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Complex phenotypic profiles leading to ozone sensitivityin Arabidopsis thaliana mutants
KIRK OVERMYER1,*, HANNES KOLLIST1,2,*, HANNELE TUOMINEN1,†, CHRISTIAN BETZ3,CHRISTIAN LANGEBARTELS3, GUNNAR WINGSLE4, SAIJALIISA KANGASJÄRVI5, GÜNTER BRADER6,PHIL MULLINEAUX7,‡ & JAAKKO KANGASJÄRVI1
1Plant Biology, Department of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland; 2Institute ofTechnology, University of Tartu, Tartu 50411, Estonia; 3Institute of Biochemical Plant Pathology, Helmholtz Center München,D-85764 Oberschleissheim, Germany; 4Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology,Swedish University of Agricultural Sciences, S-90187 Umeå, Sweden; 5Plant Physiology and Molecular Biology, Departmentof Biology, University of Turku, FI-20014 Turku, Finland; 6Genetics, Department of Biological and Environmental Sciences,University of Helsinki, FI-00014 Helsinki, Finland; and 7Department of Disease and Stress Biology, John Innes Centre,Colney, Norwich, NR4 7UH, United Kingdom
ABSTRACT
Genetically tractable model plants offer the possibility ofdefining the plant O3 response at the molecular level. To thisend, we have isolated a collection of ozone (O3)-sensitivemutants of Arabidopsis thaliana. Mutant phenotypes andgenetics were characterized. Additionally, parameters asso-ciated with O3 sensitivity were analysed, including stomatalconductance, sensitivity to and accumulation of reactiveoxygen species, antioxidants, stress gene-expression and theaccumulation of stress hormones. Each mutant has a uniquephenotypic profile, with O3 sensitivity caused by a uniqueset of alterations in these systems. O3 sensitivity in thesemutants is not caused by gross deficiencies in the antioxi-dant pathways tested here. The rcd3 mutant exhibits mis-regulated stomata. All mutants exhibited changes in stresshormones consistent with the known hormonal roles indefence and cell death regulation. One mutant, dubbedre-8, is an allele of the classic leaf development mutantreticulata and exhibits phenotypes dependent on light con-ditions. This study shows that O3 sensitivity can be deter-mined by deficiencies in multiple interacting plant systemsand provides genetic evidence linking these systems.
Key-words: reticulata; chloroplast; genetics; leaf organogen-esis; oxidative stress; reactive oxygen species; stomata.
INTRODUCTION
Plant response to the air pollutant ozone (O3) is complexand involves many biological processes at several levels. O3
acts in the apoplast and requires access to this compart-ment via stomata. Thus, stomatal aperture determines theeffective O3 dose seen by a given plant (Kollist et al. 2007).Intriguingly, stomata are also the first line of defenceagainst some types of pathogens. Stomatal regulation isintegrated with innate immunity-signalling pathways(Melotto et al. 2006) via connections in reactive oxygenspecies (ROS), salicylic acid (SA) and nitric oxide signal-ling, processes that are also central to the O3 response.Once in the leaf, O3 rapidly reacts with components of thecell wall, apoplastic fluids and plasma membrane (Kan-gasjärvi et al. 1994). O3 breaks down to form other ROS,such as H2O2, superoxide anion (O2
•-) and singlet oxygen(Kangasjärvi et al. 1994). In turn, these ROS induce theactive production of further ROS by the plant itself,termed the oxidative burst (Wohlgemuth et al. 2002).These direct and indirect O3-induced ROS signals thentrigger downstream responses. Antioxidant pathways havean important role of protecting against ROS damage andmodulating ROS signals (Langebartels et al. 2000). Mostnotable among these systems are ascorbic acid, glu-tathione and the enzymes of the Halliwell–Asada–Foyerpathway, such as superoxide dismutase (SOD) and ascor-bate peroxidase (APX). The presence of ascorbic acid inthe apoplast is an important ROS scavenger able to dimin-ish the effects of O3, especially early in the exposure(Conklin & Barth 2004). The perception of O3-inducedoxidative stress results in the reprogramming of transcrip-tion (Brosché et al. 2007). Plant stress hormones play anintegral role in the O3 response. SA, jasmonic acid (JA),ethylene (ET) and abscisic acid (ABA) are importantregulators of O3 responses at several different levels(Overmyer, Brosché & Kangasjärvi 2003). All of thesehormones induce defence responses. SA and ET promotelesion formation, and JA is involved in lesion containment(Overmyer et al. 2000; Tuominen et al. 2004). ABA isinvolved in the regulation of stomata (Kangasjärvi, Jaspers& Kollist 2005). The complex interaction of these signals is
Correspondence: K. Overmyer. Fax: +358-9-19159552; e-mail:[email protected]
*These authors contributed equally to this study.†Current address: Umeå Plant Science Centre, Department ofPlant Physiology, Umeå University, S-90187 Umeå, Sweden‡Current address: Department of Biological Sciences, University ofEssex, Wivenhoe Park, Colchester, CO4 3SQ, UK
Plant, Cell and Environment (2008) 31, 1237–1249 doi: 10.1111/j.1365-3040.2008.01837.x
© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd 1237
required for an effective defence response and, when outof balance, determines the extent of lesion development(Overmyer et al. 2003).
Physiological, stomatal, biochemical, antioxidant, geneexpression and stress hormone signalling responses are allintegrated to produce the various effects observed in plantssubjected to O3 or other oxidative stresses. Largely, thesevarious responses have been studied in isolation with onlyone or a few of these systems under study at a time. Thereare examples of more comprehensive papers: a study of sixsensitive/tolerant pairs of clones, cultivars or populations ofplants demonstrated that of the eight parameters investi-gated, only increased ET evolution provided a consistentexplanation for sensitivity or tolerance (Wellburn &Wellburn 1996). Puckette,Weng & Mahalingam (2007) havestudied the O3 sensitivity of 38 accessions of the modellegume Medicago by assaying a large number of antioxidantparameters. Furthermore, the accumulated body of workwith the tobacco O3-sensitive (BelW3) and -tolerant (BelB)pair (Heggestad 1991) has contributed to our understand-ing of O3 responses, especially at the biochemical level.However, all of these studies are limited by the complexgenetic heterogeneity between the sensitive/tolerant culti-vars or lines.
