Comparison of mitochondrial ascorbate peroxidase in the cultivated tomato, Lycopersicon esculentum, and its wild, salt-tolerant relative, L. pennellii- a role for matrix isoforms in

Plant, Cell and Environment

(2004)

27

, 237–250

© 2004 Blackwell Publishing Ltd

237

Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2004? 2004

272237250Original Article

Mitochondrial ascorbate peroxidase isoforms in tomatoV. Mittova

et al.

Correspondence: Frederica L. Theodoulou. Fax: +44 1582 763010;e-mail: [email protected]

Comparison of mitochondrial ascorbate peroxidase in the cultivated tomato,

Lycopersicon esculentum

, and its wild, salt-tolerant relative,

L. pennellii

– a role for matrix isoforms in protection against oxidative damage

V. MITTOVA

1

, F. L. THEODOULOU

2

, G. KIDDLE

2

, M. VOLOKITA

1

, M. TAL

3

, C. H. FOYER

2

& M. GUY

1

1

Albert Katz Department of Drylands Biotechnologies, Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Sede Boqer Campus 84990, Israel,

2

Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK and

3

Department of Life Science, Ben-Gurion University of the Negev, PO Box 653, Beer Sheva 84105, Israel

ABSTRACT

Mitochondria require robust antioxidant defences to pre-vent lipid peroxidation and to protect tricarboxylic acidcycle enzymes from oxidative damage. Mitochondria fromwild, salt-tolerant tomato,

Lycopersicon pennellii

(Lpa) didnot exhibit lipid peroxidation in response to high salinity(100 m

M

NaCl), whereas those isolated from cultivatedtomato,

L. esculentum

(Lem), accumulated malondialde-hyde. The activity, intraorganellar distribution and saltresponse of mitochondrial ascorbate peroxidase (mAPX)differed dramatically in the two species. In Lem mitochon-dria, the majority (84%) of mAPX was associated withmembranes, being located either on the inner membrane,facing the intermembrane space, or on the outer membrane.Total mAPX activity did not increase substantially inresponse to salt, although the proportion of matrix APXincreased. In contrast, 61% of Lpa mAPX activity wassoluble in the matrix, the remainder being bound to thematrix face of the inner membrane. Salt treatment increasedthe activity of all mAPX isoforms in Lpa, without alteringtheir intramitochondrial distribution. The membrane-bound isoforms were detected in mitochondria of both spe-cies by western blotting and found to be induced by salt inLpa. These observations suggest that matrix-associatedAPX isoforms could act in concert with other mitochondrialantioxidants to protect against salt-induced oxidative stress.

Key-words

: active oxygen species; H

2

O

2

; lipid peroxidation;oxidative stress; salinity.

Abbreviations

: AA, ascorbic acid; AOS, active oxygen spe-cies; APX, ascorbate peroxidase; CAT, catalase; CCO,cytochrome

c

oxidase; DHA, dehydroascorbate; EST,expressed sequence tag; GL, galactono-

g

-lactone; GLDH,galactono-

g

-lactone dehydrogenase; GPX, glutathione per-

oxidase; HEPES, [

N

-(2-hydroxyethyl)piperazine-

N

¢

-(2-ethanesulfonic acid)]; Lem,

Lycopersicon esculentum

; Lpa,

Lycopersicon pennellii

; mAPX, mitochondrial APX; MDA,malondialdehyde; MDHA, monodehydroascorbate; Mn-SOD, manganese-dependent superoxide dismutase; TBS,Tris buffered saline.

INTRODUCTION

Until recently, the role of mitochondria as a source of activeoxygen species (AOS) in plant cells was largely unexplored.However, it is now widely accepted that superoxide radicalsproduced during respiration make an important contribu-tion to the oxidative load experienced by the whole plantcell (Møller 2001). Respiratory complexes I and III arereported to be major sites of mitochondrial superoxide rad-ical production (Møller 2001); impaired complex I functionin the CMSII mutant, for example, results in specificchanges in antioxidant gene expression and alters antioxi-dant enzyme activities in the cytosol, peroxisomes and chlo-roplasts as well as in mitochondria (Dutilleul

et al

. 2003).The concept that enhanced activities of key antioxidantenzymes in the mitochondrial matrix are instrumental insalt tolerance was explored in the study described in thismanuscript. This hypothesis is based on observations thatmitochondrial AOS production is increased as a result ofhigh salinity (Hernández

et al

. 1993; Gómez

et al

. 1999) asconstraints are imposed on electron transport throughmitochondrial complexes I and II (Hamilton & Heckathorn2001).

Mitochondria contain three so-called ‘first line’ defenceswhich act to limit production of AOS by the electron trans-port chain: the alternative oxidase, internal NADH dehy-drogenase and uncoupling protein (Møller 2001). If theseavoidance mechanisms are overwhelmed, antioxidantenzymes are required to detoxify AOS since several keymitochondrial proteins are especially sensitive to oxidativedamage (Sweetlove

et al

. 2002; Taylor, Day & Millar 2002),either to direct inhibition by AOS (Verniquet

et al

. 1991) or

238

V. Mittova

et al

.

© 2004 Blackwell Publishing Ltd,

Plant, Cell and Environment,

27,

237–250

to modification by lipid peroxidation products (Millar &Leaver 2000).

The titre of AOS in any cellular compartment is governedby the antioxidant system, comprising the ascorbate–glu-tathione cycle, superoxide dismutase and a number of otherantioxidant enzymes (Noctor & Foyer 1998). Both AOSand antioxidants are involved in redox signal transduction(Foyer & Noctor 2003) since both act as sensors of theoxidative load on plant cells. Relatively little is knownabout the intrinsic complement of antioxidants in plantmitochondria or about how mitochondrial antioxidantsrespond to stress, although it is of note that ascorbate issynthesized in the mitochondria via the action of galactono-

g

-lactone dehydrogenase, an enzyme associated with theinner mitochondrial membrane and regulated by electrontransport (Ôba

et al

. 1995; Siendones

et al

. 1999; Bartoli,Pastori & Foyer 2000; Millar

et al

. 2003).It is now well established that superoxide formed via

leakage of electrons from complexes I and III is rapidlydismuted to H

2

O

2

by matrix Mn-superoxide dismutase(Bowler

et al

. 1991). H

2

O

2

is a potent inhibitor of aconitasein plants (Verniquet

et al

. 1991) and has also been shownto damage other tricarboxylic acid (TCA) cycle compo-nents in mammals (Tretter & Adam-Vizi 2000; Nulton-Per-sson & Szweda 2001). Furthermore, H

2

O

2

removal isrequired to prevent formation of the highly toxic hydroxylradical. The mechanisms of H

2

O

2

removal in plant mito-chondria are still not well characterized. In plant cells, H

2

O

2

is scavenged by ascorbate peroxidases and catalases (Noc-tor & Foyer 1998). Catalases are largely restricted to theperoxisomes while the ascorbate peroxidase (APX) familyof isoforms maintains H

2

O

2

contents at non-toxic concen-trations in all of the other compartments of the cell. Therole of other H

2

O

2

-utilizing enzymes, which have muchlower flux rates, is less clear. In mammalian mitochondria,H

2

O

2

detoxification is undertaken by the glutathione per-oxidases (GPX; Huang & Philbert 1996). Although severalGPX homologues have been identified in plants, they arerelatively unimportant in H

2

O

2

detoxification. Moreover,these enzymes have only been localized in chloroplasts todate, although a cytosolic location is also probable (Churin,Schillings & Borner 1999). Non-specific peroxidase activityhas been detected in mitochondria (Prasad, Anderson &Stewart 1995) but this is not considered to fulfil a role inH

2

O

2

detoxification. Components of the peroxiredoxin/thioredoxin/thioredoxin reductase system have been iden-tified in plant mitochondria recently (Laloi

et al

. 2001;Sweetlove

et al

. 2002), but it is probable that these operatein redox signalling, rather than in the detoxification of AOS(Møller 2001; Foyer & Noctor 2003).

To date, APX isoforms have been well characterized inchloroplasts, microbodies and cytosol (Chen & Asada 1989;Shigeoka

et al

. 2002). Chloroplasts contain thylakoidbound, lumen and stroma soluble forms of APX (Kie-selbach

et al

. 2000; Shigeoka

et al

. 2002) and other formsare associated with the membranes of glyoxysomes and leafperoxisomes (Yamaguchi, Mori & Nishimura 1995; Zhang

et al

. 1997). The isoforms are encoded by distinct genes and

differ in size, specificity for electron donor, and sensitivityto inactivation (Chen & Asada 1989; Yoshimura

et al

.1998).

