 saturation). Pellets were stored at -24°C. A11 chromatographic steps were carried out at 4°C using a modular FPLC system (Pharmacia).

Measurement of Rubisco Denaturation

Denaturation was assessed by a modified method based on the protein solubility in high-salt buffer as described by Davies and Delsignore (1987). Proteins were mixed with a solution containing 100 mM phosphate buffer (pH 5.5) and 3 M KC1 and placed on ice for 60 min. After centrifugation

at 50008 for 10 min, the remaining soluble proteins were quantified by Bradford (1976).

RESULTS

Effect of Light lntensity and Oxygen Concentration

Since lipid peroxidation is one of the first consequences of oxidative damage in chloroplasts, MDA production has been used as a standard indicator for oxidative stress (Gut- teridge and Halliwell, 1990). Isolated, intact chloroplasts showed light- and oxygen-dependent MDA production, indicating formation of AO (Fig. 1A).

Concomitantly, increasing light intensity and oxygen concentration caused an increase in the amount of the LSU of Rubisco associated with the insoluble fraction of chloroplasts (Fig. 1B). This suggests that oxidative stress causes changes in Rubisco structure, resulting in the formation of insoluble aggregates and/ or membrane association.

When chloroplasts were exposed to 100 or 500 W m-’ light intensities, western blot analysis showed additional immunoreactive bands between the LSU and the SSU of Rubisco (Fig. 2). The most prominent fragment had an apparent molecular mass of 36 kD (Fig. 2). The appearance of bands with a similar mobility has been reported previously (Mae et al., 1989; Mitsuhashi et al., 1992; Bushnell et al., 1993), and these bands were considered degradation products of the LSU. To further confirm the identity of this band as a degradation product, the 36-kD polypeptide was purified from polyacrylamide gels and its N-terminal sequence was determined as Val-Ala-Tyr-X-X-X-Leu. It very likely represents residues 101 to 107 of the LSU (Knight et al., 1990). The combination of immunological cross-reaction with a specific antibody and partia1 sequence analysis indicates that the 36-kD fragment is a LSU breakdown product. By separate analysis of subchloroplas- tic fractions, the degradation products were associated mainly with the insoluble fraction, but some fragments remained in the supernatant (data not shown).

The participation of oxygen in the degradative process under high-light intensity was tested by incubating chloroplasts under 500 W mp2 with a continuous passage of air consisting of either 100% oxygen or 100% nitrogen. In a nitrogen atmosphere, the LSU was not degraded (Fig. 2). In an oxygen atmosphere, increases in both the LSU associated with membranes (Fig. 1B) and that associated with degradation products (Fig. 2) were observed.

Since contamination with proteases from other subcellular compartments is conceivable, peripheral proteins bound to the organelles were digested with thermolysin as described by Miyadai et al. (1990). Protease-treated chloroplasts were subsequently repurified in a Perco11 gradient. Such a treatment did not modify the degradation pattern of the LSU when the organelles were subjected to oxidative treatments (data not shown). This result and the absence of marker enzymes for other compartments (see “Materials and Methods”) indicate that the degradation of the LSU was unlikely due to extrachloroplastic proteolytic activity.



792 Desimone et al. Plant Physiol. Vol. 111, 1996

Figure 1. Effect of light intensity, oxygen con-centration, and herbicides on lipid peroxidation(A and C) and Rubisco association with the in-soluble fraction of chloroplasts (B and D). A andB, Isolated intact chloroplasts (50 /ng Chl mL~1)were exposed for 1 h to light intensities of 0, 5,100, or 500 W rrT2. In addition, two sampleswere incubated under 100% O2 or 100% N2

atmospheres during illumination (500 W rrT2).C and D, Isolated, intact chloroplasts were re-suspended in medium containing 100 JAM MVand/or 100 /MM DCMU and exposed to 0, 5, 100,or 500 W m~2 light intensities for 1 h in air.Data points and error bars represent the mean ±so of two independent determinations.

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Effect of MV and DCMUTo determine a connection between light-driven electron

transport, activation of oxygen, and LSU degradation, MVwas introduced into the system to serve as an electronacceptor of PSI and as an electron donor for oxygen.DCMU, as a specific inhibitor of electron flow at PSII,should block formation of AO in the light if it is generatedvia linear electron transport.