The application of genetics, through the use of geneticallytractable model systems, like Arabidopsis thaliana, can beused to identify new links at the molecular level in the plantresponse to O3. Forward genetic screens for O3-sensitiveArabidopsis mutants have been used by several groups andhave made a significant contribution to plant biology. Theradical-induced cell death (rcd1) mutant, isolated in our lab,is sensitive to O3 and extracellular O2
•- but not H2O2 (Over-myer et al. 2000). RCD1 encodes a novel protein (Ahlforset al. 2004) that has been shown to interact with a number ofnuclear transcription factors (Belles-Boix et al. 2000). Uponstress treatment, RCD1 is localized also outside of thenucleus and interacts with proteins at the plasma membrane(Katiyar-Agarwal et al. 2006). RCD1 defines a small genefamily whose members are also involved in stress responses(Borsani et al. 2005). Together, these studies indicate thatRCD1 plays a central role in regulating the response tomultiple stresses involving ROS. In another screen, theO3-sensitive jasmonate-insensitive (oji) mutant has strength-ened our understanding of the role of jasmonate signallingin O3 response (Kanna et al. 2003). Finally, a subgroup ofmutants from the sensitive to O3 (soz) screen was renamed tovitamin c (vtc) when it was discovered that they were ascor-bate (AA) deficient (Conklin,Williams & Last 1996;Conklinet al. 2000). This work has resulted in the clarification of therole of AA in plant responses to O3 and other stresses.Importantly, these mutants have facilitated definition ofplant AA biosynthesis pathways (Smirnoff, Conklin &Loewus 2001). Taken together, these O3-sensitive mutantsdemonstrate the power of forward genetics in resolving thecomplex molecular pathways and networks involved inoxidative stress response.
As illustrated by several recent reviews (Overmyer et al.2003; Conklin & Barth 2004; Kangasjärvi et al. 2005),
significant progress has been made at many levels of O3
research. However, O3 and other oxidative stress responsesremain inadequately defined at the molecular level, and it islikely that novel factors involved in plant O3 responseremain unknown. Further research is required to identifynew players and to link seemingly unrelated plant systems.In this paper, we describe the further application of forwardgenetics to questions of O3 response. We have isolateda collection of additional O3-sensitive mutants based onthe appearance of O3-induced cell death lesions in theO3-tolerant Columbia-0 (Col-0) accession of A. thaliana.Multiple parameters, previously known to be associatedwith O3 sensitivity, were analysed in order to assess the basisand complexity of O3 sensitivity.
MATERIALS AND METHODS
Mutant screening and genetic mapping
Arabidopsis thaliana ecotype Col-0 seeds weremutagenized and screened for O3 sensitivity as described inLangebartels et al. (2000). Mutants were crossed to deter-mine allelism. Populations derived from crosses with Col-0and Landsberg erecta (Ler) were used for segregationand mapping studies, respectively. Linkage analysesof F2 recombinant populations were perfomed usingcleaved amplified polymorphic sequence and microsatellitemarkers (http://www.tair.org).
Microscopy, chlorophyll measurements anddetection of ROS
Cell density was quantified from images of second and thirdtrue leaves of 15-day-old plants taken with an inverted con-focal microscope (Zeiss LSM510 META, Germany) using a20 ¥ 0.50 water objective. Fluorescence was excited at543 nm with a HeNe diode laser, and detected with a 560-nm-long pass emission filter. For chlorophyll content mea-surements, leaf discs were extracted in 80% acetone with0.01% MgCO3, and absorbance at 646 and 663 nm was mea-sured with Shimadzu UV2100 (Shimadzu Corp., Kyoto,Japan) spectrophotometer. Accumulation of H2O2 wasanalysed by using 3,3’-diaminobenzidine 4 HCl (DAB)according to Schraudner et al. (1998). O2
•- was detectedby nitroblue tetrazolium (NBT) staining according toWohlgemuth et al. (2002).
Treatments with O3, superoxide and hydrogenperoxide (H2O2)
Three-week-old plants were exposed to a 6 h pulse of O3
as described in Langebartels et al. (2000). The O3 con-centrations used in experiments varied between 250 and350 nL L-1. Because of the stochastic physiological natureof O3 response and variation in batches of plants, test expo-sures with control sensitive and tolerant (rcd1 and Col-0)plants were used to determine the concentration requiredfor a typical and consistent response in a given experimen-tal period. Times of measurement refer to hours after the
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© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1237–1249
onset of exposure. Clean-air controls refer to plants kept inambient non-filtered indoor air with an O3 concentration of<10 nL L-1. To generate radicals in the apoplastic space,detached leaves were infiltrated either with superoxide(O2
•-)-generating system xanthine and xanthine oxidase(XXO; 0.5 mm/0.05 unit mL-1; Sigma, St Louis, MO, USA)or with H2O2-generating system glucose and glucoseoxidase (GGO; 2.5 mm/2.5–250 units mL-1; Calbiochem,San Diego, CA, USA) in 10 mm sodium phosphate buffer,pH 7.0 (Jabs, Dietrich & Dangl 1996). Cell death was quan-tified as ion leakage from rosette leaves into 18 MW waterfor 1 h, measured with conductivity meter (Mettler ToledoGmbH, Greifensee, Switzerland) and expressed as a % oftotal ions, quantified after boiling.
Stomatal conductance and antioxidant assays
Stomatal conductance was measured from the abaxial leafside with a steady-state diffusion porometer (AP4; Delta-T,Cambridge, UK). Poromoter was calibrated with a calibra-tion plate daily prior to measurements. Some of the keymeasurements were repeated using the adaxial leaf surfacewith similar results. APX and SOD activity determinationswere performed as described in Marklund (1985) andFoyer, Dujardyn & Lemoine (1989), respectively. AA anddehydroascorbate were analysed by GC–MS as described(Wingsle & Moritz 1997). GSH was extracted in 0.1 m HClby grinding a freeze-dried rosette in 1 mL of acid and incu-bating on ice for 30 min. Cleared supernatant was used tomeasure the GSH and GSSG contents by a derivatizationmethod (Newton, Dorian & Fahey 1981; Creissen et al.1999).
