Proc. Natl. Acad. Sci. USAVol. 93, pp. 9970-9974, September 1996Plant Biology

Environmental stress sensitivity of an ascorbic acid-deficientArabidopsis mutant

(ozone/ultraviolet B/sulfur dioxide/reactive oxygen detoxification/vitamin C)

PATRICiA L. CONKLIN, ELIZABETH H. WILLLAMS, AND ROBERT L. LAST*Boyce Thompson Institute for Plant Research at Cornell University and Section of Genetics and Development, Cornell University, Tower Road, Ithaca, NY14853-1801

Communicated by Andre T Jagendorf, Cornell University, Ithaca, NY, May 17, 1996 (received for review April 2, 1996)

ABSTRACT L-ascorbic acid (vitamin C) is a powerfulreducing agent found in millimolar concentrations in plants,and is proposed to play an important role in scavenging freeradicals in plants and animals. However, surprisingly little isknown about the role of this antioxidant in plant environ-mental stress adaptation or ascorbate biosynthesis. We reportthe isolation of sozi, a semi-dominant ozone-sensitive mutantthat accumulates only 30% of the normal ascorbate concen-tration. The results of genetic approaches and feeding studiesshow that the ascorbate concentration affects foliar resistanceto the oxidizing gas ozone. Consistent with the proposed rolefor ascorbate in reactive oxygen species detoxification, lipidperoxides are elevated in sozi, but not in wild type followingozone fumigation. We show that the sozi mutant is hypersen-sitive to both sulfur dioxide and ultraviolet B irradiation, thusimplicating ascorbate in defense against varied environmen-tal stresses. In addition to defining the first ascorbate defi-cient mutant in plants, these results indicate that screening forozone-sensitive mutants is a powerful method for identifyingphysiologically important antioxidant mechanisms and signaltransduction pathways. Analysis of sozi should lead to moreinformation about the physiological roles and metabolism ofascorbate.

Free radicals can damage macromolecules by oxidative pro-cesses, leading to cancer and other diseases associated withaging (1). Antioxidants act to detoxify reactive oxygen species(ROS) such as hydrogen peroxide (H202), superoxide (02-),hydroxyl radical (-OH-), and organic hydroperoxides. En-zymes active in ROS removal include superoxide dismutases,catalases, peroxidases, and glutathione S-transferases (1).Plants also synthesize abundant small molecule antioxidantsincluding AsA (L-ascorbic acid or Vitamin C), glutathione,a-tocopherol (Vitamin E), and carotenoids (2). Althoughthere is increasing evidence that these plant-derived antioxi-dants are important components of the human diet, relativelylittle is known about their specific functions in plants.

Microbial and animal mutants altered in antioxidant en-zymes and signal transduction processes played a pivotal rolein testing the importance of specific antioxidant detoxificationpathways, evaluating the proposed role of ROS in cellularprocesses and dissecting ROS signal transduction. For exam-ple, bacterial mutants altered in response to growth underenhanced active oxygen conditions identified key antioxidantenzymes and proteins that regulate their synthesis (3). Yeastmutants deficient in glutathione are hypersensitive to H202,implicating glutathione in ROS detoxification (4). A mamma-lian cell line expressing a mutant p2lras was recently used todemonstrate a key role for this G protein in ROS signaltransduction (5).

A collection of oxidative stress-sensitive plant mutantswould permit a critical assessment of the roles of antioxidantsystems and elucidation of ROS signal transduction pathways.With this goal in mind, Arabidopsis thaliana mutants withaltered sensitivity to the anthropogenic oxidizing air pollutant03 (ozone) (6) are being identified. We describe here asemi-dominant monogenic 03 sensitive mutant (sozl, sensitiveto ozone), which is deficient in ascorbic acid (AsA). Thisdeficiency also causes sensitivity to other ROS-generatingcompounds such as SO2 (sulfur dioxide) and UV-B (ultravi-olet-B) light.