When chloroplasts were exposed to light in the presenceof MV, light-intensity-dependent lipid peroxidation (Fig.1C) and association of up to 50% of the Rubisco with theinsoluble fraction (Fig. ID) were observed. The amount ofdegradation products increased during the 1st h underweak light intensity (Fig. 3). At higher intensities (100 and

kDa

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Light (W-m) 0 5 100 500 500 500Oxygen cone. 21% 0% 100%Figure 2. Effect of light intensity and oxygen concentration on deg-radation of the LSU of Rubisco. Isolated, intact chloroplasts (50 /^gChl mL~') were exposed to light intensities of 0, 5, 100, or 500 Wm~2 and oxygen concentrations of 0, 21, and 100%. After 1 hsamples of chloroplasts were mixed with sample buffer and boiled.The proteins were resolved in SDS-PAGE, transferred to PVDF mem-branes, and immunoblotted with anti-Rubisco.

500 W m 2), degradation products were rapidly formed,reaching a maximum at 20 min (Fig. 3). Under extremeoxidative conditions (MV, 100 and 500 W m'2), aggrega-tion products with molecular weights larger than the LSUand insoluble complexes on the loading site of the gel weredetected by western blotting (Fig. 3 and data not shown).DCMU suppressed the effects observed in light treatmentswith or without MV; neither lipid peroxidation (Fig. 1C)nor degradation of Rubisco was observed (Fig. 4). Thesedata strongly suggest that LSU degradation is stimulatedby reduction of oxygen at PSI.

Effect of Antioxidants

To find out which oxygen-reduction species might beinvolved in LSU degradation, broken chloroplasts were

LSU .».Dfef

Time (min) 0Light (W. m)

10 20 30 60 10 20 30 60100

Figure 3. Degradation of the LSU of Rubisco in the presence of MVis dependent on time and light intensity. Isolated, intact chloroplasts(50 ng Chl mL"1) were resuspended in medium containing 100 JIMMV and exposed to light intensities of 5 or 100 W m"2. After 0, 10,20, 30, or 60 min, samples of chloroplasts were mixed with electro-phoresis buffer and boiled. The proteins were resolved in SDS-PACE,transferred to PVDF membranes, and immunoblotted with anti-Rubisco. www.plantphysiol.orgon March 15, 2020 - Published by Downloaded from

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Degradation of Rubisco under Oxidative Stress 793

LSU ^

MVDCMU

Figure 4. Suppression of light- and MV-induced degradation of theLSU of Rubisco by addition of DCMU. Isolated, intact chloroplastswere resuspended in treatment medium with ( + ) or without (-) 100/HM DCMU. After 10 min in the dark on ice, MV ( + ) or medium (-)was added to a final concentration of 100 /AM and exposed to 100 Wm"2 for 1 h.

incubated under oxidative conditions (MV, 100 W m~2) inthe presence of specific antioxidative enzymes and scaven-gers of AO.

Addition of exogenous SOD (100 units mL"1), an en-zyme that degrades O2~ but produces H2O2, could notprevent LSU degradation, suggesting that O2~ is not di-rectly involved (Fig. 5, lane 3). In contrast, catalase (100units mL"1) suppressed LSU degradation, indicating a par-ticipation of H2O2 and/or its reduction derivative OH inthe degradative process (Fig. 5, lane 4). Mannitol (100 mM)(Fig. 5, lane 5) or other OH scavengers, such as DMSO (10mM) or ethanol (10 HIM) (data not shown), did not inhibitdegradation, suggesting that formation of OH is not re-quired for LSU degradation. However, addition of EDTA(Fig. 5, lane 6) or other chelators such as EGTA or 1,10-phenanthroline (data not shown), which remove ions thatcatalyze the Fenton reaction, prevented LSU degradation.

Effect of ADP-FeCl3-Ascorbate

The data presented above indicate that a light-intensity-dependent increase of AO stimulates degradation of theLSU. However, light might affect the degradative processin other ways, e.g. by regulating a proteolytic systemthrough ATP production or through pH modification. Toinvestigate whether AO accelerates degradation of Rubiscoin the absence of light, intact chloroplasts, broken chloro-plasts, and isolated Rubisco were incubated in darkness,together with an AO generating system consisting of ADP-FeCl., and 1 mM sodium ascorbate.

1 2 3 4 5 6

Figure 5. Effect of antioxidants on degradation of the LSU ofRubisco. Broken chloroplasts were incubated in light (100 W rrT2)for 0 (lane 1) or 1 h (lanes 2-6) in medium consisting of 50 mMTris-HCI (pH 7.8), 5 mM MgCI2, 100 JUM MV, and the followingadditional effectors: buffer (lane 2), 100 units mL'1 SOD (lane 3),100 units mL~' catalase (lane 4), 100 mM mannitol (lane 5), or 10 mMEDTA (lane 6).