Plant hormones and gene expression
Ethylene and 1-aminocyclopropane-1-carboxylic acid(ACC) were measured as described (Langebartels et al.2000). ABA, SA and JA were quantified with the vapour-phase extraction method described by Schmelz et al. (2003)using 100 ng of 13C1–SA, 50 ng of dihydrojasmonic acid and20 ng of D6–ABA from Icon Isotopes (Summit, NJ, USA) asinternal standard for each sample. GC–MS analysis wasperformed on a Trace-DSQ from Thermo (Sweden) asdescribed previously (Brader et al. 2007). Expression of74 defence-related genes was studied by a custom-madecDNA array analysis as described in Tuominen et al. (2004).
Statistical analysis
Data in all figures are presented as means � SD. In Figs 2, 3and 7, means marked by different letters are statisticallysignificant (P < 0.01) by two-way analysis of variance plusTukey’s test. In Fig. 5, only rcd3 was significantly differentfrom Col-0 by the same test. In all experiments, five plantswere sampled (n = 5), except Fig. 7 (n = 4) and Table 2(n = 3). Each replicate (sample) represented a differentindividual plant, not the leaves from the same plant. All
experiments were performed at least three times withsimilar results; representative results are shown.
RESULTS
Genetic characterization of O3-sensitiveradical-induced cell death (rcd) mutants
We have isolated a series of O3-sensitive mutants (Over-myer et al. 2000), including a new allele of a mutant isolatedpreviously in other screens (Barth & Conklin 2003;González-Bayón et al. 2006). These mutants, which eachhave their own characteristic pattern of O3-induced damage(Fig. 1), were assigned the designation rcd2 through rcd4.
The mode of inheritance was determined by back-crossing to the parental Col-0. All mutants exhibited wild-type phenotypes in the F1 generation. Segregation ratios inthe F2 generation and models of heritability are presentedin Table 1a. The F2 segregation of rcd4 indicated that its O3
sensitivity was the result of two independent recessivemutations. The observed 1:3:7:21 ratio is variation of the1:3:3:9 model. It is 4:12 (i.e. 1:3) segregation superimposed
Figure 1. Ozone (O3)-induced damage in Col-0, re-8, rcd3, rcd4rcd6. Three-week-old plants were exposed to 250 nL L-1 for 6 h.Photos were taken at 24 h. Collapsed tissue on rcd4 rcd6 isindicated with a black arrow.
Phenotypic profiles leading to ozone sensitivity 1239
© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1237–1249
upon a 1:3:3:9 (1:3:4 + 3:12 + 9 = 1:3:7:21). This is the resultof a cross where one locus is a heterozygote in a doublemutant with two recessive loci. Thus, it is referred to as rcd4rcd6 double mutant. While the segregation ratios observedare consistent with two recessive loci, because of the com-plexity of this segregation pattern, other models may still bepossible. In isolation, the rcd4 and rcd6 single mutants haveonly subtle O3-sensitive phenotypes; leaf wrinkling in rcd4and slight chlorosis in rcd6. Because of the weak singlemutant phenotypes, all subsequent experiments used thehomozygote rcd4 rcd6 double mutant. Genetic complemen-tation tests indicated that these mutants all representindependent loci (Table 1b). The rcd2 mutant was mappedto BAC T28M21 on the bottom of chromosome II, whichcoincided with the localization of the phenotypically similarmutant lower cell density1-1 (lcd1-1) (Barth & Conklin2003). Lack of genetic complementation in >20 F1 plants ofrcd2 ¥ lcd1-1 indicated that rcd2 is allelic to lcd1-1, whichwas subsequently shown to be an allele of reticulata (re)(González-Bayón et al. 2006). Sequencing of At2g37860revealed that rcd2 has single G to A transition at the splicesite of the fifth intron that results in a predicted truncatedprotein. Therefore, the rcd2 mutant is referred to as
reticulata-8 (re-8). The rcd3 mutant has been mappedbetween markers SNP132 and SGCSNP9742 in chromo-some I. The rcd4 rcd6 mutant loci have been mapped tochromosome 1 showing 12.96 � 1.96% recombination to
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Figure 2. Chlorophyll concentration (a) and cell density (b) of3-week-old re-8 and Col-0 wild-type plants grown under normal(250 mmol m-2 s-1) and low (30 mmol m-2 s-1) light intensity with aphotoperiod 12/12 h (light/dark). In addition, chlorophyll contentand cell density of plants grown under normal light but shorterday length (8/16) are shown. Phenotype development of allother mutants in this study was independent of photoperiod(not shown).
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Figure 3. Quantification of ozone (O3)- (a–c) and superoxide(O2
•-) and H2O2- (d) induced leaf damage on the basis of relativeion leakage (% total ions). (a) Leaves from 3-week-old plantswere collected after 250 nL L-1 O3 exposure for 6 h. (b) Same as(a) except that plants were grown and exposed to O3 under lowlight conditions (30 mmol m-2 s-1). (c) Same as (b) except thatlight conditions were normal (150 mmol m-2 s-1) during O3
exposure. To test the effect, O2•- and H2O2, leaves from
clean-air-grown plants were infiltrated with xanthine andxanthine oxidase or glucose and glucose oxidase, respectively. Ionleakage was measured 24 h after the infiltration.
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© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1237–1249
the marker ciw12 and to the bottom of chromosome 2showing 12.50 � 2.08% recombination to marker athBIO2.