MATERIALS AND METHODSPlant Culture.A. thaliana (ecotype Col-0) used in this study

were grown in "Cornell Mix" soil (7) in a constant-environment chamber, except where noted. Conditions in thischamber were as described (8). All biochemical analyses usedplants grown under a 16-hr photoperiod.Mutant Screen. Wild-type Columbia (Col-O) Arabidopsis

seed was mutagenized with ethyl methanesulfonate as de-scribed (9). Twenty-thousand M2 seeds were stratified for 4days at 4°C in 0.1% agar and sown at a density of 40/cm2.Plants were grown under a 24-hr photoperiod for 14 days andthen exposed to 250 ppb (parts per billion) 03 for 8 hr (8). The03-treated plants were screened visually for individuals withenhanced damage after 24 hr. Putative mutants were trans-planted several days later and allowed to self-pollinate toobtain M3 progeny for further studies.

Genetic Analysis. To determine dominance, F1 seed (SOZ1/sozl) was obtained by pollination of SOZ1/SOZ1 with sozl/sozl (M4) pollen, and F2 seeds were obtained by self-pollination of F1. F2 progeny from this cross were grown undera 16-hr photoperiod for 2 weeks, exposed to 250 ppb 03 for 8hr, and scored for 03 sensitivity 16 hr after the exposure.The SOZ1 locus was mapped onto the Arabidopsis genome

with 117 individuals from a polymorphic F2 mapping popula-tion generated by self-pollination of F1 seed from a crossbetween the SOZ1/SOZ1 (Ler ecotype) and sozl/sozl (iso-lated in the Col-0 background) with the use of both cleavedamplified polymorphic sequencer (10) and microsatellite (11)markers. The Kosambi mapping function was used to calculatethe map distances (12).

Biochemical Assays. For both AsA and glutathione assays,rosette tissue w-as pooled and acidic extracts were produced asdescribed (13). Glutathione (total and oxidized) was measuredin a neutralized extract with the 5,5'-dithiobis(2-nitrobenzoicacid) assay as described (14). AsA was measured in a neutral-ized extract with the AsA oxidase assay, with the oxidized form(dehydroascorbate) estimated as (AsAtotal- ASAreduced) (13).

Abbreviations: AsA, L-ascorbic acid; ppb, parts per billion; ROS,reactive oxygen species; UV-B, ultraviolet B; UV-BBE, biologicallyeffective ultraviolet B light.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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A standard curve for AsAtotal was generated with purified AsA(Sigma), with the addition of dithiothreitol to a final concen-tration of 4 mM. Dithiothreitol was also added to the samplesfor the standard curve because the oxidized dithiothreitolformed from reduction of dehydroascorbate absorbs UV light(15). Relative AsA concentrations for sozl and wild type,obtained by the spectrophotometric method, were confirmedby HPLC analysis of the bis(dinitrophenyl)hydrazone deriva-tive of dehydroascorbate (16).

Malondialdehyde lipid peroxidation products were assayedas 2-thiobarbituric acid-reactive substances (7). Malondialde-hyde levels are expressed on a dry weight basis, because 03causes severe dehydration of sozl. Average freshweight:dry-weight ratios were derived from 2-3 replicate samples unlessnoted otherwise in the legend of Table 1.S02 and UV-B Treatments. Five-week-old plants grown on

a 16-hr photoperiod were exposed to 800 ppb SO2 for 6 hr andphotographed 24 hr later. A Model 43 Pulsed Fluorescent SO2Analyzer (Thermo Environmental, Franklin, MA) was used tomeasure SO2. UV-B experiments were performed on soilgrown plants under a 24-hr photoperiod as described (7). Seedswere allowed to germinate under Mylar filtered light, and 4days after planting were exposed to continuous 0.9 kJ biolog-ically effective UV-B (UV-BBE) m-2 h-1 (17) for 17 days byfiltration through Pyrex glass, or kept under mylar for, UV-Bcontrols.

RESULTSMutant Screen. Two-week-old wild-type A. thaliana plants

(ecotype Col-0) are resistant to 03 fumigation. For example,the wild type exhibits little sign of 03 pathology followingexposure to 400 ppb for 8 hr (Fig. 1A). This intrinsic tolerancefacilitated the search for 03-sensitive mutants, which wereidentified by exposure to 250 ppb 03 for 8 hr. One day aftertreatment, fumigated plants were screened visually for thosewith enhanced chlorosis, lesions, or tissue collapse. Only 4putative mutants out of 20,000 M2 plants screened appearedunusually debilitated by fumigation, and 3 of these exhibitedheritable 03 sensitivity. One of these mutants (sozl) waschosen for intensive analysis because it showed dramatic leafdamage (Fig. 1A). Exposure of sozl to .200 ppb 03 for 8 hrcauses tissue collapse and death of all fully expanded rosetteleaves. Although the affected leaves do not recover from 03treatment, the toxicity is not systemic and newly emergingleaves are not noticeably harmed. The relative insensitivity ofimmature organs presumably results from lack of access of thegas to tissues that lack functional stomata. The 03-sensitivephenotypes of the two other mutants isolated in this screen aredistinct from that of sozl. Upon 03 exposure, the secondmutant (which is not allelic to sozl) becomes chlorotic,whereas the third develops large well-defined lesions on allfully expanded leaves.