There have been a number of recent reports indicatingthat APX activity is present in mitochondria. A completeascorbate–glutathione enzyme cycle and membrane-boundAPX isoforms have been detected in leaf mitochondria ofpea (Jiménez

et al

. 1997, 1998a) and tomato (Mittova

et al

.2000). Pea leaf mitochondrial APX was located on theintermitochondrial face of the inner membrane or on theouter bounding membrane (Jiménez

et al

. 1998a, b). APXhas also been demonstrated in legume nodule mitochon-dria (Iturbe-Ormaetxe

et al

. 2001). In this case, APX waslocated on the inner membrane, with lesser amountsdetected in the matrix. A mitochondrial APX isoform withan apparent molecular mass of 31 kDa was recently puri-fied to homogeneity from potato tuber mitochondria (DeLeonardis, Diperro & Diperro 2000).

In a number of previous studies, we have explored themetabolic basis for salt tolerance in the wild salt-tolerant

Lycopersicon pennellii

(Lpa), compared with the relativesalt sensitivity in the cultivated tomato,

L. esculentum

(Lem) (Shalata

et al

. 2001; Mittova

et al

. 2002a, b, 2003).This work demonstrated that activities of all the ascorbate-glutathione cycle enzymes (including APX) are detectablein leaf and root mitochondria from both species (Mittova

et al

. 2000) and that salt-induced increases in the activity ofAPX and other antioxidative enzymes in the mitochondriawere associated with salt tolerance and the prevention ofsalt-induced oxidative damage in Lpa (Mittova

et al

. 2003).The results obtained in the present study elucidate the dif-ferential effects of salt stress on mitochondrial APX iso-forms in the two tomato species and indicate for the firsttime that mitochondrial matrix APX isoforms may play arole in the protection of these important organelles againstthe harmful effects of AOS caused by high salinity.

MATERIALS AND METHODS

Plant material

Plants of the cultivated tomato species

Lycopersicon escu-lentum

Mill. cv. M82 (Lem) and its wild relative

Lycopersi-con pennellii

acc. Atico (Lpa) were grown in a greenhousewith day/night temperatures of 20 to 10

∞

C (winter); 30 to20

∞

C (summer) and light intensity, at noon time, rangingfrom 400 (winter) to 1000 (summer)

m

mol m

-

2

s

-

1

. Theplants were grown hydroponically, with six plants per con-tainer of 8 L aerated Hoagland solution. Salt treatmentstarted at the stage of about four true leaves by increasingthe NaCl concentration by 25 m

M

per day to a final concen-tration of 100 m

M

. Plants were analysed after 14 d at100 m

M

NaCl.

Assay for lipid peroxidation

Lipid peroxidation was determined as the amount of mal-ondialdehyde (MDA,

e

= 155 m

M

-

1

cm

-

1

), a product of lipid

Mitochondrial ascorbate peroxidase isoforms in tomato

239

© 2004 Blackwell Publishing Ltd,

Plant, Cell and Environment,

27,

237–250

peroxidation, according to (Draper & Hadley 1990). Inorder to reduce reaction of thiobarbituric acid with oxi-dized products of amino acids, including proline whichaccumulate in response to salt stress (Gutteridge & Halli-well 1990; Zhang & Blumwald 2001), MDA content wasdetermined in the pellets (150 000

g

, 40 min) of osmoticallyshocked mitochondria. The means

±

SD of three indepen-dent experiments were presented. Differences betweentreatments and control values were significant according toStudent’s

t

-test at

P

<

0.001.

Purification of organelles

Organelles were isolated from fully expanded leaves ofcontrol and salt-stressed plants (14 d after the completionof salt treatment) by differential and density gradient cen-trifugation as described in (Mittova

et al

. 2000). Briefly,leaves and roots (20 g each) were chopped with 5 volumesof medium per g FW using a Wareing blender in a mediumcontaining 50 m

M

HEPES [

N

-(2-hydroxyethyl)piperazine-

N

¢

-(2-ethanesulfonic acid); pH 7.5], 5 m

M

g

-caproic acid,30 m

M

sodium ascorbate, 0.3% bovine serum albumin(BSA) (w/v), 0.4

M

sucrose, 10 m

M

NaCl, 10 m

M

mercapto-ethanol, 2 m

M

ethylenediaminetetraacetic acid (EDTA)and 1% (w/v) polyvinylpyrrolidine. The homogenates werefiltered through two layers of Miracloth (Calbiochem, LaJolla, CA, USA) (‘crude extract’). The crude chloroplastfraction from leaves was sedimented by centrifugation at370

g

for 10 min, using a Sorvall HB4 rotor. The crudechloroplast fraction was purified by a 10 to 80% Percollgradient in the presence of ascorbate, according to (Bar-tlett, Grossman & Chua 1982).

The 370

g

supernatant was re-centrifuged at 12 000

g

for15 min and the pellet collected and re-suspended in: 20 m

M

HEPES-KOH, pH 7.5, 5 m

M

sodium ascorbate, 330 m

M

sorbitol, 10 m

M

NaCl, 2 m

M

EDTA. 0.6–0.9 mL of theresulting mitochondria- and peroxisome-enriched samplewas fractionated on a 25 to 57% (w/w) sucrose gradient at68 000

g

in a Beckman SW-41 rotor for 3.5 h. Ascorbate(5 m

M

) was added to the sucrose density gradient. Fractionsof 0.5 mL were collected from the bottom of the gradientand analysed for the respective enzyme marker activities.The fraction showing the highest activity of the mitochon-drial or peroxisomal marker enzymes and the two fractionsadjacent to the peak from both sides were collected andpooled for further study.

Enzyme assays

APX activity (EC 1.11.1.11) was determined according to(Jiménez

et al

. 1997) and corrections were made for low,non-enzymatic oxidation of ascorbate by H

2

O

2

. Catalaseactivity (CAT; EC 1.11.1.6) was determined according toRao, Paliyath & Ormrod (1996); cytochrome

c

oxidase(CCO; EC 1.9.3.1) according to Schwitzguebel & Siegent-haler (1984). Chlorophyll was assayed according to Arnon(1949) and protein according to Bradford (1976) using BSAas standard.

Enzyme latency determination

Intactness of mitochondria was determined by measuringthe latency of cytochrome c oxidoreductase activity inintact mitochondria as compared with that of 0.02% (v/v)Triton X-100-treated mitochondria (Schwitzguebel &Siegenthaler 1984). APX latency in mitochondria wasdetermined by measuring its activity in mitochondria sus-pended in APX assay media supplemented with 0.3 M man-nitol with or without 0.02% (v/v) Triton X-100. The latencywas calculated according to the formula: [100 - (activityassociated with intact organelle/activity associated withpermeabilized organelle) ¥ 100].

Preparation of mitochondrial membrane and soluble fractions

Sucrose density gradient-purified mitochondria wereloaded on Sephadex G-25 columns (1 mL; Sigma-Aldrich,Poole, Dorset, UK), pre-equilibrated with 20 mM HEPES-KOH buffer (pH 7.2). The void volume of each column wascollected by centrifugation (700 g for 5 min). This proce-dure caused an osmotic shock to the mitochondria asjudged by the loss of their intactness. The recovery of mito-chondrial enzymes was estimated by determination of CCOactivity before and after the gel filtration step. The Sepha-dex G-25-eluted mitochondrial protein was re-suspended(1 : 2 v/v) in a medium containing 50 mM HEPES-KOHbuffer (pH 7.2), 1 mM EDTA, 2 mM ascorbate and 10% (v/v) glycerol. The preparations were kept on ice for 40 min,vortexed every 10 min and then centrifuged at 150 000 g for30 min in a Kontron TBF 80.2 rotor. The pellets (mem-branes) were then separated into two and re-suspended inthe above buffer with/without 0.2 M KI. The same proce-dure was repeated for membranes re-suspended in KI-supplemented buffer. Both the supernatants and mem-branes were assayed for APX activity and protein content.The percentage of APX activity in the mitochondrial mem-brane or soluble (matrix) fractions was calculated by divid-ing the recovered APX activity in each fraction by the totalactivity in the lysed mitochondrial preparation.

