Changes in the antioxidative systems in mitochondria during ripeningof pepper fruits

Ana Jiménez *, Juana M. Gómez, Eusebio Navarro, Francisca Sevilla

Nutrition and Plant Physiology Department, Centro de Edafología y Biología Aplicada del Segura (CSIC), Apdo. 4195, 30080 Murcia, Spain

Received 19 March 2002; accepted 7 May 2002

Abstract

The presence of enzymes of the ascorbate–glutathione cycle was studied in mitochondria purified from green and red pepper (Capsicumannuum L.) fruits. All four enzymes, ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (MDHAR; EC 1.6.5.4),dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) were present in the isolated mitochondria ofboth fruit ripening stages. The activity of the reductive ascorbate–glutathione cycle enzymes (MDHAR, GR and DHAR) was higher inmitochondria isolated from green than from red fruits, while APX and the antioxidative enzyme superoxide dismutase (SOD; EC 1.15.1.1)were higher in the red fruits. The levels of ascorbate and L-galactono-γ-lactone dehydrogenase (GLDH; EC 1.3.2.3) activity were foundto be similar in the mitochondria of both fruits. The higher APX and Mn-SOD specific activities in mitochondria from red fruits might playa role in avoiding the accumulation of any activated oxygen species generated in these mitochondria, and suggests an active role for theseenzymes during ripening. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: Ascorbate; Glutathione; Mitochondria; Pepper fruit; Ripening

1. Introduction

Activated oxygen species (AOS) are the primary media-tors of oxidative damage in plants and mainly includesuperoxide radicals (O2

–•), H2O2, and hydroxyl radicals(•OH) [13]. These activated oxygen species can rapidlyattack all types of biomolecules to cause membrane dete-rioration, lipid peroxidation and DNA mutation, leading toirreparable metabolic and structural dysfunctions and celldeath [13]. Reactions involving activated oxygen speciesare an intrinsic feature of senescence and fruit ripeningsince they promote the process of oxidative deterioration

that contributes to a general deterioration of cellular me-tabolism [9,39]. For this to be the case, there must be abalance between the induction of AOS and their removal byantioxidant systems. It is likely, therefore, that the antioxi-dant systems, which are distributed in cell organelles[6,7,11,20]play an important role in both the senescenceand the ripening process. Superoxide dismutase (SOD),catalase (CAT), peroxidases and the ascorbate–glutathione(ASC–GSH) cycle enzymes: ascorbate peroxidase (APX),mono- and dehydroascorbate reductase (MDHAR, DHAR)and glutathione reductase (GR), are components of theantioxidative enzymes. The non-enzymatic antioxidantsascorbate (ASC) and glutathione (GSH), together with theenzymatic components, ultimately scavenge H2O2 at theexpense of NADPH or NADH[1].

During fruit ripening, chloroplast differentiation intochromoplasts is characterized by a breakdown in chloro-phyll, important structural modifications and the accumula-tion of polyunsaturated lipids and carotenoids[35]. It hasbeen reported that ‘in vivo’ transient oxidative stress playsa key role in the induction of chromoplast carotenoidbiosynthesis and in the transformation of chloroplasts tochromoplasts[4]. It has been shown that the formation of

Abbreviations: AOX, alternative oxidase; APX, ascorbate peroxidase;ASC, ascorbate: reduced form; CAT, catalase; CCO, cytochromec oxidase;DHA, ascorbate: oxidized form (dehydroascorbate); DHAR, dehydroas-corbate reductase; GLDH, L-galactono-γ-lactone dehydrogenase; GR,glutathione reductase; GSH, glutathione: reduced form; GSSG, gluta-thione: oxidized form; MDHA, monodehydroascorbate; MDHAR, mono-dehydroascorbate reductase; Mn-SOD, manganese-containing superoxidedismutase; OHPR, hydroxypyruvate reductase; TPI, triose 3-phosphateisomerase

* Corresponding author.E-mail address: ajimenez@cebas.csic.es (A. Jiménez).

Plant Physiol. Biochem. 40 (2002) 515–520

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O2–• and the accumulation of H2O2 increase during fruit

ripening, as indicated by the accumulation of lipid peroxi-dation and protein oxidation products [17,26]. Changes inenzymatic and non-enzymatic components of the plantantioxidant defences have also been described during fruitripening but, with some exceptions, the exact subcellularlocalization of such changes has not been identified [17,35].