Table 1. Effect of 03 on lipid peroxide accumulation

Genotype 03, hr FWT/DWT* MDAtSOZ1 0 12.4 ± 0.4 82.8 ± 12.8

4 9.9 ±0.6 87.7± 8.1sozl 0 10.7 ± 1.1 67.9 ± 16.1

4 5.2 ± 0.5 135.8 ± 15.6

Fifteen-day-old plants were exposed to 400 ppb 03 for 4 hr. Tissuewas collected for analysis prior to (0 hr) and immediately after (4 hr)exposure. Average fresh weight:dryweight (FWT/DWT) ratios andmalondialdehyde (MDA) values are shown for three independentexperiments with SE.*For the sozl 4-hr sample, a single DWT value was used to obtainFWT/DWT in one of the three 03 treatments.tMDA, ,umol of 2-thiobarbituric acid-reactive substances/g DWT.

A

C

FIG. 1. Response of sozl to 03, SO2, and UV-B. Plants exposed to03 and SO2 were photographed 24 hr after the exposure ended. (A)Representative 15-day-old sozl/sozl, SOZ1/SOZ1, and SOZl/sozl(F1) individuals exposed to 400 ppb 03 for 8 hr. Also shown is anunexposed sozl/sozl homozygous mutant plant (sozl ctl). (B) Wild-type and sozi mutant exposed to 800 ppb SO2 for 6 hr. Individualrosette leaves from four different 5-week-old individuals are shown foreach genotype. (C) Wild type and sozl exposed for 17 days to 0.9UV-BBE m-2 h-1 (UV-B) or UV-B deficient Mylar-filtered light (ctl).Single rosette leaves from four different individuals are shown for eachgenotype and treatment.

Genetic Analysis of sozi. The 03 sensitivity of sozl isconferred by a semi-dominant monogenic mutation, as seenwhen heterozygous sozi/SOZl F1 progeny were challengedwith 400 ppb of 03 for 8 hr. The F, exhibited an 03 sensitivityintermediate between that of SOZI/SOZI wild-type andsozl/sozl mutant plants, producing 03 induced lesions withoutthe massive tissue collapse characteristic of the homozygousmutant (Fig. 1A). Unlike wild type, the F1 plants weresomewhat wilted immediately after 03 exposure. As furtherevidence of this semi-dominance, three phenotypic classes (03sensitive, 03 semi-resistant, 03 resistant) were identified insegregating F2 progeny from a self-cross of the heterozygote.In this F2 population, the 03-sensitivity segregated in anapproximately 3:1 03-resistant plus semi-resistant:03-sensitiveratio (358:101, P > 0.10), consistent with a monogenic trait. Asshown in Fig. 24, the SOZ1 locus maps to the bottom ofchromosome 2, 15.9 centimorgans below cleaved amplifiedpolymorphic sequence marker m429 (10) and 16.8 centimor-gans centromere distal to microsatellite marker ngal68 (11).

sozi Is Impaired in ROS Detoxification. Based upon thesevere 03 damage to sozi plants, we hypothesized that thismutant is defective in ROS detoxification. This view wasstrengthened by the observation that, when detached leaveswere floated in increasing concentrations of H202 in an

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ngal68 m429 sozl15.9cM16.8cM

BE

* sozi/sozi

sozl/sozlSOZ1/soz 1

3

(3:

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2

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o1 2 3 4 5 1 23 4 5 6 7 8 910

03 Resistant 03 Sensitive

C.