Triton X-114 partitioning to separate integral and peripheral membrane proteins

Mitochondrial membrane fractions were prepared asdescribed above and re-suspended in a solution containing10 mM Tris-HCl pH 7, 0.3 M sucrose. Samples were diluted100 times with 0.1 M Na2CO3, pH 11, incubated on ice for30 min and centrifuged at 15 000 g for 1 h. The supernatantwas precipitated with 5 volumes of acetone at -20 ∞C andcentrifuged at 10 000 g for 15 min. The pellet was re-sus-pended in a solution containing 2% (v/v) Triton X-114 in10 mM Tris-HCl pH 7.6, 150 mM NaCl and incubated on icefor 15 min. Samples were centrifuged at 10 000 g for 10 minand the supernatant was incubated at 37 ∞C for 5 min. Fol-lowing centrifugation at 1000 g for 10 min, the upper, deter-gent-depleted and lower, detergent-enriched phases were

240 V. Mittova et al.

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 237–250

collected separately and used for sodium dodecyl sulphate(SDS)-polyacrylamide gel electrophoresis (PAGE).

Native gel electrophoresis and APX activity staining

Mitochondria were osmotically disrupted in medium con-taining 50 mM HEPES, pH 7.2, 1 mM EDTA and 2 mM

ascorbate. Protein samples were separated on 10% non-denaturing polyacrylamide gels (Laemmli 1970). APX wasdetected according to Mittler & Zilinskas (1993). The den-sitometric analysis was made using the National Institutesof Health (NIH; Bethesda, MD, USA) IMAGE program.The active bands were excised from the gel, re-suspendedin 50 mL 0.125 M Tris pH 6.8, 0.1% (w/v) SDS, incubated at37 ∞C overnight and centrifuged at 10 000 g for 15 min. Thesupernatant was mixed (1 : 1) with sample buffer contain-ing 62.5 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 5% (v/v) b-mercaptoethanol, 10% (v/v) glycerol, 0.002% (w/v)bromophenol blue, denatured at 95 ∞C for 4 min and usedfor SDS-PAGE and western blotting (see below).

Preparation of APX antiserum

Protein sequences of cytosolic, peroxisomal and plastidicAPX were aligned using the Genetics Computer Group(GCG) program PILEUP, permitting identification ofconserved peptide sequences conserved in cytosolic butabsent from plastidic APX isoforms. Sequences wereassessed for hydrophilicity, presence of beta-turns and easeof peptide synthesis. Following selection based on thesecriteria, a peptide corresponding to the N-terminus ofmaize cAPX (Q41772) (6-PTVNEDYLKAVDKAKRKL-RGLIAEKNCA-33) was synthesized and coupled to key-hole limit haemocyanin by Sigma Genosys (Pampisford,Cambs., UK) and used to immunize rabbits. This work wascarried out in accordance with the Animals (Scientific Pro-cedures) Act 1986, Project Licence No. PPL 70/4356.

SDS-PAGE and western blotting

Protein samples were denatured at 95 ∞C for 4 min in samplebuffer, containing 62.5 mM Tris-HCl (pH 6.8), 2% (w/v)SDS, 5% (v/v) b-mercaptoethanol, 10% (v/v) glycerol,0.002% (w/v) bromophenol blue. Following electrophoresisin 12% denaturing polyacrylamide gels, proteins wereelectro-transferred to polyvinylidene fluoride (PVDF;Immobilon-P; Millipore Co. Bedford, MA, USA) or nitro-cellulose membranes (Hybond C extra; Amersham Phar-macia Biotech, Little Chalfont, Bucks., UK) at 100 V for1.5 h. Blots were blocked for 24 h at 4 ∞C in 5% (w/v) BSAdissolved in Tris-buffered saline (TBS) and then incubated(24 h, room temperature) with polyclonal antibodies raisedagainst maize cytosolic APX. The antibodies were diluted1 : 5000 for crude extract preparations and 1 : 3000 fororganellar preparations. The membranes were washed fivetimes with TBS over a 12-h period, then incubated (1 h,room temperature) with horseradish peroxidase-conjugated

secondary antibody diluted 1 : 50 000 and washed six times(10 min each in TBS at room temperature), and developedusing ECL substrate (Amersham-Pharmacia Biotech),according to the manufacturers’ instructions. The intensityof the decorated bands was calculated using the SIGMAGEL

program (SPSS Science, Chicago, IL, USA).

Data mining and sequence analysis

The tomato gene index (Quackenbush et al. 2001) wassearched for APX sequences using BLAST and gene productname searches. Tentative consensus sequences were exam-ined for open reading frames, and protein sequences encod-ing APX isoforms were generated using the GCG program,TRANSLATE. Subcellular location was predicted using fourprograms: TARGETP (Emanuelsson et al. 2000), PSORT

(Nakai & Kanehisa 1992), PREDOTAR and MITOPROT

(Claros & Vincens 1996) and by homology comparisonswith APX isoforms for which the subcellular location hasbeen experimentally determined. Sequences were exam-ined manually for determinants required for targeting ofperoxisomal APX (Mullen & Trelease 2000). The predictedpeptide sequence was compared with that of the peptideused to raise the antiserum, using the GCG program, BEST-

FIT. For full-length sequences, the theoretical Mr was cal-culated using the GCG program, PEPTIDESORT.

RESULTS

Lipid peroxidation in response to salt stress

The effect of salt stress on mitochondrial lipid peroxidationin the relatively salt-sensitive L. esculentum (Lem) and thesalt-tolerant L. pennellii (Lpa) tomato species was assessedby measurement of MDA. Treatment with 100 mM NaClover 14 d of growth increased MDA content in mitochon-dria of Lem from 22.2 ± 2.4 to 44.3 ± 1.6 nmol mg-1 protein.In contrast, the MDA content of Lpa mitochondria wasreduced from 30.9 ± 4.4 nmol mg-1 protein in controls to22.8 ± 2.0 nmol mg-1 protein following salt-treatment. Val-ues represent the means of three independent experiments±SD and differences from controls were significant(according to Student’s t-test) at P < 0.01. We were inter-ested in investigating the molecular mechanisms preventingsalt-induced lipid peroxidation in Lpa. Since a preliminarystudy revealed that APX activity was increased followingsalt treatment of Lpa (Mittova et al. 2003) we decided toconduct a detailed characterization of mitochondrial APXin response to salt stress in the two tomato species.

Localization of APX by organelle fractionation

APX was localized in tomato leaf organelles using differ-ential and density gradient centrifugation. Chloroplastswere removed from crude homogenates by low-speed cen-trifugation, and mitochondria and peroxisomes were sepa-rated by sucrose density gradient centrifugation (Fig. 1).The resultant mitochondrial fraction contained only low

Mitochondrial ascorbate peroxidase isoforms in tomato 241

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 237–250

contamination by peroxisomes, as judged by the presenceof the marker enzyme catalase (10–15%), and thylakoidsas judged by the amount of chlorophyll present (5%;Fig. 1). These values are within an acceptable range forpurified mitochondrial fractions (see Jiménez et al. 1998b).APX activity was present in both peroxisomes and mito-chondria but was considerably higher in mitochondria(Fig. 1), being enhanced substantially in the purified frac-tion (Table 1). This is broadly similar to the increase in the

mitochondrial marker, CCO relative to the crude leafhomogenates (Table 1). Differences in the relative degreeof purification between APX and CCO can be attributed tothe presence of different APX isoforms in organelles (Mit-tova et al. 2000) and the cytosol (Webb & Allen 1995).Following exposure to salt stress, APX activity increased2.5-fold in Lpa leaf mitochondria. In marked contrast, saltstress had no effect on APX activity in isolated Lem leafmitochondria (Table 1).

Figure 1. Distribution of ascorbate per-oxidase (APX) and marker enzyme activi-ties of mitochondria and peroxisomes in sucrose density gradients. The cultivated tomato L. esculentum and the wild species L. pennellii were grown for 14 d under con-trol conditions (closed symbols) and salt stress (open symbols). Enriched mitochon-dria and peroxisome fractions were obtained by differential centrifugation from leaves and were separated further on a linear sucrose-density gradient. Marker enzymes: cytochrome c oxidase (CCO) for mitochondria; catalase for peroxisomes; chloroplast contamination was estimated by chlorophyll. Enzyme activities are expressed as mmol ml-1 min-1. Protein and chlorophyll contents are expressed as mg ml-1.

242 V. Mittova et al.

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 237–250

Location of mitochondrial APX isoforms under normal and salt stress conditions

The distribution of APX in mitochondrial compartmentswas investigated following fractionation by osmotic shockand centrifugation. In Lem, the membrane-enriched pelletfraction contained 84% of the total mitochondrial APXactivity (Table 2). Some (36%) of the bound APX wasreleased in the presence of chaotropic agent, indicating thisAPX was only loosely associated with the mitochondrialmembranes. The ratio of soluble to membrane-bound APXactivity was changed in leaves of Lem plants that had beenexposed to salt stress. In this case, the proportion of solubleAPX activity was increased significantly (1.7-fold) in rela-tion to the membrane-bound form. However, the propor-tion of loosely bound APX activity (30%) was similar tountreated controls. The intramitochondrial distribution ofAPX was different in Lpa. Lpa mitochondrial APX waslargely soluble (61%) and no loosely bound APX activitycould be detected. The activities of both the Lpa soluble

and membrane-bound APX isoforms increased signifi-cantly in response to salt stress (Table 2).