Clean-air phenotypes of rcd mutants
Both visual and microscopic examinations of leaves of themutants revealed that rcd4 rcd6 shows no morphological
difference to Col-0 wild type, while rcd3 has slightly broaderleaves. In contrast, re-8 clearly differs from wild type.The characteristic visual phenotype of re-8 is a reticulatepattern of pale coloration (Fig. 1). We measured chloro-phyll content and cell density of re-8 and Col-0 plants grownunder various light conditions. Mutant re-8 plants had sig-nificantly lower chlorophyll content (Fig. 2a) and reducedcell density in the palisade parenchyma (Fig. 2b) whengrown under standard light intensity [photosyntheticphoton flux density (PPFD), 250 mmol m-2 s-1] with a pho-toperiod of 12/12 h (light/dark). However, these phenotypeswere absent when plants were grown under low light(<30 mmol m-2 s-1; Fig. 2a,b). Furthermore, when re-8 wasgrown under short-day length (8/16 h light/dark), it wasvisually indistinguishable from Col-0 and had the samechlorophyll content. Furthermore, the cell density in plantsgrown under normal (12/12 h light/dark) day with differentlight intensities (PPFD between 30 and 250 mmol m-2 s-1)did not differ between re-8 and Col-0 (Fig. 2b). Thus, thecell density phenotype of re-8 seems to be the result ofphotoperiod-specific light intensity-dependent processesaffecting leaf development.The re-8 mutant is also tempera-ture sensitive; it is stunted considerably when grown underlow temperature.An additional allele, re-7 (SALK_037307),has phenotypes identical to re-8 (data not shown). No othermutants in this study exhibited light period dependency forphenotype development (data not shown).
Timing and pattern of O3-induced damageformation in mutants
The timing of O3-induced damage formation was similar forall mutants. Dark water-soaked lesions appeared at 12 h,they turned brown and collapsed and turned into drylesions with distinct borders by 24 h.The pattern of affectedtissue differed between mutants (Fig. 1). Lesions on re-8were found only within the intervascular tissue. The rcd4rcd6 mutant had randomly distributed lesions. In rcd3,damage was concentrated along the vascular bundles.Frequently, and especially at higher O3 concentrations (asshown in Fig. 4), these mutants can exhibit a second modeof damage formation where large areas of confluent lesions
Table 1. Genetics of the rcd mutants: (a) inheritance of theozone (O3)-sensitive phenotypes showing segregation in the F2
generation of a back-cross between the respective mutants andtheir wild-type Col-0 parental background; all crosses wereperformed using the mutant plant as the pollen donor, and (b)mutant allelism (genetic complementation) tests
(a)
Mutant F2 segregation Ratio/Model X2 P =
re-8 61/209a 1:3/single recessive 0.763 0.382rcd3 72/218a 1:3/single recessive 0.004 0.946rcd4 rcd6 8/33/69/194b 1:3:7:21c/two recessive 1.19 0.754
(b)
Crossd 03 Sensivity (sensitive/total tested)
rcd3 ¥ rcd4 rcd6 4/69re-8 ¥ rcd4 rcd6 1/38rcd3 ¥ re-8 0/8
Reported is the number of O3-sensitive individuals divided by thetotal number of individuals tested. Plants tested were the F1
progeny of cross between the O3-sensitive mutants as indicated.Crosses were done as listed above (acceptor ¥ donor).aTwo phenotype classes are reported as the number of plantsscored as sensitive (lesion bearing)/tolerant.bFour different phenotype classes are reported as the numberof plants scored as sensitive/intermediate sensitive type1/intermediate sensitive type 2/tolerant. Plants of the intermediatesensitive classifications did not exhibit lesion per se. Intermediatetype 1 plants were characterized by leaf wrinkling, and intermedi-ate type 2 by chlorotic spots.cThe ratio 1:3:7:21 is variation of the 1:3:3:9 model. It is 4:12 (i.e.1:3) segregation superimposed upon a 1:3:3:9 (1:3:4 + 3:12 + 9 =1:3:7:21). This is the result of a cross where one locus is a heterozy-gote in a double mutant with two recessive loci.dCrosses are listed here in the order: pollen acceptor ¥ pollendonor.
Table 2. Basal antioxidants of ozone (O3)-sensitive mutantsa
GenotypeAscobic acid(mg g-1 FW)
Glutathione(mmol g-1 DW)
APX activity(U mg-1 prot.)
SOD activity(U mg-1 prot.)
Col-0 734.5 � 82.1 3.69 � 2.25 0.051 � 0.015 0.280 � 0.145re-8 880.1 � 51.9 3.42 � 1.86 0.039 � 0.017 0.330 � 0.047rcd3 771.6 � 77.5 3.32 � 2.11 0.029 � 0.008 0.221 � 0.075rcd4 rcd6 667.1 � 65.8 2.65 � 1.57 0.039 � 0.009 0.171 � 0.025
aThe basal concentration of total ascorbate (AA), total glutathione and the basal activities of ascorbate peroxidase (APX) and superoxidedismutase (SOD) were determined in re-8, rcd3, rcd4 rcd6 and Col-0 wild type from 3-week-old plants grown in clean-air control conditions.None of the mutants exhibited significant differences from Col-0. Means are expressed � SD and are not statistically different (P > 0.1) byanalysis of variance.
Phenotypic profiles leading to ozone sensitivity 1241
© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1237–1249
collapse entire tissues. In this case, visible damage wasapparent as a region of turgor loss and tissue collapse asearly as 3 h.
Increased sensitivity to O3 and superoxide,but not to H2O2
To quantify the observed O3 sensitivity and assess theresponse to O2
•- and H2O2, we used ion leakage, an indica-tor of plasma membrane damage, as a measure of celldeath. Consistent with their O3 sensitivity, all mutantsexhibited ion leakage markedly higher than Col-0 afterexposure to 250 nL L-1 O3 for 6 h (Fig. 3a).