Despite the increased respiratory activity shown by somefruits during ripening, studies on the energy metabolism ofmitochondria from fruits are few. Several studies haveaddressed the ‘ in vitro’ performance of mitochondria iso-lated from fruits during ripening or as influenced byexposure to stress conditions, including high temperature,irradiation and controlled-atmosphere storage conditions[16,29]. However, little is known about the activated oxy-gen function of mitochondria in fruit ripening. This aspect isimportant considering that mitochondria are a major sourceof reactive oxygen in plant tissues [5]. Recent studies haveshown that mitochondria from green bell pepper fruits maybe a major source of O2

–• when this chilling-sensitive plantis exposed to low temperatures [32], and studies of the roleof the ASC–GSH cycle of mitochondria and peroxisomes ofpea leaves suggest that during senescence oxidative injuriescould be accelerated in the former because a depression oftheir antioxidant systems results in enhanced H2O2 produc-tion [19].

In an attempt to characterize the antioxidant systemsoperating in mitochondria isolated from pepper (Capsicumannuun L.) fruits at two ripening stages, we have investi-gated the presence and activities of the enzymes involved inthe ASC–GSH cycle and the antioxidative enzyme super-oxide dismutase. In addition, the content of the solubleantioxidant ascorbic acid and the activity of the mitochon-drial enzyme L-galactono-γ-lactone dehydrogenase(GLDH), a membrane-bound protein that is responsible forthe last step of the ascorbic acid synthesis [3], weredetermined.

2. Results and discussion

2.1. Isolation of pepper fruit mitochondria

When fruits of green and red peppers were subjected tocell fractionation by differential centrifugation, the activityof the marker enzymes indicated a relatively clean separa-tion of mitochondria from other organelles and the cytosolicfraction (Fig. 1, data not shown for red fruits). Analyzing theactivity of different organelle marker enzymes (Table 1), wefound that the percentage of peroxisomal contamination wasabout 1.4 and 1.1% in mitochondria isolated from the greenand red peppers, respectively, while the percentage of thechloroplast and cytosol marker was around 0.1% in themitochondria from the green peppers and 0.04% in those

Fig. 1. Analysis of the Percoll density-gradient used for the isolation ofmitochondria from green peppers. Protein content is expressed as mg ml–1.Chlorophyll (mg ml–1) was used as the chloroplast marker, and CCO andOHPR expressed as nkatals ml–1, were used as markers for mitochondriaand peroxisomes, respectively.

Table 1Total activity of marker enzymes in crude extract and in isolated mitochondria from green and red pepper fruits. Total activity is expressed as nkatals. Dataare the mean ± S.E. of three independent experiments. The percentage activity was calculated relative to the total activity in the crude extract

CCO OHPR TPI

Total activity Percentage activity Total activity Percentage activity Total activity Percentage activity

Green pepperCrude extract 400 ± 82 100 352 ± 33 100 11 008 ± 215 100Mitochondria 123 ± 12 31 5 ± 1 1.4 15 ± 3 0.1

Red pepperCrude extract 120 ± 15 100 452 ± 48 100 11 236 ± 273 100Mitochondria 62 ± 5 51 5 ± 1 1.1 5 ± 1 0.04

516 A. Jiménez et al. / Plant Physiol. Biochem. 40 (2002) 515–520

from the red fruit. The yield of mitochondrial protein from100 g of green and red pepper fruits was approximately1 mg.

About 30–50% of the total CCO activity found in thecrude extracts was recovered in the isolated mitochondria.Analysis of the specific activity of this enzyme (Table 2)pointed to higher activity in the mitochondria isolated fromthe green peppers than from the red fruit. These resultssuggest a higher CCO-dependent respiration in mitochon-dria from green pepper than in red. Some fruits exhibitrespiratory increases during ripening, although pepper fruitsshow a non-climacteric-type ripening physiology [22,31].The absence of higher cytochrome c oxidase activity in redmitochondria may reflect that behavior. However, it must betaken into account that not only the CCO-dependent path-way but also the cyanide-resistant, alternative respirationpathway may be contributing to total respiratory activity inboth fruits.