Latency experiments confirmed that the mitochondriawere largely intact as demonstrated by measurement ofCCO activity (Table 3). Measurement of NADH cyto-chrome c reductase gave similar values (87–94% intactness;data not shown) and high intactness (90–94%) for glu-tathione reductase activity was demonstrated for identicalmitochondrial preparations in a previous publication (Mit-tova et al. 2000). The latency of APX activity in mitochon-dria isolated from control and salt-treated Lpa plants wasof a similar magnitude to cytochrome c reductase (> 80%;Table 3), indicating that APX is predominantly an enzymeof the mitochondrial matrix in Lpa. In marked contrast,Lem mitochondrial APX showed only a low degree oflatency (15%), indicating that about 85% of the APX wasoutside the matrix compartment and accessible to externalsubstrates. The degree of latency significantly increasedtwo-fold in mitochondria from leaves of Lem plantsexposed to salt stress and correlated with a similar increase

Table 1. Effect of salt stress on activity of ascorbate peroxidase (APX) in mitochondria isolated from leaves of control and salt (100 mM NaCl)-treated plants of the cultivated tomato L. esculentum (Lem) and wild salt-tolerant species L. pennellii (Lpa)

Fraction

Lem Lpa

Control NaCl Control NaCl

CCO APX CCO APX CCO APX CCO APX

Homogenate 0.14 ± 0.01 0.28 ± 0.06 0.12 ± 0.02 0.23 ± 0.03 0.12 ± 0.01 0.24 ± 0.01 0.13 ± 0.01 0.41 ± 0.02Crudemitochondria 0.30 ± 0.02 0.37 ± 0.011 0.25 ± 0.02 0.35 ± 0.04 0.26 ± 0.03 0.24 ± 0.02 0.22 ± 0.02 0.49 ± 0.03Mitochondria 1.68 ± 0.23 1.42 ± 0.38 2.21 ± 0.31 1.64 ± 0.20 1.26 ± 0.14 1.6 ± 0.48 1.15 ± 0.17 4.01 ± 0.53

Enzyme activities are expressed as mmol min-1 mg-1 protein. The results are the means of at least three independent experiments ± SD. CCO,cytochrome c oxidase.

Treatmentand fraction

Osmotic shock Osmotic shock + KI

Specificactivity

% of totalactivity

Specificactivity

% of totalactivity

L. esculentumControl

soluble 0.53 ± 0.07 16 ± 3 1.46 ± 0.54 52 ± 6membrane 2.32 ± 0.16 84 ± 8 1.33 ± 0.15 48 ± 4

NaClsoluble 0.90 ± 0.13 31 ± 3 1.65 ± 0.18 61 ± 6membrane 1.95 ± 0.22 69 ± 7 1.09 ± 0.15 39 ± 4

L. pennelliiControl

soluble 1.58 ± 0.13 61 ± 6 1.65 ± 0.20 64 ± 4membrane 0.98 ± 0.12 39 ± 2 0.91 ± 0.10 36 ± 3

NaClsoluble 3.95 ± 0.34 72 ± 4 4.19 ± 0.51 78 ± 5membrane 1.51 ± 0.17 28 ± 3 1.13 ± 0.12 22 ± 1

APX activity is expressed as mmol min-1 mg-1 protein. Percentage of activity was calculatedas described in Materials and Methods. The results are the means of at least two independentexperiments ± SD.

Table 2. Intraorganellar localization of ascorbate peroxidase (APX) in mitochon-dria isolated from leaves of control and salt (100 mM NaCl)-treated plants of the culti-vated tomato L. esculentum (Lem) and wild, salt-tolerant species L. pennellii (Lpa)

Mitochondrial ascorbate peroxidase isoforms in tomato 243

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 237–250

in total soluble mitochondrial APX activity under theseconditions (Table 3).

The presence of different mitochondrial APX isoformswas investigated further by native gel electrophoresis.

When mitochondria from unstressed Lem and Lpa leaveswere subjected to activity staining following nativePAGE, three bands of APX activity (i, ii and iii) weredetected in Lem and Lpa extracts (Fig. 2). Analysis of the

Table 3. Latency of ascorbate peroxidase (APX) and cytochrome c oxidase (CCO) in mitochondria isolated from leaves of control and salt (100 mM NaCl)-treated plants of the cultivated tomato L. esculentum (Lem) and wild, salt-tolerant species L. pennellii (Lpa)

Lem Lpa

Control NaCl Control NaCl

APX CCO APX CCO APX CCO APX CCO

Mitochondria 1.20 ± 0.20 0.11 ± 0.01 0.85 ± 0.13 0.17 ± 0.02 0.18 ± 0.02 0.18 ± 0.01 0.51 ± 0.02 0.12 ± 2.00Mitochondria + 1.42 ± 0.36 1.75 ± 0.15 1.56 ± 0.21 1.49 ± 0.18 1.44 ± 0.26 1.53 ± 0.11 3.89 ± 0.45 1.29 ± 0.14Triton X-100Latency (%) 15 ± 2 94 ± 10 33 ± 4 89 ± 10 88 ± 10 89 ± 9 84 ± 8 90 ± 10

Enzyme activities are expressed as mmol min-1 mg-1 protein. Latency values were calculated as described in Material and Methods. Theresults are the means of at least three independent experiments ± SD.

Figure 2. Isoenzyme pattern of APX activ-ity in mitochondria isolated from leaves of control and salt (100 mM NaCl)-treated plants of the cultivated tomato L. esculen-tum and wild, salt-tolerant species L. pennel-lii. (a) Total mitochondrial proteins (100 mg) were separated on a non-denaturing poly-acrylamide gel and stained for APX activity. Samples: (–) mitochondria isolated from control plants; (+) mitochondria isolated from salt-stressed plants. (b) densitometric recording of APX staining of mitochondria from control and salt-stressed plants, analy-sed by NIH IMAGE. Activity bands were numbered (i–iii) according to their migra-tion rates.

244 V. Mittova et al.

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 237–250

matrix fraction from control mitochondria revealed thatthe band denoted as APXiii is a soluble matrix form (datanot shown). The bands denoted as APXi and ii are proba-bly membrane-associated since they did not enter thenative gel efficiently (Fig. 2, and see below). Taking intoaccount the fact that enzyme activity measured by gelstaining methods may not always bear a linear quantita-tive relationship to the amount of protein loaded, the fol-lowing observations were made. The relative amount ofAPXiii activity increased in mitochondria from leaves ofboth species following exposure to salt stress (Fig. 2).Under these conditions, the relative activities of the mem-brane-bound APXi and ii decreased in Lem and in con-trast, increased in Lpa. These results are consistent withthe APX measurements in mitochondrial subfractionspresented in Table 2.

Western blot analysis of mitochondrial APX

Western blotting was used to investigate the presence ofAPX isoforms in different subcellular compartments oftomato leaves. A polyclonal antiserum prepared against apeptide from maize cytosolic APX revealed two polypep-tides with estimated molecular mass of 27 and 29 kDa incrude tomato leaf extracts (Fig. 3a). Comparison with pre-viously published work suggested that the 27 kDa bandrepresents a cytosolic protein and the larger protein repre-sents peroxisomal APX (Yamaguchi et al. 1995; Gadea,Conejero & Vera 1999); the identity of the 29 kDa band asperoxisomal APX was subsequently confirmed by fraction-ation (data not shown). A single mitochondrial polypeptideof approximately 34 kDa was detected in the isolated mito-chondrial fraction from leaves of both species (Fig. 3a). Saltstress did not appear to have a marked effect on thispolypeptide in Lem (Fig. 3b). In contrast, the abundance ofthis polypeptide was lower in mitochondria isolated fromcontrol Lpa plants but increased 3.2-fold in response to saltstress (Fig. 3b). Although the chloroplastic APX isoformscomprise the majority of total APX activity in tomato leafcells (Asada 1996; Noctor, Veljovic-Jovanovic & Foyer2000), the antiserum raised against the maize cytosolic APXdid not cross-react with any of the tomato chloroplasticAPX isoforms (Fig. 3b).

