Physiological, biochemical and molecular responses to a combination of drought and ozone in Medicago truncatula

Physiological, biochemical and molecular responses to acombination of drought and ozone in Medicago truncatula

NIRANJANI J. IYER1, YUHONG TANG2 & RAMAMURTHY MAHALINGAM1

1Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater and 2Department of PlantBiology, Samuel Roberts Noble Foundation, Ardmore, Oklahoma, USApce_12008 706..720

ABSTRACT

Drought and tropospheric ozone are escalating climatechange problems that can co-occur. In this study, weobserved Medicago truncatula cultivar Jemalong that issensitive to ozone and drought stress when applied singly,showed tolerance when subjected to a combined applica-tion of these stresses. Lowered stomatal conductance maybe a vital tolerance mechanism to overcome combinedozone and drought. Sustained increases in both reducedascorbate and glutathione in response to combined stressmay play a role in lowering reactive oxygen species andnitric oxide toxicity. Transcriptome analysis indicated thatgenes associated with glucan metabolism, responses to tem-perature and light signalling may play a role in dampeningozone responses due to drought-induced stomatal closureduring combined occurrence of these two stresses. Geneontologies for jasmonic acid signalling and innate immunitywere enriched among the 300 differentially expressed genesunique to combined stress. Differential expression of tran-scription factors associated with redox, defence signalling,jasmonate responses and chromatin modifications may beimportant for evoking novel gene networks during com-bined occurrence of drought and ozone. The alterations inredox milieu and distinct transcriptome changes in responseto combined stress could aid in tweaking the metabolomeand proteome to annul the detrimental effects of ozone anddrought in Jemalong.

Key-words: agriGO; ascorbate; combined stress; glu-tathione; microarray; reactive oxygen species; stomatalconductance.

INTRODUCTION

In field conditions, plants face several distinct environmen-tal extremes either simultaneously or at different timesduring the growing season (Tester & Bacic 2005). Forexample, drought and heat stress represent the conditionscommonly encountered by plants growing in arid regions(Mittler et al. 2001; Moffat 2002). Analysis of these twostresses in Arabidopsis and tobacco plants showed thatcombination of heat and drought affected plants differently

when compared with drought or heat stress applied indi-vidually with reference to photosynthesis, respiration, sto-matal conductance and leaf temperature (Rizhsky, Liang &Mittler 2002; Rizhsky et al. 2004). In the wake of globalclimate change caused by increasing levels of CO2, severalstudies reported on the interactive effects of CO2 and tro-pospheric ozone (Gupta et al. 2005; Burkey et al. 2007;Kontunen-Soppela et al. 2010; Gillespie et al. 2012).

Ozone, the most abundant air pollutant, reduces plantbiomass (Heagle 1989) by affecting allocation of assimilatesand induces senescence process in plants (Pell, Schlag-nhaufer & Arteca 1997; Miller,Arteca & Pell 1999). Highestozone concentrations usually occur around midday andduring summer season (Lorenzini, Nali & Panicucci 1994),in conjunction with high light and/or drought. Studies oncombined effects of high light and ozone in Phaseolus vul-garis indicated that the former exacerbated the detrimentaleffects of ozone on photosynthesis (Guidi, Tonini & Solda-tini 2000). Interactive effects of ozone and drought havebeen well studied in tree species. Birch saplings grownunder field conditions showed extensive ozone injury,reduced height and lowered stomatal density underrestricted water supply conditions (Paakkonen et al. 1998).Field experiments on combined ozone and drought inNorway spruce and beech revealed that ozone affectedgrowth and biomass very differently in two different clonesof these trees (Dixon, Le Thiec & Garrec, 1998, Karlssonet al. 1997). In the needles of conifers, the impact of com-bined ozone and drought seems to vary depending on thespecies. For example, drought prevents ozone injury in pon-derosa pine (Beyers, Riechers & Temple 1992; Temple et al.1993), while ozone retards the ability of Aleppo pine totolerate drought (Alonso et al. 2001). Modelling studies onthe interactive effects of drought and ozone using yieldforecasting predicted a general drought-induced reductionof crop sensitivity to ozone and it varied between crops,regions and years (King 1988).

Since the flux of ozone into the plant is through thestomata, it has been argued that differences in sensitivity ofplants to this pollutant are partly due to differences instomatal conductance (Reich 1987). Based on the ozoneuptake models, drought-induced stomatal closure wouldlimit ozone uptake into leaves and hence protect plantsfrom ozone stress (Panek & Goldstein 2001; Panek, Kurpius& Goldstein 2002; Grunehage & Jager 2003). However, this

Correspondence: R. Mahalingam. Fax: +405-744-7799; e-mail:[email protected]

Plant, Cell and Environment (2013) 36, 706–720 doi: 10.1111/pce.12008

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simplistic model was challenged by observations that ozonecaused stomatal ‘sluggishness’ that leads to incomplete sto-matal closure and hence exacerbated the effects of drought(Grulke et al. 2003, 2005; Karnosky et al. 2005). The pres-ence of night-time ozone in rural locations (McCurdy 1994)could also greatly reduce the biomass of plants (Winneret al. 1989), especially if ozone hindered opening of guardcells after a dark exposure (Torsethaugen, Pell & Assmann1999). These studies showed that physiological impacts ofozone and water deprivation together on plants arecomplex and merits attention given that concentrations ofozone in the troposphere will continue to increase in thefuture (Ashmore 2005; Ainsworth, Rogers & Leakey 2008).