As other re-8 phenotypes were light dependent, the O3
sensitivity of this mutant was also tested under varied lightregimes. Growing plants under low light reduced stomatalconductance about twofold: conductance values for Col-0,re-8 and rcd1, respectively, were 311.1 � 48.3, 369.4 � 100.3,460.6 � 87.3 under normal light and 121 � 38, 146 � 30,188 � 57 under low light. To compensate for reduced O3
influx and maintain an equal effective O3 dose, the concen-tration of O3 was doubled to 500 nL L-1. Under these con-ditions, cell death was absent in re-8 and Col-0, while theO3-sensitive control, rcd1, still displayed its characteristicsensitivity (Fig. 3b). The presence of O3 lesions in rcd1 butnot in re-8 supports the assertion that O3 tolerance in re-8under low light was caused by a response to light intensityrather than reduced stomatal conductance. Accordingly,
when re-8 and Col-0 plants were grown under low lightintensity but treated with O3 under normal light intensity,significant cell death was evident in re-8 (Fig. 3c). This sug-gests the involvement of light-dependent processes as afactor causing O3 lesions in re-8.
O3 exerts its effect on plants via degradation into variousROS including O2
•- and H2O2. To determine the ROSsensitivity of these mutants, plants were infiltrated with
Col-0
re-8
rcd3
rcd4rcd6
Control Ozone Lesions
re-8 Low light
Figure 4. Accumulation of hydrogen peroxide (H2O2), stained with diaminobenzidine in Col-0, re-8, rcd3 and rcd4 rcd6 plants grownunder standard growth conditions (control) and after treatment with 300 nL L-1 ozone (O3) for 6 h. In addition, leaves from re-8 plantsgrown under low light intensity (30 mmol m-2 s-1) were stained (re-8 low light). Last panel illustrates typical O3-induced lesions of studiedgenotypes. At least 10 leaves per genotype were stained; representative results are shown.
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Figure 5. Leaf stomatal conductance of re-8, rcd3, rcd4 rcd6and Col-0 wild type exposed to 250 nL L-1 of ozone (O3) for 6 h.
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exogenous ROS-generating systems XXO for O2•-, and
with GGO for H2O2 (Jabs et al. 1996; Alvarez et al. 1998),and ion leakage was measured at 12 h. XXO caused amarked increase in ion leakage in all plants. However,the increase was always significantly higher in mutants(Fig. 3d). Infiltration of leaves with GGO did not causeincreased cell death (Fig. 3d). Direct treatment of leaveswith various concentrations of H2O2 confirmed this result;mutants and Col-0 exhibited similar sensitivity to H2O2 overa wide range of concentrations (Supplementary Fig. S1).These results indicate that these O3-sensitive mutants arehypersensitive to O3 and O2
•-, but not H2O2. However, wedo not exclude the possibility that other ROS derived fromO2
•- or multiple ROS, such as O2•- and H2O2, could be
required together for the observed ROS hypersensitivity.
Reactive oxygen species production in rcdmutants and Col-0
To elucidate the type and spatial distribution of ROS pro-duced in rcd mutants, we infiltrated leaves with DAB orNBT, indicative of H2O2 and O2
•-, respectively (Jabs et al.1996; Thordal-Christensen et al. 1997). No DAB precipitatecould be detected in most clean-air-grown plants (Fig. 4).Clean-air-grown re-8 displayed markedly higher DAB pre-cipitation, which was always located around the vasculature(Fig. 4). Such DAB precipitate was absent in re-8 plantsgrown under low light (PPFD < 30 mmol m-2 s-1) and indi-cates that standard growth light of 250 mmol m-2 s-1 resultsin light-dependent perivascular H2O2 accumulation in re-8.
To analyse O3-induced production of ROS, plants wereexposed to a standard O3 treatment, and leaves weresampled at 8 h (2 h after the 6 h treatment), excluding thedetection of ROS from direct O3 breakdown products. There-8 mutant displayed only somewhat higher O3-inducedDAB precipitate around the vasculature (Fig. 4). Stainingof O3-exposed re-8 with NBT revealed intense formation ofdark blue precipitate at the borders of spreading lesions(Supplementary Fig. S2). This indicates that O2
•- is themajor ROS accumulating adjacent to expanding lesions inre-8. In contrast, rcd3, rcd4 rcd6 and Col-0 seemed to accu-mulate primarily H2O2 after O3 treatment (Fig. 4), and sitesof accumulation coincided with the pattern of leaf injury inthese genotypes.Although Col-0 is extremely tolerant to O3
and rarely forms visible damage, some spot-like injury canoccur, especially at the higher end of our concentrationrange (�300 nL L-1). Under these conditions, damage isalways greater in the sensitive genotypes. In Col-0, H2O2
production occurs only with the appearance of such damageand is located on the borders of these spot-like lesions(Fig. 4). Similarly, H2O2 accumulation occurred in develop-ing lesions and at the border of lesions in rcd3 and rcd4rcd6. Notably higher deposition of NBT precipitate inO3-treated rcd3 and rcd4 rcd6 indicates that also O2
•- accu-mulation was induced in these genotypes (data not shown).However, O2
•- accumulation was randomly distributed,while H2O2 accumulation was more distinctly related withlesion formation in rcd3 and rcd4 rcd6.
Stomatal regulation
Measurements of stomatal conductance before, during(2 h, 4 h) and after (6 h) of 250 nL L-1 O3 exposure (Fig. 5)revealed some differences in stomatal behaviour. Col-0,re-8 and rcd4 rcd6 responded similarly to O3, closing theirstomata by the end of the exposure. However, conductancewas already significantly higher prior to O3 exposure inrcd3. Furthermore, rcd3 exhibited no O3-induced closureafter 3 h and only a modest decrease by the end of theexposure. This indicates that, of the O3-sensitive mutantsdescribed here, altered stomatal behaviour accounts for O3
sensitivity only in rcd3.
O3 sensitivity of rcd mutants is not caused byreduced antioxidant capacity
To address whether O3 sensitivity of these mutants is causedby a deficiency in antioxidant capacity, such as that seen insoz/vtc mutants (Conklin et al. 1996), the core antioxidantpathway was analysed. Basal AA was similar in all mutantsand wild type (Table 2). Concentrations of second majorantioxidant, glutathione, were also similar in Col-0 andmutants (Table 2). Additionally, the basal activities of twomajor antioxidant enzymes APX and SOD were assayedand found not to significantly differ from wild-type Col-0(Table 2). Taken together, this indicates that O3 sensitivityof these mutants is not likely caused by a gross inability todetoxify ROS within these systems.