In order to identify which of the mitochondrial isoformswere recognized by the antiserum, APX bands were excisedfrom native gels (Fig. 4a) and subjected individually towestern blotting. Putative membrane-bound isoforms i andii reacted with the antiserum, but the matrix isoform iii didnot (Fig. 4b). Blotting of the matrix fraction and of thenative gel gave identical results (not shown). Triton X-114partitioning of a mitochondrial membrane fraction demon-strated that the immunoreactive protein partitioned in thedetergent fraction, thereby confirming that membraneAPX is mainly tightly bound (Fig. 4c). The small proportionof loosely bound APX detected in Lem (Table 2) was eithersufficiently hydrophobic to be detergent soluble or was notof sufficient abundance to be detected in the aqueousfraction.

In silico analysis of tomato APX isoforms

The recognition of mitochondrial membrane-bound APXby the antipeptide antiserum provided a potential startingpoint for its molecular identification. To date, only twotomato APX sequences have been published; these encodecytosolic and chloroplastic isoforms (Table 4; Gadea et al.1999; Kieselbach et al. 2000). A third, full-length proteinsequence (accession number Q8LSK6) is deposited in thedatabase but its intracellular location has not yet beendetermined. To identify other tomato APX genes, thetomato expressed sequence tag (EST) collection (Quack-enbush et al. 2001) was searched for sequences with homol-ogy to APX and cytochrome c peroxidase. The predicted

Figure 3. Western blot analysis of APX in tomato leaf extracts. (a) Crude and mitochondrial fractions were isolated from untreated leaves of the cultivated tomato L. esculentum (Lem) and wild, salt-tolerant species L. pennellii (Lpa). Crude extract (40 mg protein) and mitochondrial protein (mit; 50 mg protein) were loaded onto 10% (w/v) denaturing acrylamide gels, transferred to nitro-cellulose membranes and probed with antibodies raised to maize cytosolic APX. (b) Chloroplasts and mitochondria were iso-lated from leaves of control and salt (100 mM NaCl)-treated Lem and Lpa plants. Chloroplastic or mitochondrial proteins (30 mg) were loaded onto 10% (w/v) denaturing acrylamide gels, trans-ferred to PVDF membranes and probed with antibodies raised against maize cytosolic APX. Chl, chloroplasts; Mit, mitochondria. The positions of molecular mass standards (kDa) are shown on the left-hand side of the blots.

Mitochondrial ascorbate peroxidase isoforms in tomato 245

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 237–250

protein sequences were subjected to a pair-wise compari-son with the peptide sequence used to raise the antiserum.In addition to comparisons with APX isoforms of knownsubcellular location, computer programs were used to pre-dict the putative locations of the tomato APX isoforms andthe results are presented in Table 4.

Of the tomato APX sequences identified, four encodeproteins which in theory would be strongly recognized bythe maize APX antiserum (> 90% amino acid identity).These proteins include the previously identified cytosolicisoform (Gadea et al. 1999) and two closely related, novelsequences which encode proteins with a calculated molec-ular weight of approximately 27 kDa, consistent with theband observed in western blots of crude and cytosolic frac-tions (Fig. 3). The protein encoded by TC124067 is verysimilar to cytosolic APX but appears to be truncated at the

C-terminus (Table 4). On the basis of sequence compari-sons, two further tomato sequences are also likely to berecognized by the antiserum (50–57% amino acid identityover 28 residues). Homology matching suggests that theserepresent putative peroxisomal APXs.

Of all the tomato APX peptide sequences examined,only Q8LSK6 was consistently predicted to contain a mito-chondrial transit peptide (by TARGETP, PREDOTAR andMITOPROT) and to have a molecular weight of >30 kDa.However, sequence analysis predicted that this proteinwould not be recognized by the antiserum used in the cur-rent study. This putative mitochondrial APX was very sim-ilar to plastidic APX isoforms, with 87 and 82% amino acididentity to tobacco stromal and thylakoid APX, respec-tively. In agreement with this, none of the putative chloro-plast APX sequences was sufficiently similar to the peptideantigen to be recognized by the antiserum, which is consis-tent with our western data for isolated chloroplasts (Fig. 3).

DISCUSSION

Strategies for coping with salt stress in plants, as in animals,involve the deployment of a range of appropriate changesin gene expression, metabolism and physiology. Many ofthese responses are directed at amelioration of the defencesystems that prevent oxidative damage and lipid peroxida-tion. Robust mitochondrial antioxidant defences arerequired to prevent compromise of the TCA cycle and elec-tron transport chain under conditions such as salt stress(Sweetlove et al. 2002). The results presented in this manu-script show that mitochondrial APX activity is induced byhigh salt and suggest that this could have an importantfunction in preventing oxidative stress in the mitochondria.Analysis of APX enzyme activities and protein allowed theresolution of three tomato leaf mitochondrial APX iso-forms. The data obtained in this study allow us to draw thefollowing conclusions.

Tomato mitochondrial APX exists in soluble and membrane-bound forms

In the salt-sensitive tomato species, Lem, a high percentage(84%) of the mitochondrial APX activity was precipitatedby centrifugation following osmotic shock (Table 2) andlatency experiments indicated that the majority (85%) ofLem leaf mitochondrial APX had access to its substrateswhen the organelles were assayed intact (Table 3). Thissuggests that most of the Lem APX protein is anchoredeither on the intermembrane face of the inner membraneor on the outer bounding membrane. The remaining 15%of APX activity is soluble in the matrix. These results areconsistent with previous observations for pea mitochon-drial APX (Jiménez et al. 1998a, b). A different localizationpattern was found for mitochondrial APX in the salt-tolerant tomato species, Lpa. Similar to potato tuber mito-chondrial APX (De Leonardis et al. 2000), the majority ofLpa mitochondrial APX (61%) was soluble in the matrixand the remainder was localized on the matrix face of the

Figure 4. Western blot analysis of APX isoforms. (a) Total mito-chondrial proteins (100 mg) extracted from untreated leaves of L. esculentum (Lem) and L. pennellii (Lpa) were separated in a non-denaturing polyacrylamide gel and stained for APX activity. (b) Individual activity bands (marked i, ii, iii) were excised and pro-teins extracted into 0.125 M Tris-HCl, pH 6.8, 0.1% (w/v) SDS prior to separation in 12% denaturing gels. Proteins were transferred to nitro-cellulose membrane and probed with antiserum raised to maize cytosolic APX. (c) Mitochondrial membrane fractions were subjected to Triton X-114 partitioning and the upper, detergent-depleted and lower, detergent-enriched phases separated and anal-ysed by Western blotting as in (b).

246 V. Mittova et al.

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 237–250

inner membrane. Although H2O2 is relatively mobile, thefunction of each type of APX depends on its compartmen-tation. Our experimentally derived model for the locationof mitochondrial APX isoforms is shown in Fig. 5.

The localization of APX either in or facing the matrix is correlated with defence against the build-up of H2O2 in mitochondria

The amount, location, and regulation of mitochondrialAPX clearly differ between plant species. Salt treatmentaffected mitochondrial APX isoforms differently in Lemand Lpa. In Lem, the total mitochondrial APX activity didnot increase in response to salt (Table 1), however, theintramitochondrial distribution of APX was altered inresponse to salt treatment, with soluble matrix APXincreasing slightly at the expense of membrane-boundforms (Fig. 2; Tables 2 & 3). In contrast, salt treatment sub-stantially increased the activity of all three Lpa mitochon-drial APX isoforms (Table 2, Fig. 2) but did not alter theintramitochondrial distribution of APX (Tables 2 & 3).

The salt-induced increase in mitochondrial APXobserved in Lpa is reminiscent of that reported in salt-stressed pea plants (Gómez et al. 1999). In the latter, how-ever, the increase in mitochondrial APX was restricted tomembrane-associated forms and was apparently insuffi-cient to prevent oxidative damage (Gómez et al. 1999).Lipid peroxidation was decreased in mitochondria from

salt-treated Lpa plants, suggesting that these organelles aremore resistant to salt-dependent oxidative stress than theirLem counterparts, in which lipid peroxidation wasincreased at high salinity. These results are consistent withthe up-regulation of mitochondrial matrix APX in salt-treated Lpa plants as judged by levels of activity (Fig. 2;Tables 2 & 3) and also reflect the up-regulation of a numberof other antioxidant enzymes (Mittova et al. 2003).Although this correlation could be fortuitous, we suggestthat the presence of an inducible APX either in the matrixor facing the mitochondrial matrix would therefore appearto be important in defence against the build-up of H2O2

produced by the action of matrix Mn-SOD. Although sol-uble matrix APX increases slightly following salt treatmentof Lem, the level appears to be insufficient to provide pro-tection against oxidative stress.