2.2. Antioxidant enzymes in fruit mitochondria

Mitochondria are one of the major organelles that pro-duce activated oxygen species (AOS), and the main target ofAOS-induced damage, as observed in various stress situa-tions, including chilling, senescence and salt-stress[12,19,25,28]. During ripening strong oxidative damagetakes place, and in this situation the mitochondrial antioxi-dant systems could help in limiting the increase in activatedoxygen species. Thus, and as a first step, we measured thechanges in some of the components of the antioxidantsystem including the ASC–GSH cycle as well as superoxidedismutase enzyme in the mitochondria isolated from greenand red pepper fruits (Table 3). Similar to that previouslydescribed in mitochondria from pea leaves [18] and in thoseisolated from leaves and roots of different tomato species

[27], all the enzymes involved in that cycle were found inmitochondria isolated from both ripening stages of thefruits, together with Mn-SOD, the mitochondrial SODisoenzyme [18,22]. The specific activities of the enzymes,APX and Mn-SOD, were found to be lower (about 3-fold)in mitochondria from green peppers than in red fruits,whereas the other enzymes (MDHAR, DHAR and GR)were higher in the green fruit mitochondria, (about 1.5, 1.9and 3.2-fold, respectively). In both, green and red fruitmitochondria, MDHAR specific activity seems to be impor-tant in regenerating mitochondrial ascorbate, while DHARand GR have a minor role in this respect. This moreimportant role of MDHAR activity compared to DHARactivity was clearer in red fruits, in which its specificactivity was three times higher than that of DHAR. More-over, the much lower activity of GR in red pepper mito-chondria, further supports this suggestion. These resultscontrast with the situation in mitochondria from pea leaves,where DHAR activity was higher than MDHAR [14]. Anincrease of APX, GR and SOD during the chloroplast tochromoplast transition has previously been described inpepper fruits [35]. In the case of APX, it was found thatdifferent isoforms appeared or disappeared in the total fruitextract during the transition, implying the existence ofisoenzymes that are involved in the ripening process indifferent organelles [35]. More especially, a comparison ofthe SOD patterns obtained from chloroplasts and chro-moplasts points to significant differences, mainly due to amarked increase in one chloroplast APX isoenzyme. Thus,the higher level of APX activity in mitochondria from redpepper fruits agrees with the pattern described for thisenzyme in chloroplasts and could also contribute to thedifferences found in APX activity of total fruit extracts ofpepper fruits during ripening [35]. Changes in SODisozymes have also been reported during the tomato fruitripening [24,30] in which the plastidial SOD accumulatesduring chromoplast formation [24].

2.3. Ascorbate metabolism in fruit mitochondria

Analysis of the ascorbate content in mitochondria (Table4) revealed that the total amount of ascorbic acid detected inthe mitochondria was similar in both green and red fruitsand that there was no DHA detectable in the organelles. Thehigh MDHAR activity in mitochondria from both fruitscould be responsible for the prevention of the accumulationof DHA in these organelles. In pea leaf mitochondria, which

Table 2Specific activity of Cytochrome c oxidase in crude extract and in isolatedmitochondria from green and red pepper fruits. Data are the mean ± S.E. ofthree different experiments

CCO activity(nkatals mg–1 prot.)

Purification index

Green pepperCrude extract 0.92 ± 0.14 1Mitochondria 51.7 ± 7.5 56

Red pepperCrude extract 0.86 ± 0.14 1Mitochondria 19.3 ± 2.6 22

Table 3Specific activity of the ascorbate–glutathione cycle enzymes and Mn-superoxide dismutase in mitochondria isolated from green and red pepper fruits. Dataare the mean ± S.E. of four different experiments and asterisks indicate statistically significant differences between green and red pepper fruits at the levelof P < 0.05 *; P < 0.01 **

(nkatals mg–1 prot.) (U mg–1 prot.)

APX MDHAR DHAR GR Mn-SOD

Mitochondria of green pepper 7.94 ± 0.42 4.37 ± 0.42 1.75 ± 0.22 2.85 ± 0.87 102.7 ± 8.9Mitochondria of red pepper 23.21 ± 2.36 ** 2.94 ± 0.46 * 0.92 ± 0.25 * 0.89 ± 0.23 * 337.0 ± 22.3 (**)