O3-induced accumulation of stress hormones
We analysed O3-induced synthesis of the gaseous hormoneET and the levels of its immediate precursor ACC. Addi-tionally, we determined the levels of three other stress hor-mones, SA, JA and ABA, in O3-exposed plants. O3 exposureresulted in the increased accumulation of all of these hor-mones to some extent in all genotypes. Generally, thehormone response was either faster or of higher magnitudein the O3-sensitive mutants (Figs 6 & 7).
All the observed changes of ACC and ET productionwere O3 specific as clean-air control plants show no changes(Fig. 6). O3 caused a rapid increase in ACC synthesis at 2 hin all genotypes studied. Importantly, this increase was sig-nificantly higher in the mutants than in Col-0 (Fig. 6a,b). Incontrast to Col-0, in which ACC quickly returned to controllevels, all mutants maintained high ACC concentrationsuntil the 24 h time-point. Similarly, O3 triggered ET evolu-tion in all genotypes (Fig. 6c,d). ET evolution lasted longerand was significantly higher in re-8 and rcd3 (Fig. 6c,d).SA accumulation was similar in all plants by 8 h post-fumigation. However, SA induction was higher in re-8 at1.5 h and in all O3-sensitive mutants at 3 h (Fig. 7a). Low-level accumulation of JA was seen in O3-exposed Col-0 andrcd3 at time-points after 3 h. At 3 and 8 h, the O3-sensitivere-8 and rcd4 rcd6 accumulated high levels of JA (Fig. 7b).ABA levels were also induced by O3 in all plants; however,only at the latest time point of 8 h (Fig. 7c).
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O3-induced gene expression
DNA macroarray hybridization was utilized to follow theexpression of 74 selected stress-related and hormone sig-nalling marker genes (for the complete data set and a list ofgenes’ AGI codes, see Supplementary Table S1 and Fig. S3)at 8 h post-O3 exposure and unexposed controls in Col-0and the three O3-sensitive mutants. Expression of antioxi-dant genes was enhanced; however, largely similar to Col-0,indicating an intact transcriptional antioxidant response(Fig. 8a). O3-induced expression of these genes was some-what higher in mutants, consistent with their heightenedoxidative stress. One notable difference is the reduced basallevel and lack of O3 induction of chloroplastic iron SOD inre-8 (Fig. 8a). Consistent with the induction of cell death byO3, pathogen response (PR) genes, especially those respon-sive to SA, are more highly expressed in mutants (Supple-mentary Fig. S3 and Table S1). Generally, markers for theO3-induced stress hormones, SA, JA, ET and ABA are all
more highly expressed in the mutants (SupplementaryFig. S3 and Table S1). However, the O3 response of ABAmarker genes (RAB18, ERD10, COR47) is weak in rcd4rcd6 as compared with Col-0 (Fig. 8b), and the level of someJA-responsive genes (PDF1.2, LOX2, AOS) is depressed inre-8 under clean-air conditions (Fig. 8c).
DISCUSSION
We present here the characterization of three O3-sensitivercd mutants. These mutants exhibit O3 sensitivity deter-mined by multiple factors indicating that sensitivity is theresult of the complex interplay of multiple systems. Somefactors were common to all mutants, while others wereunique to individual mutants.
AA is an important ROS scavenger involved in O3
responses, as demonstrated by the O3 sensitivity of AA-deficient mutants (Conklin et al. 1996). In order to test ifthe O3 sensitivity of rcd mutants could be explained by a
Figure 6. Ozone (O3)-induced synthesisof ethylene (ET) and its immediateprecursor 1-aminocyclopropane-1-carboxylic acid (ACC) from the leavesof three O3-sensitive mutants (re-8,rcd3, rcd4 rcd6) and Col-0. ACCconcentrations (a, b) and ET evolution(c, d) were measured from plantsexposed to 250 nL L-1 O3 for 6 h (filledsymbols) and to clean-air control (blank).
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1244 K. Overmyer et al.
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gross deficiency in ROS detoxification, this study monitoredthe status of several key ROS detoxification systems, at thegene expression and anitioxidant metabolite levels. A largeblock of 12 antioxidant pathway genes was studied (Supple-mentary Fig. S2). These genes exhibit O3 responsivenessequal to or greater than that of wild-type plants (Supple-mentary Fig. S2), with one exception, FeSOD, as notedearlier. This indicates the transcriptional antioxidantresponse for these genes is intact and functional. No differ-ence was found in AA levels, basal APX activity, basal totalSOD activity and glutathione levels (Table 2). We concludethat ROS sensitivity of these mutants is not because of grossdeficiencies in these key ROS detoxification systems.
All of the rcd mutants exhibited an oxidative burst;however, each mutant has its own unique pattern of ROS
accumulation (Fig. 4), underscoring their mechanisticdifferences in damage formation. This study showed nocorrelation between damage (Fig. 3a) and the type orextent of ROS accumulation. Wohlgemuth et al. (2002) alsoobserved a similar disjuncture between damage, site of pro-duction and reactive species produced in O3-exposed Ara-bidopsis. This observation suggests that a given ROS signalcan summon different outcomes depending on the source,localization and context of other ROS signals in the cell.Accordingly, the rcd1 mutant is sensitive to apoplastic O2
•-
and O3 but more tolerant to chloroplast O2•- produced by
paraquat (Ahlfors et al. 2004; Fujibe et al. 2004). In the Ara-bidopsis lsd1 mutant, which is a model for cell death regu-lation, ROS from NADPH oxidases RBOHD and RBOHFare required for the negative regulation of cell death(Torres, Jones & Dangl 2005).This underscores the multipleroles of ROS and the importance of the timing and locationof their production. Taken together, these facts support theconcept of an ‘ROS signature’ that has been previouslysuggested (Mahalingam & Fedoroff 2003).This refers to thefact that signals from different ROS sources that are spa-tially and temporally separated are integrated to determinea specific output.