Mitochondrial membrane-bound APX is immunologically related to cytosolic and peroxisomal isoforms, but matrix APX is not

As predicted, a polyclonal antiserum raised against a con-served region of cytosolic APX recognized a polypeptideof 27 kDa in crude leaf homogenates. The antiserum alsodetected a 29-kDa protein in these extracts and in purifiedperoxisomes (Fig. 3a). Comparison of the predicted aminoacid sequences of putative tomato peroxisomal APX iso-forms with the peptide sequence used to raise the antiserum

Table 4. Tomato APX sequences

TIGR TC number and/oraccession number Putative locationa Full-length?

Recognized by antibody in silico?b

Predicted Mr

(kDa)c

TC124064 cytosol yes YES 27.3Y16733 Gadea et al. (1999)TC117677 thylakoid lumen yes NO 37.7 (pre)Q9THX6 Kieselbach et al. (2000) 34.1 (mature)TC116683 mitochondrion? yes NO 42.2 (pre)Q8LSK6 38.8 (mature)TC116883 cytosol yes YES 27.5TC124065 cytosol yes YES 27.3TC124066 cytosol? yes? YES 13.3TC124067 cytosol? no NDd

TC116499 peroxisome no Possible (50% id)TC116500 peroxisome no NDd

TC131526 peroxisome no Possible (57% id)TC116680 chloroplast no NOTC116681 chloroplast no NOTC131316 chloroplast no NDd

The tomato gene index (Quackenbush et al. 2001) was searched for APX sequences using BLAST and gene product name searches. Tentativeconsensus (TC) sequences were examined for open reading frames, and protein sequences were generated using the GCG program,TRANSLATE.aPutative location is based on homology with APX isoforms whose locations have been experimentally determined and on analysis oftargeting sequences. Subcellular location was predicted using four programs: TARGETP, PSORT, PREDOTAR and MITOPROT. Sequences wereexamined manually for determinants required for targeting of peroxisomal APX (Mullen & Trelease 2000).bThe predicted peptide sequence was compared with that of the peptide used to raise the antiserum, using the GCG program, BESTFIT.cTheoretical Mr was calculated using the GCG program, PEPTIDESORT. The size of mature protein was calculated after removal of transitpeptide, as predicted by TARGETP.dNot determined; full length cDNA sequence not available.

Mitochondrial ascorbate peroxidase isoforms in tomato 247

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 237–250

provided an explanation for this result (Table 4). The anti-bodies raised against the maize cytosolic APX also detecteda single mitochondrial polypeptide of approximately34 kDa in Western blots (Fig. 3a & b). Detergent partition-ing and western analysis of excised APX activity bandsdemonstrated that this 34 kDa band represented the twomembrane-bound isoforms (Fig. 4). Moreover, the antibod-ies against the maize cytosolic APX did not cross-react withany chloroplastic protein (Fig. 3b), excluding the possibilitythat the 34 kDa polypeptide is a chloroplastic contaminant.

The L. esculentum EST database was analysed in anattempt to identify clones encoding mitochondrial APXisoforms. Many mitochondrial proteins possess a cleavableN-terminal targeting sequence which is required for importand which can be recognized in the primary proteinsequence using bioinformatic tools (Nakai & Kanehisa1992; Claros & Vincens 1996; Emanuelsson et al. 2000).Four such programs: TARGETP, PSORT, PREDOTAR andMITOPROT, were used to predict the putative subcellularlocation of tomato APX proteins, resulting in the identifi-

Figure 5 Antioxidant defences of plant mitochondria in salt tolerant and salt sensitive tomato. Ascorbate (AA) is synthesized from galactono-g-lactone (GL) by the inner membrane-bound flavoprotein, galactono-g-lactone dehydrogenase (GLDH; black oval symbol). AA is exported to the cytosol and imported into the matrix by as yet uncharacterized mechanisms (marked ?). Active oxygen species (AOS) are predominantly generated by Complexes I and III of the electron transport chain. In the absence or failure of first-line protective mechanisms, superoxide (O2

–) which may be produced on both sides of the inner mitochondrial membrane, leads to lipid peroxidation. In non-stress conditions, superoxide is efficiently dismuted to hydrogen peroxide (H2O2) by Mn-superoxide dismutase (Mn-SOD) in the mitochondrial matrix. H2O2 can be converted to the highly toxic hydroxyl radical or exert direct effects. If matrix H2O2 is not detoxified, the TCA cycle is compromised due to inhibition of aconitase. Moreover, the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes of the TCA contain lipoic acid and are especially sensitive to oxidative damage from ROS-generated products of lipid peroxi-dation. NAD-dependent malic enzyme and the glycine decarboxylase complex are also major targets of oxidative damage in mitochondria (not shown). Ascorbate peroxidase constitutes a major route of H2O2 detoxification in mitochondria: H2O2 reacts with ascorbate to yield water and monodehydroascorbate (MDHA), which is rapidly dismuted to dehydroascorbate (DHA). MDHA and DHA are then re-reduced to ascorbate by the respective reductase activities. In the salt-tolerant tomato species, Lycopersicon pennellii, matrix H2O2 is detoxified by soluble ascorbate peroxidase (APX) and inner membrane-bound APX which faces the matrix (grey ovals). A smaller proportion of the inner membrane-bound APX faces the intermembrane space. The activities of all three forms are up-regulated significantly under salt stress. In the salt-sensitive tomato species, Lycopersicon esculentum, the majority of APX is membrane bound, being accessible to ascorbate in either the cytosol, or the intermembrane space (open ovals). A smaller proportion exists as a soluble matrix activity which increases in response to salt stress, but much less dramatically than in L. pennellii. For simplicity, mitochondrial glutathione reductase has been omitted. Antioxidant roles for thioredoxin- and glutaredoxin-based systems are possible but have not yet been firmly established in plants and may be more active in AOS-mediated signalling rather than detoxification.

APX

APX

APX

L. pennellii

– –

248 V. Mittova et al.

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 237–250

cation of a sequence (Q8LSK6) containing a putative mito-chondrial transit peptide. The molecular weight of themature protein was calculated to be approximately 38 kDa.Although its intracellular location remains to be deter-mined, this protein is a possible candidate mitochondrialAPX. The sequence is not predicted to contain any trans-membrane helices and interestingly, primary sequencecomparisons indicated that this protein would not be rec-ognized by the antiserum used in this study, which suggeststhat it does not represent a membrane-bound mitochon-drial APX isoform. It is nevertheless possible that antibodybinding occurs in vitro, given that re-naturation on westernblots could lead to the presentation of non-linear epitopes,although if this is the case, it is difficult to explain why theantiserum does not also recognize chloroplastic APX iso-forms which are highly homologous to Q8LSK6.

As the tomato EST database is currently incomplete, theArabidopsis genome sequence provides a useful resourcefor the in silico investigation of possible mitochondrialAPX isoforms. In a recent survey of Arabidopsis APXgenes, no sequences were annotated as mitochondrial (Jes-persen et al. 1997). We re-examined the complete APX con-tent of Arabidopsis using location prediction programs andnone of the putative Arabidopsis APX sequences was pre-dicted to contain a mitochondrial transit peptide (data notshown). In a recent proteomic investigation of Arabidopsismitochondria however, 20–30 proteins were not predictedto contain mitochondrial targeting sequences, dependenton the programme used (Millar et al. 2001). Although manyof these proteins are known to be mitochondrial, they arepredominantly located in the outer membrane and inter-membrane space and therefore do not require a cleavable,N-terminal extension for import (Millar et al. 2001). Ourdata suggest that APX is associated with these compart-ments in Lem and therefore would not require a classicalpre-sequence. However, this does not explain the apparentabsence of genes encoding matrix APX and APX bound tothe inner mitochondrial membrane. An alternative andmore plausible explanation is that APX isoforms are tar-geted to mitochondria by other mechanisms.

There are now several examples of proteins which are co-targeted to chloroplasts and mitochondria in vivo (Creissenet al. 1995; Chow et al. 1997; Cleary et al. 2002) and it hasbeen demonstrated very recently that an APX isoform isdual-targeted to the chloroplast stroma and to the mito-chondrial matrix in Arabidopsis (Chew, Whelan & Millar2003). In tomato, the membrane-bound APX isoform isunlikely to be dual-targeted since it was detected in westernblots and chloroplastic APX isoforms were not recognizedby the antiserum (Fig. 3b). However, the soluble matrixAPX isoform was not recognized by the antiserum, suggest-ing it could indeed be dual targeted and thus encoded by achloroplastic APX gene. Alternatively, the putative mito-chondrial protein, Q8LSK6, could be a matrix APX. Atpresent therefore, the molecular identities of membrane-bound and soluble mitochondrial APX isoforms remain tobe determined for L. esculentum and L. pennellii. Since theintramitochondrial distribution and regulation of APX iso-

forms differ even between two closely related tomato spe-cies, it is possible that the complement of mitochondrialAPX isoforms and their genetic organization vary in differ-ent plants. To this end, current work is focused on the useof proteomics to identify cDNAs encoding membrane-bound and soluble mitochondrial APX isoforms in severalplant species.