In intact chloroplasts, addition of ADP-FeCl3 producedRubisco degradation only at concentrations above 1 mM(Fig. 6A). This is probably due to the poor diffusion of ironions through envelope membranes. Lysed chloroplasts(protein concentration approximately 1 mg mL"1) showedthe typical degradation pattern after 1 h of incubation with10 JU.M ADP-FeCl3, reaching a maximal effect around 100IJ.M (Fig. 6B). This effect could be suppressed by simulta-neous addition of EDTA to the reaction medium. Thesedata support the notion that high-light intensity promotesRubisco degradation in chloroplasts via an increase in AO.

In parallel, isolated Rubisco (1 mg mL"1) was incubatedunder the same conditions used above. A decrease in theprotein solubility in salt solution proportional to the ADP-FeCl3 concentration was observed, indicating that the pro-tein structure was modified (Fig. 7). Additional studiesrevealed a change in the protein mobility by IEF and cross-linking of SH groups of Rubisco in experiments carried outaccording to the methods described in Mehta et al. (1992)(data not shown). However, no fragmentation productswere detected (Fig. 6C), indicating that AO alone does notfragment LSU, so other factors coming from chloroplastsmust be required for its degradation.

A Intactchloroplasts

B Brokenchloroplasts

C IsolatedRubisco

li-

ADP-FeCl, (nM) 0 1 10 100 1000 1000+

EDTA

Figure 6. Effect of ADP-FeCI3 on degradation of the LSU of Rubisco.Intact chloroplasts (A), broken chloroplasts (B), and isolated Rubisco(C) were resuspended in medium consisting of 50 mM Tris-HCI, pH7.8, and 5 HIM MgCI2 (and 330 mM sorbitol for intact chloroplasts).After addition of ADP-FeCl, to the indicated final concentrations and1 mM sodium ascorbate, the samples were incubated under the sameconditions (25°C for 1 h) in darkness. The last lanes show suppres-sion of Rubisco degradation when 10 mM EDTA was added in theresuspension medium before incubation. www.plantphysiol.orgon March 15, 2020 - Published by Downloaded from

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794 Desimone et al. Plant Physiol. Vol. 11 1, 1996

'*O I

ADP-FeC13 1 10 50 100 500 1000 1000 yM EDTA - - - - - - 10 mM

Figure 7. Solubility in high-salt medium of isolated Rubisco after treatment with AO. lsolated Rubisco (1 mg mL-') was incubated at 25OC in the presence of 1 , 1 O, 50, 100, 500, or 1 O00 p~ ADP-FeCI, and 1 mM sodium ascorbate. After 1 h (or at start as shown in the last bar), 10 m M EDTA were added and mixed with a solution containing 100 mM phosphate buffer, pH 5.5, and 3 M KCI. After 1 h on ice, the samples were centrifuged at 12 ,OOOg for 5 min. The remaining soluble protein was determined according to Bradford (1 976). Data points and error bars represent the mean 2 SD of three independent determinations.

DI SC U SSI ON

The present work documents the participation of AO in the degradation of Rubisco under oxidative-stress conditions. The predominant source of AO in plants is the light- dependent reduction of O, at PSI, particularly when NADE'+ pools are reduced (Asada, 1994). We produced similar conditions in isolated chloroplasts using standard strategies to promote or prevent the production of AO and studied their effect on degradation of the LSU of Rubisco.

Experiments with light intensities (Fig. 2), oxygen concentrations (Fig. 2), MV (Fig. 3), and DCMU (Fig. 4) clearly indicate that an increase in the amount of AO generated by reduction of O, stimulated the degradation of the LSU. A direct relationship between the amount of degradation products detected and the intensity of the stress factor assayed was observed in a11 experiments. Under mild stress conditions (e.g. 5 W m-' plus MV), degradation of the LSU occurred slowly but increased with the incubation time. On the other hand, strong stress (e.g. 100 W m-' plus MV) produced a rapid increase in the appearance of products without further increments (Fig. 3), which suggest that a rapid, AO-mediated inhibition of electron flow (probably at PSII) prevented reduction of additional oxygen.