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FIG. 2. Map location of sozl and cosegregation of both 03sensitivity and ascorbate deficiency with the mutation. (A) Mapposition of sozl relative to selected physical markers on chromosome2. Arrowhead points toward the centromere. (B) Total AsA wasdetermined in 14-day-old self-pollinated progeny from either true-breeding 03-resistant (SOZI/SOZJ, solid bars) or -sensitive (sozllsozl, shaded bars) F2 plants from the cross (SOZ1/SOZ1 x sozi /sozl).(C) Total AsA was determined in 14-day-old SOZIISOZ1, sozl /sozl,and two independently generated pools of F1 plants (SOZ1/sozl,crosshatched bars)

aqueous solution, sozl leaves became bleached at a lowerconcentration of H202 than did wild type (data not shown). Animpaired ability to adapt to elevated ROS would result inincreased oxidative damage to cellular components includinglipids. To test this proposal, the accumulation of lipid perox-ides in both sozl and wild type was measured using a malon-dialdehyde assay (18, 19). Lipid peroxide levels in sozl were

increased -2 times after 4 hr exposure to 400 ppb 03, and incontrast remained virtually unchanged in SOZI (Table 1). Thissuggests that, whereas wild-type plants are able to successfullyadapt to the enhanced ROS following 03 fumigation, freeradical detoxification is compromised in sozl. It is interestingto note that sozl plants do not experience enhanced lipidperoxide accumulation when grown in a growth chamber withcharcoal-filtered air (Table 1).

sozi Is Ascorbate Deficient. Plants contain millimolar con-

centrations of the soluble antioxidants ascorbic acid andglutathione. Consistent with a role for AsA in 03 adaptation,we found that 2-week-old sozl plants contain only '30% of thewild-type level of total AsA (Table 2). As seen in Table 2, theabsolute level of oxidized AsA (in the form of dehydroascor-bate) is similar in the two genotypes. However, a greater

Table 2. Total and oxidized ascorbate and glutathione in sozl

DHA% of

Genotype Total AsA DHA total Glutathione GSSG*

SOZ1 42.7 ± 1.7 5.5 ± 2.8 -13% 5.3 ± 0.8 0.16 ± 0.02sozl 11.9 ± 0.6 2.8 ± 0.5 -24% 5.0 ± 0.3 0.10 ± 0.05

Two-week-old plants were harvested for analysis. Average values(,mmol/g dryweight) from five (for glutathione) or six (for AsA)independent extractions of each genotype with SD shown. DHA,dehydroascorbate. GSSG, oxidized glutathione.*GSSG is expressed as ,umol of glutathione equivalents/g dry weight.

percentage of the AsA pool is oxidized in sozl than in SOZ1.This altered ratio of reduced to oxidized AsA is presumably theresult of the smaller AsA pool in sozl. Despite the proposedrole of glutathione as a reductant for oxidized AsA (2), thepresence of a diminished AsA pool in sozl does not affect theaccumulation of reduced or oxidized glutathione (Table 2).Ascorbate Deficiency Correlates with 03 Sensitivity. A

number of approaches were taken to test whether the 03sensitivity of sozl is caused by AsA deficiency. First, we askedwhether the sozl AsA deficiency and 03 sensitivity are theresult of the same mutation by monitoring both phenotypes ina segregating population from a genetic cross (Fig. 2B).True-breeding F3 families (derived from self-pollination of F2individuals from the cross SOZI/SOZI X sozifsozl) wereassayed for total AsA and scored for 03 sensitivity. As wouldbe expected if one genetic locus causes both defects, a perfectcorrelation was observed between 03 sensitivity and AsAdeficiency in 15 F3 families.

If AsA plays an important role in mediating 03 tolerance inArabidopsis, there should be a correlation between theamount of AsA that accumulates and the degree of stressresistance. This idea was tested in two ways. First, it was foundthat F1 SOZl/sozl heterozygous plants, which display anintermediate 03 sensitivity (Fig. 1A), accumulate a concen-tration of total AsA intermediate to that of SOZI/SOZI andsozl/sozl homozygotes (Fig. 2C). The heterozygote reproduc-ibly accumulates -75% of the wild-type concentration, whichis 250% that in sozl. The assertion that AsA plays an importantrole in 03 tolerance is strengthened by the observation thatAsA levels correlate with 03 resistance during Col-0 wild-type(SOZ1/SOZl) development. AsA levels fall between the agesof 2 and 6 weeks, whereas 03 injury, measured as lesions of . 1mm in diameter, increases during this developmental timecourse, as shown in Fig. 3.To further test the hypothesis that 03 sensitivity in Arabi-

dopsis is influenced by AsA concentration, this antioxidantwas artificially elevated in sozl. Both wild-type and mutantplants were watered with H20 (control), AsA, or L-galactono-1,4-lactone (a proposed AsA precursor). Forty-eight hoursafter this treatment plants were either harvested for AsAanalysis or exposed to 03. Pretreatment with AsA or L-galac-tono-1,4-lactone caused elevation ofAsA in both sozl and wildtype (Fig. 4A) and this restored 03 resistance to sozl (Fig. 4B).This result further strengthens the hypothesis that the sozl 03sensitivity is caused by an AsA deficit.