CONCLUSION

In conclusion, we have demonstrated that APX isoformsare present in the membranes and matrix of mitochondriaand that they are differentially regulated by salt stress. Weshow that interspecific differences in the amount and loca-tion of mitochondrial APX activity (Fig. 5) coincide withvariations in salt tolerance between Lpa and Lem. We sug-gest that APX isoforms present in, or facing the matrix inLpa mitochondria act in concert with matrix-located Mn-SOD to protect the TCA cycle enzymes from salt-inducedoxidative inactivation and consequent loss of function(Fig. 5), in addition to lowering the risk of harmful lipidperoxidation reactions. The isolation of cDNAs encodingmitochondrial APX isoforms in the near future will allowtesting of this hypothesis using L. esculentum/L. pennelliiintrogression lines (Eshed & Zamir 1995) and transgenicplants.

ACKNOWLEDGMENTS

This work was supported by the Dr Herman KesselResearch Fund, in memory of Mr C.J.J. Van Kensbury,Israel, and V.M. was supported by a J. Blaustein fellowshipand a short-term EMBO fellowship. Rothamsted Researchreceives grant-aided support from the BBSRC of the UK.

REFERENCES

Arnon D. (1949) Copper enzymes in isolated chloroplasts.Polyphenol oxidase in Beta vulgaris. Plant Physiology 24, 1–8.

Asada K. (1996) Radical production and scavenging in chloro-plasts. In Photosynthesis and Environment (ed. N.R. Baker),pp. 123–150. Kluwer Academic Publisher, Dordrecht, TheNetherlands.

Bartlett S.G., Grossman A.R. & Chua N.H. (1982) In vitro synthe-sis and uptake of cytoplasmically-synthesized chloroplast pro-tein. In Methods in Chloroplast Molecular Biology (eds M.Edelman, R.B. Hallick & N.H. Chua), pp. 1081–1091. ElsevierBiomedical Press, Amsterdam, The Netherlands.

Bartoli C.G., Pastori G.M. & Foyer C.H. (2000) Ascorbate biosyn-thesis in mitochondria is linked to the electron transport chainbetween complexes III and IV. Plant Physiology 123, 335–343.

Bowler C., Slooten L., Vandenbranden S., De Rycke R., Botter-man J., Sybesma C., Van Montagu M. & Inzé D. (1991) Manga-nese superoxide dismutase can reduce cellular damage mediatedby oxygen radicals in transgenic plants. EMBO Journal 10,1723–1732.

Bradford M.N. (1976) A rapid and sensitive method for the quan-tification of microgram quantities of protein utilizing the princi-ple of protein-dye binding. Analytical Biochemistry 72, 248–254.

Chen G.X. & Asada A.K. (1989) Ascorbate peroxidase in tealeaves- occurrence of 2 isozymes and the differences in their

Mitochondrial ascorbate peroxidase isoforms in tomato 249

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 237–250

enzymatic and molecular properties. Plant Cell Physiology 30,987–998.

Chew O., Whelan J. & Millar A.H. (2003) Molecular identificationof the ascorbate-glutathione cycle in Arabidopsis mitochondria:implications for dual-targeted antioxidant defences in plants.Journal of Biological Chemistry 273, 46869–46877.

Chow K.S., Singh D.P., Roper J.M. & Smith A.G. (1997) A singleprecursor protein for ferrochelatase-I from Arabidopsis isimported in vitro into both chloroplasts and mitochondria. Jour-nal of Biological Chemistry 272, 27565–27571.

Churin Y., Schilling S. & Borner T. (1999) A gene family encodingglutathione peroxidase homologues in Hordeum vulgare (bar-ley). FEBS Letters 459, 33–38.

Claros M.G. & Vincens P. (1996) Computational method to pre-dict mitochondrially imported proteins and their targetingsequences. European Journal of Biochemistry 241, 779–786.

Cleary S.P., Tanm F.C., Nakrieko K.A., Thompson S.J., Mul-lineaux P.M., Creissen G.P., von Stedingk E., Glaser E., SmithA.G. & Robinson C. (2002) Isolated plant mitochondria importchloroplast precursor proteins in vitro with the same efficiencyas chloroplasts. Journal of Biological Chemistry 277, 5562–5569.

Creissen G., Reynolds H., Xue Y. & Mullineaux P. (1995) Simul-taneous targeting of pea glutathione reductase and of a bacterialfusion protein to chloroplasts and mitochondria in transgenictobacco. Plant Journal 8, 167–175.

De Leonardis S., Dipierro N. & Dipierro S. (2000) Purification andcharacterization of an ascorbate peroxidase from potato tubermitochondria. Plant Physiology and Biochemistry 38, 773–779.

Draper H.H. & Hadley M. (1990) Malondialdehyde determinationas index of lipid peroxidation. Methods in Enzymology 186, 421–431.

Dutilleul C., Garnier M., Noctor G., Mathieu C., Chetrit P., FoyerC.H. & DePaepe R. (2003) Leaf mitochondria modulate wholecell redox homeostasis, set antioxidant capacity, and determinestress resistance through altered signaling and diurnal regula-tion. Plant Cell 15, 1–16.

Emanuelsson O., Nielsen H., Brunak S. & von Heijne G. (2000)Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. Journal of Molecular Biology300, 1005–1016.

Eshed Y. & Zamir D. (1995) An introgression line population ofLycopersicon pennellii in the cultivated tomat enables the iden-tification and fine mapping of yield-associated QTL. Genetics141, 1147–1162.

Foyer C.H. & Noctor G. (2003) Redox sensing and signaling inchloroplasts and mitochondria. Physiologia Plantarum 119, 355–364.

Gadea J., Conejero V. & Vera P. (1999) Developmental regulationof a cytosolic ascorbate peroxidase gene from tomato plants.Molecular and General Genetics 262, 212–219.

Gómez J.M., Hernández J.A., Jiménez A., del Rio L.A. & SevillaF. (1999) Differential response of antioxidative enzymes of chlo-roplasts and mitochondria to long-term NaCl stress of peaplants. Free Radicals Research 31, S11–S18.

Gutteridge J.M. & Halliwell B. (1990) The measurement andmechanism of lipid peroxidation in biological systems. Trends inBiochemical Science 15, 129–135.

Hamilton E.W. & Heckathorn S.A. (2001) Mitochondrial adapta-tions to NaCl. Complex I is protected by anti-oxidants and smallheat shock proteins, whereas complex II is protected by prolineand betaine. Plant Physiology 126, 1266–1274.

Hernández J.A., Corpas F.J., Gómez M., del Rio L.A. & SevillaF. (1993) Salt-induced oxidative stress mediated by activatedoxygen species in pea leaf mitochondria. Physiologia Plantarum89, 103–110.

Huang J. & Philbert M.A. (1996) Cellular responses of cultured

cerebellar astrocytes to ethacrynic acid-induced perturbation ofsubcellular glutathione homeostasis. Brain Research 711, 184–192.

Iturbe-Ormaetxe I., Matamoros M.A., Rubio M.C., Dalton D.A.& Becana M. (2001) The antioxidants of legume nodule mito-chondria. Molecular Plant Microbe Interaction 14, 1189–1196.

Jespersen H.M., Kjaersgardm I.V., Ostergaard L. & Welinder K.G.(1997) From sequence analysis of three novel ascorbate perox-idases from Arabidopsis thaliana to structure, function and evo-lution of seven types of ascorbate peroxidase. BiochemicalJournal 326, 305–310.

Jiménez A., Hernández J.A., del Rio L.A. & Sevilla F. (1997)Evidence for the presence of the ascorbate-glutathione cycle inmitochondria and peroxisomes of pea leaves. Plant Physiology114, 275–284.

Jiménez A., Hernández J.A., Ros Barcelo A., Sandalio L.M., delRio L.A. & Sevilla F. (1998a) Mitochondrial and peroxisomalascorbate peroxidase of pea leaves. Physiologia Plantarum 104,687–692.