The first reduction product of molecular oxygen via the Mehler reaction is O,-, which is further converted to H,O, by SOD in vivo (Asada and Takahashi, 1987). Addition of exogenous SOD to lysed chloroplasts caused no significant changes in the degradation of Rubisco (Fig. 5), indicating that O,- itself is not directly involved. On the other hand, scavenging of H,O, by exogenously added catalase sup-

presses degradation (Fig. 5 ) . This result indicates that H,O, and/or OH. participate in the chain of events leading to the degradation of Rubisco. Formation of OH. in vivo probably occurs by reduction of H,O, in a metal-catalyzed reaction (Asada and Takahashi, 1987). The effectiveness of low ADP-FeCl, concentrations in inducing the typical degradation pattern (Fig. 6) and the ability of chelating agents to suppress Rubisco degradation (Fig. 5 ) suggest that OH. production is necessary for this process. On the other hand, relatively high mannitol concentrations (100 mM) do not protect Rubisco (Fig. 5). Each protein, however, shows a different sensitivity to OH scavengers depending on its specific metal binding site (Stadtman, 1993). Although inhibition of degradation by chelating agents might be due to remova1 of metals catalyzing the Fenton reaction, it must be taken into account that these compounds are also strong inhibitors of metalloproteases.

The evidence presented here confirms and extends pre- vious observations (Casano et al., 1990; Mehta et al., 1992; Landry and Pell, 1993) about the participation of AO in the degradation of Rubisco. However, the role of AO in the degradative process of Rubisco remains open. Some authors propose that certain modifications of Rubisco increase its susceptibility to proteolysis by chloroplastic enzymes (Moreno et al., 1995), as was similarly demonstrated for other biological systems (Wolff et al., 1986; Goldberg, 1992; Rivett, 1993). For example, the degradation of Rubisco has been correlated with protein denaturation and with cross-linking of SH groups (Garcia-Ferris and Moreno, 1994) when plants or chloroplasts are exposed to oxidative stress. Furthermore, studies with isolated Rubisco have demonstrated that AO produces structural changes on the protein (Eckardt and Pell, 1995), and that these modifications lead to proteolysis by externally added proteases such as trypsin (Pefiarrubia and Moreno, 1990). A stimulation of Rubisco degradation in chloroplasts by addition of 0.1% SDS (Mae et al., 1989) or by a pH lower than 5.0 (Nettleton et al., 1985; Casano et al., 1994) was reported. These observations are in agreement with the susceptibility hypothesis in that both factors cause Rubisco denaturation. In accordance, we found a relationship between AO production (Fig. l , A and C), aggregation of Rubisco (measured as loss of solubility of the protein, Fig. 1, B and D), and LSU degradation (Figs. 2 4 ) . However, on the basis of the data presented here, it is not possible to establish whether AO-mediated denaturation of Rubisco precedes its degradation under oxidative stress or whether these are concomitant events. To clarify this point, future experiments should test whether an untreated chloroplast preparation can preferentially degrade Rubisco that was previously modified by AO.

To our knowledge, a chloroplastic proteolytic system capable of discriminating between native and denatured proteins has not been described. A potential candidate for this function is the homolog of the ATP-dependent Clp A/P protease from Esckerickia coli that was recently found in chloroplasts (Maurizi et al., 1990; Shanklin et al., 1995). In E. coli this protease is responsible primarily for the degradation of abnormal proteins, but its function in chlo-



Degradation of Rubisco under Oxidative Stress 795

roplasts remains unknown (Vierstra, 1993). Another protease (EP1) purified from pea chloroplasts (Liu and Jagen- dorf, 1986; Bushnell et al., 1993) has biochemical characteristics similar to the protease that is thought to be involved in the degradation of the LSU of Rubisco described here: (a) Isolated EP1 degrades the LSU of isolated Rubisco in vitro, forming a single degradation product of 36 kD. In a11 of our experiments, a polypeptide accumu- lated with a similar molecular mass (Fig. 2); (b) in accordance with our results (Fig. 5) , EP1 is totally inhibited by chelating agents; (c) EP1 is active in a nonreducing envi- ronment (100% inhibition with 5 mM DTT). In our assays, degradation of Rubisco occurred under oxidizing conditions.

The identification of a possible protease(s) involved in Rubisco degradation under oxidative stress is our present effort. However, the elucidation of this point seems insuf- ficient to explain the whole degradative process of Rubisco, because we described only a partia1 degradation of the LSU to a polypeptide of 36 kD. It is important to know whether similar degradation products accumulate in vivo and which sequence of steps leads to the complete breakdown of Rubisco into reutilizable free amino acids.

ACKNOWLEDCMENTS

We thank Thomas Steger-Hartmann and Fernando Pitossi for constructive comments and suggestions on the original manu- script, Dr. Drumm-Herrel for the gift of antibodies against Rubisco, and Dr. Carlos Dotti (European Molecular Biology Lab- oratory, Heidelberg, Germany) for the N-terminal sequence deter- mination of the degradation product.

Received December 18, 1995; accepted April 8, 1996. Copyright Clearance Center: 0032-0889/96/111/0789/08.

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