Sensitivity of sozi to Other Abiotic Stresses. The AsAdeficient sozl mutant was used to assess the importance ofAsAin adaptation to SO2 and UV-B, which both generate ROS (7,20). To assess S02 sensitivity, 5-week-old plants were exposedto 800 ppb SO2 for 6 hr. Under these conditions sozl developedlarge spreading bifacial necrotic lesions typical of SO2 injurywithin 24 hr of the treatment, whereas SOZI plants were nearlyuninjured (Fig. 1B). Exposure to a chronic high dose of UV-B(0.9 kJ UV-BBE m-2.h-1) for 17 days caused severe growthretardation in both wild-type and mutant plants. However,unlike wild type, sozl leaves became chlorotic (Fig. 1C), aphenotype that was consistently seen in three independent

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A4-

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t 2-

1.0

E--

u-

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A

2 3 4 5 6Age (weeks)

Age (weeks) 2 3 4 5 6ISOZi (% injury) 4 3 53 27- 47

FIG. 3. AsA and 03 resistance both decline during development inwild type. (A) Total AsA was determined in SOZ1 rosette tissue at1-week intervals during development. (B) Plants were exposed to 400ppb of 03 for 8 hr and scored for injury 16 hr after the exposure. 03injury was measured as the percentage of plants (n = 25) with03-induced lesions of -1 mm in diameter.

UV-B treatments. Collectively, these results show that AsA isimportant in the detoxification of ROS generated by a varietyof diverse sources.

DISCUSSIONThe A. thaliana sozl mutant described above is hypersensitiveto the air pollutant 03, and has enhanced lipid peroxidationproducts following fumigation. Several lines of evidence havebeen presented that suggest the failure to adapt to oxidativestress is caused by a deficiency in L-ascorbic acid (vitamin C).First, the SOZ1 gene dosage correlates with both AsA accu-mulation and 03 tolerance in 2-week-old plants (Figs. 1A and2C), and the two phenotypes cosegregate in a genetic cross(Fig. 2B). Second, developmental studies of wild-type Arabi-dopsis indicate a correlation between enhanced 03 sensitivityand reduced AsA concentration as the plants age (Fig. 3).Finally, both the sozl 03 sensitivity and AsA deficiency arereversed by feeding with L-galactono-1,4-lactone or AsA (Fig.4).

Several other aspects of our results are worthy of note. Thesozl mutation is pleiotropic, showing that AsA also plays a rolein adaptation to the ROS generated by SO2 and UV-B (Fig. 1B and C). It is also surprising that this AsA deficient mutanthas a phenotype very similar to wild type when propagated ina growth chamber under standard Arabidopsis culture condi-tions with a 16-hr photoperiod and charcoal-filtered air (Fig.1A). This suggests that the residual AsA in sozl is sufficient tofulfill its roles as a scavenger of ROS and a reducing agentunder controlled environment growth conditions. However,the rosette leaves of sozl are elongated when grown under thesame conditions with constant illumination (Fig. 1C; "ctl"plants). Finally, the screen for 03-sensitive mutants is influ-enced by the fumigation conditions and is rather subjective.For example, a greater percentage of sensitive mutants wererecovered in a more recent screen using a higher dose of 03and progeny testing those M2 plants with less visible injury.