A. Jiménez et al. / Plant Physiol. Biochem. 40 (2002) 515–520 517

contain relatively high DHAR activity, the DHA contentwas found to represent up to 8–10% of the total mitochon-drial ascorbate [18]. In addition, the activity of GLDH wasfound not to be significantly different in mitochondria fromthe green and red fruits (Table 4). This enzyme, which islocated in the inner mitochondrial membrane, is involved inthe last step of ascorbate synthesis in mitochondria anddonates electrons to the electron transport chain betweencomplex III and IV [3,37]. The maintenance of GLDHactivity levels in the mitochondria from both green and redfruits suggests that ascorbate biosynthesis does not decreaseas fruit mature and that this activity contributes to maintaina similar ascorbate level in the mitochondria from bothfruits. However, the level of reduced ascorbate in red peppermitochondria was similar to that in green despite thespecific APX activity and APX/MDHAR ratio in red fruits,being about three- and eight-fold higher than those observedin green fruit mitochondria (Table 3). High mitochondrialAPX activity in red fruits may have the effect of allowingincreased scavenging of H2O2, which, together with thehigh mitochondrial Mn-SOD activity, could contribute toprevent AOS accumulation in the mitochondria. However,high APX activity would also imply higher ASC consump-tion, which would favor a lower accumulation of reducedascorbate whether or not an efficient regenerating and/orsynthesizing system were operating. Moreover, it is worthnoting that a regulatory role of ASC on APX activity hasalso been reported [1,8]. Thus, the results suggest thatGLDH is present in excess of requirements in both fruitsand that neither the availability of its substrate nor thereduction state of the cytochrome c, its physiologicalelectron acceptor [3,38], limit its activity, despite thedecreased cytochrome c oxidase activity in red fruits com-pared with that in green fruit mitochondria. However, thelack of detectable DHA and particularly the absence of anyincrease in DHA in mitochondria of red fruits suggest somespeculative possibilities.

It could also be suggested that together with its regen-eration to ascorbate inside mitochondria, a system for theexport of DHA to the cytosol, where it would then beregenerated, is operating in mitochondria. Various ascorbatetransporters have been described in plant cell membranesbut so far little is known about the presence of an ascorbatetransport in plant mitochondria [15]. It has recently beensuggested that, as in the case of other low-molecular-masssolutes that can cross the outer membrane, ascorbate canleave mitochondria without the need for a carrier [38], butthis point needs further investigation. In addition to this

possibility, a different ASC recycling pathway could beoperating in pepper fruit mitochondria [40]. In fact, likeferredoxin, NADPH and reduced glutathione, thioredoxincan act as an intermediate in re-reducing the oxidized formsof ascorbate [1,11]. In plant mitochondria, two thioredoxinsand also an NADPH-thioredoxin reductase have been iden-tified [2,23]. Nothing is yet known about the role of thisenzyme system in plant mitochondria but a function acti-vating cytrate-synthase and AOX, one of the first lines ofdefence against AOS, has been proposed [23]. Among othersituations, an enhanced alternative pathway activity hasbeen observed during fruit ripening [21]. It could be veryinteresting to identify the presence and function of athiorredoxin system in mitochondria from pepper fruits.

The expression of a number of antioxidant enzymesincluding SOD, GR and APX was found to increase inconditions that favor the enhanced production of activeoxygen species [1,12,33]. In fact, a substantial induction ofmitochondrial Mn-SOD and APX has been reported inNaCl-stress conditions when the H2O2 content of themitochondria also increased [12,16]. One may assume thatin red fruit mitochondria, the generation rate of O2

–• andH2O2 is higher than in mitochondria from green fruits. Theinduction of the antioxidative Mn-SOD and APX activitieswith no parallel decrease in ASC content, may allow themitochondria to function during ripening, because they areable to respond to increased activated-oxygen productionwith the increased synthesis of antioxidant systems thatcould partly counteract the accumulation of activated oxy-gen species. This pattern also suggests a function forMn-SOD and APX in the ripening process and is verydifferent from that found in mitochondria from dark senes-cent pea leaves, in which the peroxisomes were able tofunction longer than mitochondria in the oxidative mecha-nism of leaf senescence [9,19].

3. Conclusion

All the enzymes of the ascorbate–glutathione cycle havebeen detected in mitochondria from green and red pepperfruits. A function for Mn-SOD and APX activities in theripening process is suggested and MDHAR seems to beimportant in regenerating mitochondrial ascorbate. Themaintenance of mitochondrial GLDH activity in both fruitssuggests that ascorbate biosynthesis does not decrease asfruit mature, and that this activity contributes to maintainthe similar ascorbate content detected in mitochondria from

Table 4Reduced ascorbate content and L-galactono-γ-lactone dehydrogenase activity in mitochondria isolated from green and red pepper fruits. Data are themean ± S.E. of at least three independent experiments. ns, not significant difference between green and red pepper mitochondria values

Proteins ASC GLDH

(mg ml–1) (nmol mg–1 prot.) Total (nmol) (nkatals mg–1 prot.)