The results in Figs 6 and 7 demonstrate that SA, JA andET are induced by O3 exposure in these mutants, indicating
Col-0 re-8 rcd3 rcd4 rcd6
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Figure 7. Ozone (O3)-induced stress hormone accumulation.The accumulation of the hormones, (a) salicylic acid, (b) jasmonicacid and (c) abscisic acid, was determined at 0, 1.5, 3 and 8 hafter the beginning of a 6 h pulse of 250 nL L-1 O3 in Col-0 andO3-sensitive mutant plants.
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MSD1CSD2CSD1FSD1GR1GPX2APX1CAT3CAT1
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Figure 8. Clean air and ozone (O3)-induced gene expression.The expression of select genes is depicted as fold difference toCol-0 according to the color-coded key at the bottom for themutants re-8, rcd3 and rcd4 rcd6 (rcd4) under control clean air(CA) conditions and 8 h after the beginning of a 6 h ¥ 250 ppb O3
exposure (8 h O3). Gene abbreviations are listed on the right andinclude a block of antioxidant genes (a) abscisic acid responsegenes (b), and jasmonic acid response genes (c). Full gene namesand AGI codes can be found from Supplementary Table S1.
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that these mutants are not deficient in the biosynthesis ofthese hormones. These results are consistent with the pre-viously suggested roles of plant stress hormones in the regu-lation of O3 defence and cell death responses (Overmyeret al. 2000, 2003). In several instances, SA and ACC/ETresponses began prior to lesion appearance, suggesting thatthese are specific signalling events and not simply indicatorsof lesion formation. Significantly, Fig. 7c shows the induc-tion of ABA in response to O3 treatment in Col-0 wild-typeplants and to a greater extent in O3-sensitive mutants. Inspite of the importance of ABA for stomatal regulation, andstomata for O3 response, O3-induced ABA accumulationhas been largely neglected. Taken together, all these datasuggest that hormone signalling imbalances are involved inthe development of damage in these O3-sensitive mutants.
In addition to the mentioned features that were commonto all sensitive genotypes, characteristics unique to theirindividual sensitivity mechanisms were revealed for eachmutant.
The rcd4 rcd6 mutant phenotype requires two recessivemutations for full O3 sensitivity.The uncommon segregationratio observed in rcd4 rcd6 was from segregation at onelocus in a population from which one cross was done(Table 1). Individually, rcd4 and rcd6 are very minor O3
sensitivity loci that do not result in lesions. However,together in the double mutant, they exhibit a synergismresulting in tissue collapse.Altered ABA marker expression(Fig. 8b) suggests an impaired ABA response in the rcd4rcd6 mutant. ABA biosynthesis appears intact (Fig. 7),suggesting insensitivity to ABA. ABA insensitivity is alsoa feature of the rcd1 mutant (Ahlfors et al. 2004).
We show that rcd2 is allelic to reticulata (re) and lcd1; thisnew allele results in a disrupted splice site in the last intronand predicts production of a truncated protein. González-Bayón et al. (2006) studied seven alleles of the classicalgenetic marker re and cloned the gene showing it was allelicto lcd1. The light-dependent phenotypes and predictedchloroplast localization of RE suggest that it is involved inchloroplastic processes, although chloroplast number andfunction are not affected in lcd1/re mutants (Barth &Conklin 2003; González-Bayón et al. 2006). The charac-teristic phenotype of re-8 is a pale reticulate phenotypewhen grown under standard Arabidopsis growth conditions(PPFD 250 mmol m-2 s-1 light, 12 h photoperiod; Fig. 1).Several studies (this study; Barth & Conklin 2003;González-Bayón et al. 2006) demonstrate that this wascaused by lower cell density of leaf parenchyma in lcd1/remutant plants, implicating RE in normal leaf development.Decreased mesophyll cell density is the primary defect inlcd1, and LCD1/RE is reported to control cell division earlyin the early stages of leaf development (Barth & Conklin2003; Yu et al. 2007). Double mutant analysis with othervariegated leaf mutants suggests that RE acts in a develop-mental pathway with CUE1 but not DOV1 (González-Bayón et al. 2006). Here, we show that re-8 developmentalphenotypes are dependent on the combined effect of lightintensity and photoperiod (Fig. 2). Furthermore, its O3 sen-sitivity is light intensity dependent (Fig. 3b,c). This suggests
that RE is involved in processes driven by light dosage.Such dependence on environmental conditions for pheno-type development has been seen previously in the varie-gated mutant immutans, whose bleaching is dependent onlight levels, and variegated1, whose variegation pattern canbe suppressed at temperatures below 20 °C (Yu et al. 2007).However, the photoperiod dependency seen here has notbeen previously reported in variegated mutants. REtogether with At5g22790 comprise to a two-member genefamily, encoding proteins of unknown function bearing pre-dicted chloroplast targeting signals. It is likely that this genepair is functionally redundant, as is often the case for genesinvolved in determining variegated phenotypes (Yu et al.2007). Chloroplast envelope membrane localization hasbeen experimentally confirmed for At5g22790 (Ferro et al.2003), but not RE.
The depressed jasmonate-responsive gene expression inthis mutant under non-stressed conditions suggests a rolefor jasmonates in both the developmental and O3 sensitivityphenotypes of this mutant.
Given the O3-sensitive phenotype and the increased vas-cular H2O2 accumulation of re-8 (Fig. 4), it is possible thatRE is involved, at least indirectly, in chloroplast ROSsignalling. Consistent with this idea, re-8 was unable totranscriptionally induce chloroplastic Fe SOD (Fig. 8a) inresponse to O3-induced oxidative stress. ROS signals are acommon currency in the regulation of the chloroplast(Karpinski et al. 2003), and it has been shown that H2O2
from the chloroplast acts as a systemic stress acclimationsignal (Karpinski et al. 1999). Importantly, light signallinghas been linked to cell death control. Changes in lightquality, perceived by the photosynthetic apparatus modu-lated cell death in the lsd1 mutant, a well-known cell deathmodel (Mateo et al. 2004). Furthermore, perception ofblue light by cryptochrome is required for execution ofcell death triggered by the production of singlet oxygen(Danon, Coll & Apel 2006).