+ H20 + GaL + AsAGenotype I II I II I IISOZi 3.4 3.3 17.3 17.8 19.6 20.3sozl 0.9 1.3 19.6 21.3 20.8 23.8

Bsozl Sozi

FIG. 4. Treatment of sozl with an AsA precursor or AsA results inelevation of total AsA and 03 tolerance. Duplicate pots of 12-day-oldplants (=100 seeds per pot) were watered with 50 ml of H20, 0.25 ML-galactono-1,4-lactone (GaL), or 0.25 M AsA and allowed to grow for48 hr. (A) AsA levels of leaf tissue. Total AsA (,umol/g fresh weight)in the samples from each pot (I and II) are shown. (B) The remainderof the treated plants in each pot described in A were exposed to 330ppb 03 for 8 hr and photographed 24 hr after the 03 exposure. Arepresentative plant from each of the duplicate treatments is shown.This experiment was performed on three separate occasions, and in allexperiments pretreatment of sozl with AsA or l-galactono-1,4-lactonecaused increased 03 resistance and elevation of in planta AsA togreater than untreated wild-type levels.

AsA is thought to play a variety of critical roles in plants andanimals. As an abundant reducing agent it is able to directlydetoxify active oxygen species and is thought to maintain thereduced state of the chloroplastic antioxidant a-tocopherol. Itis also a substrate for AsA peroxidase, a component of theproposed superoxide and hydrogen peroxide detoxifyingascorbate-glutathione cycle. AsA in plants may be involved inthe synthesis of zeaxanthin, which dissipates excess light energyin the thylakoid membranes, preventing oxidative damage. Inaddition, it acts as a reductant in a number of other enzymaticreactions. AsA is also responsible for keeping prosthetic metalions in the reduced form, thereby maintaining the activity ofvarious enzymes. Thus, AsA acts both as an abundant antiox-idant free radical scavenger and a reductant in enzymaticreactions.A small pool ofAsA located outside the plant cell wall in the

apoplastic space is thought to have a major role in thedetoxification of ROS generated by 03 and was found tobecome highly oxidized during exposure to 03. This was incontrast to the redox state of the total AsA pool, which was notsignificantly altered (21). We have also found that the redoxstate of total AsA does not change significantly in either wildtype or sozl upon 03 exposure (data not shown). Cell frac-tionation experiments will be needed to reveal the subcellularlocation of the residual AsA and dehydroascorbate in sozl.

Analysis of AsA-deficient mutants such as sozl shouldprovide needed information on AsA metabolism. It is plausiblethat this mutant is defective in AsA synthesis. However, thebiosynthesis of this abundant antioxidant is not well under-stood in plants (22). In animals and several algae it is synthe-sized via a pathway in which the carbon skeleton of theprecursor D-glucose is inverted. Although there is no evidencefor the existence of the first three steps of this pathway (22),

- - sozl

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flowering plants do contain an activity for the terminal step,conversion of L-galactono-1,4-lactone to AsA (23). Our feed-ing experiments suggest that sozl can make this conversion(Fig. 4A). Data from several plant species are consistent withthe existence of a direct biosynthetic pathway in which theorder of the carbons in D-glucose is preserved (22). In thisproposed pathway, D-glucose is converted to AsA by means ofthe intermediates D-glucosone and L-sorbosone.Why is sozl not completely deficient in AsA? One possible

explanation would be that there are biochemically and/orgenetically redundant AsA biosynthetic pathways in plants.For example, both the direct and inversion pathways may beactive in Arabidopsis, with only one of these affected in sozl.Another plausible scenario is one in which there are multipleisoforms of an AsA biosynthetic enzyme (perhaps in distinctsubcellular compartments), and only one is defective in themutant. Alternatively, sozl may have enhanced catabolism ofAsA to shorter chain compounds such as oxalate and tartrate(22). It is also plausible that a defect in the ability to enzy-matically regenerate reduced AsA from its oxidized formscould lead to decreased total AsA, since both monodehy-droascorbate and dehydroascorbate are unstable under phys-iological conditions (24). A variation on this theme is that sozlmay have a defect in the active transport system proposed forshuttling oxidized AsA from the apoplast into the cell (25),thus preventing this oxidized AsA pool from being reducedwithin the cell. Future studies will clarify the nature of the sozldefect as well as provide insights into the origin and biologicalconsequences of ROS in varied stress conditions includingpathogen infection, photooxidative stress, drought, and chill-ing or in the aging process.

We thank C. J. Arntzen and S. H. Howell for critically reading themanuscript and P. King for assistance with 03 and SO2 monitoring.Work was supported by an American Cancer Society PostdoctoralFellowship (PF-3801) to P.L.C. and an National Science FoundationPresidential Young Investigator Award (DMB-9058134) to R.L.L.

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