Jiménez A., Hernández J.A., Pastori G., del Rio L.A. & Sevilla F.(1998b) Role of the ascorbate-glutathione cycle of mitochondriaand peroxisomes in the senescence of pea leaves. Plant Physiol-ogy 118, 1327–1335.

Kieselbach T., Bystedt M., Hynds P., Robinson C. & SchröderW.P. (2000) A peroxidase homologue and novel plastocyaninlocated by proteomics to the Arabidopsis chloroplast thylakoidlumen. FEBS Letters 480, 217–276.

Laemmli A.K. (1970) Cleavage of structural protein during theassembly of the head bacteriophage T4. Nature 227, 680–685.

Laloi C., Rayapuram N., Chartier Y., Grienenberger J.M., Bon-nard G. & Meyer Y. (2001) Identification and characterizationof a mitochondrial thioredoxin system in plants. Proceedings ofthe National Academy of Sciences of the USA 98, 14144–14149.

Millar A.H. & Leaver C.J. (2000) The cytotoxic lipid peroxidationproduct, 4-hydroxy-2-nonenal, specifically inhibits decarboxy-lating dehydrogenases in the matrix of plant mitochondria.FEBS Letters 481, 117–121.

Millar A.H., Mittova V., Kiddle G., Heazlewood J.L., BartoliC.G., Theodoulou F.L. & Foyer C.H. (2003) Control of ascor-bate synthesis by respiration and its implications for stressresponses. Plant Physiology 133, 443–447.

Millar A.H., Sweetlove L.J., Giege P. & Leaver C.J. (2001) Anal-ysis of the Arabidopsis mitochondrial proteome. Plant Physiol-ogy 127, 1711–1727.

Mittler R. & Zilinskas B.A. (1993) Detection of ascorbate perox-idase activity in native gels by inhibition of the ascorbate depen-dent reduction of nitroblue tetrazolium. Analytical Biochemistry212, 540–546.

Mittova V., Guy M., Tal M. & Volokita M. (2002a) Response ofthe cultivated tomato and its wild salt-tolerant relative Lycoper-sicon pennellii to salt-dependent oxidative stress: increasedactivities of antioxidant enzymes in root plastids. Free RadicalsResearch 36, 195–202.

Mittova V., Tal M., Volokita M. & Guy M. (2002b) Salt stressinduces upregulation of an efficient chloroplast antioxidant sys-tem in the salt-tolerant wild tomato species Lycopersicon pen-nellii but not in the cultivated species. Physiologia Plantarum115, 393–400.

Mittova V., Tal M., Volokita M. & Guy M. (2003) Upregulation ofthe leaf mitochondrial and peroxiosomal antioxidative systemsin reponse to salt-induced oxidative stress in the wild salt-tolerant tomato species, Lycopersicon pennellii. Plant, Celland Environment 26, 845–856.

Mittova V., Volokita M., Guy M. & Tal M. (2000) Activities ofSOD and the ascorbate- glutathione cycle enzymes in subcellu-lar compartments in leaves and roots of the cultivated tomato

250 V. Mittova et al.

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 237–250

and its wild salt-tolerant relative Lycopersicon pennellii. Physi-ologia Plantarum 110, 42–51.

MøIler I.M. (2001) Plant mitochondria and oxidative stress: Elec-tron transport, NADPH turnover, and metabolism of reactiveoxygen species. Annual Review of Plant Physiology and PlantMolecular Biology 52, 561–591.

Mullen R.T. & Trelease R.N. (2000) The sorting signals for perox-isomal membrane-bound ascorbate peroxidase are within its C-terminal tail. Journal of Biological Chemistry 275, 16337–16344.

Nakai K. & Kanehisa M. (1992) A knowledge base for predictingprotein localization sites in eukaryotic cells. Genomics 14, 897–911.

Noctor G. & Foyer C. (1998) Ascorbate and glutathione: keepingactive oxygen under control. Annual Review of Plant Physiologyand Plant Molecular Biology 49, 249–279.

Noctor G. Veljovic-Jovanovic S. & Foyer C.H. (2000) Peroxideprocessing in photosynthesis: antioxidant coupling and redoxsignaling. Philosophical Transactions of the Royal Society ofLondon Series B 355, 1465–1475.

Nulton-Persson A.C. & Szweda L.I. (2001) Modulation of mito-chondrial function by hydrogen peroxide. Journal of BiologicalChemistry 276, 23357–23361.

Ôba K., Ishikawa S., Nishikawa M., Mizuno H. & Yamamoto T.(1995) Purification and properties of 1-galactono-gamma-lactone dehydrogenase, a key enzyme for ascorbic acid biosyn-thesis, from sweet potato roots. Journal of Biochemistry 117,120–124.

Prasad T.K., Anderson M.D. & Stewart C.R. (1995) Localizationand characterization of peroxidases in the mitochondria of chill-ing-acclimated maize seedlings. Plant Physiology 108, 1597–1605.

Quackenbush J., Cho J., Lee D., Liang F., Holt I., KaramychevaS., Parvizi B., Pertea G., Sultana R. & White J. (2001) The TIGRgene indices: analysis of gene transcript sequences in highlysampled eukaryotic species. Nucleic Acids Research 29, 159–164.

Rao M.V., Paliyath C. & Ormrod D.P. (1996) Ultraviolet-B- andozone-induced biochemical changes in antioxidant enzymes ofArabidopsis thaliana. Plant Physiology 110, 125–136.

Schwitzguebel J.P. & Siegenthaler P.A. (1984) Purification of per-oxisomes and mitochondria from spinach leaf by percol gradientcentrifugation. Plant Physiology 75, 670–674.

Shalata A., Mittova V., Volokita M., Guy M. & Tal M. (2001)Response of the cultivated tomato and its wild salt-tolerant rel-ative Lycopersicon pennellii to salt-dependent oxidative stress:the root antioxidative system. Physiologia Plantarum 112, 487–494.

Shigeoka S., Ishikawa T., Tamoi M., Miyagawa Y., Takeda T.,Yabuta Y. & Yoshimura K. (2002) Regulation and function ofascorbate peroxidase isoenzymes. Journal of Experimental Bot-any 53, 1305–1319.

Siendones E., Gonzáz-Reyes J.A., Santos-Ocaña C., Navas P. &Córdoba F. (1999) Biosynthesis of ascorbic acid in kidney bean.L-Galactono-g-lactone dehydrogenase is an intrinsic proteinlocated at the inner mitochondrial membrane. Plant Physiology120, 907–912.

Sweetlove L.J., Heazlewood J.L., Herald V., Holtzapffel R., DayD.A., Leaver C.J. & Millar A.H. (2002) The impact of oxida-tive stress on Arabidopsis mitochondria. Plant Journal 32, 891–904.

Taylor N.L., Day D.A. & Millar A.H. (2002) Environmental stresscauses oxidative damage to plant mitochondria leading to inhi-bition of glycine decarboxylase. Journal of Biological Chemistry277, 42663–42668.

Tretter L. & Adam-Vizi V. (2000) Inhibition of Krebs cycleenzymes by hydrogen peroxide: a key role of [alpha]-ketoglut-arate dehydrogenase in limiting NADH production under oxi-dative stress. Journal of Neuroscience 20, 8972–8979.

Verniquet F., Gaillard J., Neuburger M. & Douce R. (1991) Rapidinactivation of plant aconitase by hydrogen peroxide. Biochem-ical Journal 276, 643–648.

Webb R.P. & Allen R.D. (1995) Isolation and characterization ofa cDNA for spinach cytosolic ascorbate peroxidase. Plant Phys-iology 108, 1325–1325.

Yamaguchi K., Mori H. & Nishimura M. (1995) A novel isoenzymeof ascorbate peroxidase localized on glyoxysomal and leaf per-oxisomal membranes in pumpkin. Plant and Cell Physiology 36,1157–1162.

Yoshimura K., Ishikawa T., Nakamura Y., Tamoi M., Takeda T.,Tada T., Nishimura K. & Shigeoka S. (1998) Comparative studyon recombinant chloroplastic and cytosolic ascorbate peroxi-dase isozymes of spinach. Archives of Biochemistry and Bio-physics 353, 55–63.

Zhang H.X. & Blumwald E. (2001) Transgenic salt-tolerant tomatoplants accumulate salt in foliage but not in fruit. Nature Biotech-nology 19, 765–768.

Zhang H., Wang J., Nickel U., Allen R.D. & Goodman H.M.(1997) Cloning and expression of an Arabidopsis gene encodinga putative peroxisomal ascorbate peroxidase. Plant MolecularBiology 34, 967–971.

Received 8 January 2003; received in revised form 27 October 2003;accepted for publication 29 October 2003