The rcd3 mutant has constitutively more open stomataand a lack of O3-induced stomatal closure (Fig. 5). Stomatalclosure allows stress avoidance, and lack of this responsemay promote O3 damage in rcd3. The higher stomatalconductance in rcd3 together with its changes in stresshormone levels and signalling illustrate the complex waychanges in multiple plant systems determine O3 sensitivity.Lesion propagation has been shown to be a hormone-dependent process, where SA and ET promote O3 lesionexpansion. This study (Figs 5 & 6a) suggests that directoxidative damage driven by higher initial or prolonged O3
influx in rcd3 may be the trigger of increased ET biosyn-thesis, which drives further lesion propagation. Thus, ashas been suggested previously (Kangasjärvi et al. 2005), thelevel of O3 influx may determine lesion initiation.
Notably, O3-induced stomatal closure occurred in Col-0plants already by 3 h; however, the accumulation of ABAfirst appears later at 8 h. This suggests a mechanism otherthan ABA is responsible for O3-induced closure, althoughother explanations, such as changes in ABA sensitivity,remain to be excluded. Interestingly, stomatal closure has
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recently emerged as a model for signal transduction, andH2O2 is an important signalling intermediate in this geneti-cally defined pathway (Pei et al. 2000; Kwak et al. 2003).This suggests that O3-derived ROS may act directly instomata to induce closure. Utilizing a new specializedapparatus, Kollist et al. (2007) have reported rapid stomatalclosure within circa 10 min of O3 exposure. This rapidresponse was dependent on the class II protein phosphataseABI2 and was apparently because of the direct perceptionof O3-derived ROS by stomata in Arabidopsis.
Work in our lab subsequent to this current study hasidentified RCD3 as At1g12480, which is expressed inguard cells and annotated as a distant homolog of fungaland bacterial dicarboxylate/malic acid transport proteins(Vahisalu et al. 2008). Electrophysiological studies indicatethat RCD3 is required for slow (s-type) anion channelcurrent.Taken together, this work contributes to our under-standing, not only of O3 responses, but also basic signaltransduction in the regulation of stomatal function.
O3-sensitive mutants provide genetic evidence that plantO3 response is under complex genetic regulation. Thenumber of mutants isolated in this low/medium saturationscreen implies that many loci are involved and suggeststhere are many O3-sensitive mutants that remain to bediscovered. It is to be expected that O3 sensitivity can beconferred by deficiencies in a number of different systems.Importantly, this study shows that O3 sensitivity, within onegenotype, can be determined by multiple factors, such ashormone responses, stomatal regulation, ROS accumula-tion, etc. This provides genetic evidence delineating pre-viously unseen relationships between these factors. Forexample, in rcd3/slac1, it is likely that increased directoxidative damage from unregulated O3 influx drives ETevolution, which further drives the accumulation of ROS.Beyond illustrating that point, this study shows that sensi-tivity screens can isolate novel mutants, and eventually theirrespective genes, which regulate a variety of known andnovel O3-induced processes. Further work with O3-sensitiveand other mutants of Arabidopsis should allow the defini-tion, at the molecular level, of pathways and networksinvolved in oxidative stress response.
ACKNOWLEDGMENTS
We acknowledge the excellent technical help of Mrs.Baldeep Kular for the GSH determinations, Cheikh Diopfor APX activity measurements and Thomas Moritz forAA determinations. GSH analyses were performed at theJohn Innes Centre in the lab of P.M. K.O. was supportedby postdoctoral grants (decisions 202828 and 115034)from the Finnish Academy, and H.K. by Estonian ScienceFoundation (grant no. 6241) and by Targeted FundingGrant from Ministry of Research and Education(SF0180071507). Work in the lab of J.K. was supportedby the Academy of Finland Centre of Excellence pro-grammes 2000–2005 and 2006–2011, and by the HelsinkiUniversity Environmental Research Centre.
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Received 22 December 2007; received in revised form 30 April 2008;accepted for publication 1 May 2008
SUPPLEMENTARY MATERIAL
The following supplementary material is available for thisarticle:
Figure S1. Induction of cell death by hydrogen peroxide(H2O2). H2O2-induced cell death was quantified as ionleakage. Detached leaves were infiltrated with buffer con-taining various concentrations of H2O2. Leaf treatment wasdone in an excess volume of buffer (10 leaves in 50 mL) to
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ensure that H2O2 was not cleared by leaf detoxificationsystems. H2O2 concentrations were monitored spectropho-tometrically before and after leaf incubation, and nevervaried by more than 5–10%. Data are displayed as means(n = 5) with error bars representing SD.Figure S2. Superoxide accumulation in re-8. Superoxideaccumulation was visualized by nitro blue tetrazolium(NBT) staining. The reaction of NBT with superoxideresults in the deposition of a dark purple formizan precipi-tate. Leaves were stained at 8 h after the beginning of a6 h ¥ 300 ppb ozone (O3) exposure. At least 10 leaves werestained in each of three independent exposures. Represen-tative results are shown.Figure S3. Clean air (CA) and ozone induced gene expres-sion for all genes on the array. The expression is depicted asfold difference to Col-0 according to the colour coded keyat the bottom for the mutants re-8, rcd3 and rcd4 rcd6 (rcd4)under control CA conditions and 8 h after the beginning ofa 6 h ¥ 250 ppb ozone exposure (8hO3). Gene abbreviations
are listed on the right. The data including full gene namesand AGI codes can be found from supplemental Table 1.Table S1. Full array raw data set. The expression of 74genes, selected for their involvement in stress and hormonesignalling, were studied by macro array hybridizationwith samples from plants grown under control clean airconditions and 8 h after the begin of a 6 h (250 ppb) ozoneexposure.
This material is available as part of the online articlefrom http://www.blackwell-synergy.com/doi/abs/10.1111/j.1365-3040.2008.01837.x(This link will take you to the article abstract)
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