Mitochondria of green pepper 1.6 ± 0.4 1.5 ± 0.1 1.2 ± 0.2 0.39 ± 0.03Mitochondria of red pepper 1.3 ± 0.3 ns 1.5 ± 0.3 ns 1.0 ± 0.2 ns 0.33 ± 0.02 ns

518 A. Jiménez et al. / Plant Physiol. Biochem. 40 (2002) 515–520

both fruits. However, the lack of detectable DHA in themitochondria of both fruits and particularly the absence ofany increase in DHA in mitochondria of red fruits suggestssome possibilities: the presence of a system for the export ofDHA to the cytosol and/or a different ASC recyclingpathway operating in pepper fruit mitochondria.

4. Methods

4.1. Plant material

California pepper (Capsicum annuum L., var Wonders)fruits were hand harvested at green and red commercialripening stages and selected for uniformity of size andshape, and freedom of defects.

4.2. Purification of cell organelles

Mitochondria were isolated from pepper fruits by differ-ential and density-gradient centrifugation, as described byRahman and Huber [34] with minor modifications: afterhomogenizing the fruit pericarp, centrifugation at 1000 × geliminates most of chloroplasts and chromoplasts. Thesupernatant was centrifuged at 16 000 × g to precipitate themitochondria, and after a careful resuspension of the pellet,a further centrifugation step at 1000 × g to eliminate evenmore of the contaminating chloroplasts or chromoplasts wascarried out. The mitochondria were sedimented from thesupernatant by centrifuging at 16 000 × g and the precipitatewas then layered onto a percoll gradient to finally separatethe mitochondria from other organelles, centrifuging at26 000 × g for 15 min in a Beckman ultracentrifuge using a50.2Ti rotor. After that, mitochondria were collected foranalysis by using a syringe or by fractioning the gradient.When the mitochondial band was taken directly it waswashed at least twice, as described by Rahman and Huber[34]. For studies of APX activity, an independent organelleisolation procedure was used, with the difference that20 mM Na-ascorbate were added to the extraction mediumand that all the other solutions contained 2 mM ascorbate toprevent possible inactivation of APX.

4.3. Enzyme assays

Unless otherwise indicated, the activities of all enzymeswere assayed in organelle samples diluted two- to five-foldwith 50 mM potassium phosphate buffer (pH 7.8), contain-ing 0.1% (v/v) Triton X-100. All the enzymatic activitycomponents of the ascorbate–glutathione cycle and thesuperoxide dismutase activity (SOD, EC 1.15.1.1) wereassayed as described by Jiménez et al. [18]. L-galactono-γ-lactone dehydrogenase (GLDH, EC 1.3.2.3) activity wasassayed as described by Siendones et al. [37]. The activitiesof hydroxypyruvate reductase (OHPR, EC 1.1.1.29) andcytochrome c oxidase (CCO, EC 1.9.3.1) were used as

markers of peroxisomes and mitochondria, respectively[18]. Triose 3-phosphate isomerase (TPI, EC 5.3.1.1) activ-ity present in chloroplasts and cytosol [36] was also assayed[10].

4.4. Determination of ascorbate

Ascorbate was extracted from purified mitochondria bymixing the samples with an equal volume of 10%m-phosphoric acid and incubating for 30 min. The mixturewas diluted with distilled water to give a final concentrationof 2% m-phosphoric acid and centrifuged at 12 000 × g for10 min [18]. ASC and DHA levels in the supernatant weredetermined by HPLC as described by Castillo and Greppin[6].

4.5. Other analytical methods

Chlorophyll and proteins were quantified as described byHernández et al. [14].

4.6. Statistical analysis

Data were analyzed and compared by Student’s t-test. Allthe changes described were relatives to the values found inthe mitochondria isolated from green pepper fruits.

Acknowledgements

The authors are grateful to D. Lapaz for technicalassistance. This work was supported by grants DGES: PB98-0493-C02-02, FEDER: IFD97-0889-C02-01 and SEN-ECA Foundation (through a grant to A Jiménez).

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