A common biochemical response in plants to stressesincluding ozone and drought is the production of reactiveoxygen species (ROS) (Mittler 2002; Mahalingam & Fedor-off 2003). In fact, ozone toxicity in plants is related to theformation of ROS (Kangasjarvi et al. 1994; Baier et al. 2005;Kangasjarvi, Jaspers & Kollist 2005) and nitric oxide (NO)(Ahlfors et al. 2009). Drought stress also induces the pro-duction of free radicals (Quartacci & Navariizzo 1992;Biehler & Fock 1996). Changes in ROS levels in turnperturb the redox homeostasis via changes in ascorbate(AsA) and glutathione (GSH) (Noctor & Foyer 1998).Apart from ROS scavenging, both AsA and GSH play arole in development via regulating cell cycle and serving ascofactors for other proteins (Potters et al. 2000; Arrigoni &De Tullio 2002; Rouhier, Lemaire & Jacquot 2008; Vivancoset al. 2010). Thus, changes in redox balance can have a sig-nificant impact on the proteome both during developmentand stress (Foyer & Noctor 2009).

ROS and NO bursts induced by stressors like ozone inturn affect interactions between phytohormones jasmonicacid (JA), salicylic acid (SA), ethylene and abscisic acid(ABA) (Rao, Koch & Davis 2000; Rao & Davis 2001; Over-myer, Brosche & Kangasjarvi 2003). Increase in ozone-induced ethylene biosynthesis that is dependent on afunctional SA signalling pathway leads to increase in ABAbiosynthesis which regulates stomatal conductance (Rao,Lee & Davis 2002; Ahlfors et al. 2004). Interestingly, ozonehas been shown to suppress ABA-induced stomatal closurevia an ethylene-dependent mechanism (Wilkinson &Davies 2009, 2010). Thus, changes in the biochemical milieuin responses to single stresses and combined stresses syner-gize or antagonize different hormonal signalling pathwaysthat ultimately trigger distinct physiological responses.

Since physiological and biochemical changes are mostlyregulated by proteins, efforts were focused on isolation andcharacterization of protein coding genes such as phospho-enolpyruvate caraboxylase and ribulose 1·5-bisphosphatecarboxylase/oxygenase activase, commonly induced bycombined stresses (Fontaine, Cabane & Dizengremel 2003).Recently, genomics approaches using microarrays toanalyse transcriptional changes to combined stresses clearlyshowed that responses of plants to combined stresses weremarkedly different when compared with response to indi-vidual stresses (Rizhsky et al. 2002, 2004;Atienza et al. 2004;Bilgin et al. 2008; Casteel et al. 2008).

In this study, we have analysed the changes in geneexpression in response to combined ozone and droughtstress in Medicago truncatula cultivar Jemalong that waspreviously identified as an ozone-sensitive line (Puckette,Weng & Mahalingam 2007). Changes in gene expressionwere juxtaposed with measurements of physiological andbiochemical traits following single and combined ozone anddrought stresses. This integrative analysis led to the identi-fication of several novel mechanisms that may be useful fordeveloping resistance to combined occurrences of ozoneand drought stress in plants.

MATERIALS AND METHODS

Plant material and growing conditions

M. truncatula cultivar Jemalong was selected for this studybased on their sensitive responses to ozone and drought(Puckette et al. 2007). Plants were grown in 10″ ¥ 10″ traysfilled with 750 g of MetroMix 200 (Scotts-Sierra Horticul-tural Products Company, Marysville, OH, USA). Excessseedlings were thinned out to maintain 40 plants per tray.Plants were maintained in two identical growth chambers(Percival, Model 2000; Perry, IA, USA) maintained at 24 °C,light intensity of 200 mmol s-1 m-2, 10 h/14 h day and nightconditions. Each tray was irrigated with 600 mL of water.Watering was done every 3 d, maintaining the moisturelevels in each tray as closely as possible. Trays were ran-domly rearranged every 3 d to minimize variation inmicroenvironment among trays in different shelves and inthe two different growth chambers. All the plants weremaintained in ambient O3 levels (approximately 40 nmol-mol-1) for until 50 d after sowing.

Stress treatments

Stress treatments were initiated when plants were 50 d old.Three different treatments were assayed: drought, ozoneand combined drought and ozone stress.

Drought treatment was initiated by withholding water tothe trays starting from day 51, for a period of 10 d. Controlplants were maintained in the same growth chamber byadding measured amounts of water.

Ozone stress was imposed on 54-day-old plants using anearlier described ozone generation setup (Puckette et al.2007) by exposing plants to 1.75 times of the ambient ozonelevels (70 nmol mol-1) for 6 h per day for 6 consecutivedays. The AOT40 value for this short-term ozone exposuretreatment was 140 ppbh and was calculated using theformula:

AOT i40 0= ( ) ×[ ]C T h h–

where Ci is the average ozone concentration used for thetreatment, T is the threshold concentration, h is the totalpossible number of hours over the investigation period andh0 is the number of measured hourly values (Mauzerall &Wang 2001).

Combined ozone and drought stress in Medicago truncatula 707

© 2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 36, 706–720

Water levels in ozone-treated plants were maintained tosame levels as in control plants. The stress treatments wererepeated three times. Symptoms on the plants were evalu-ated visually at the end of stress treatments. The totalnumber of leaves and number of symptomatic leaves oneach plant at the end of stress treatment were recorded andused for calculating the percentage of leaves showing symp-toms such as chlorosis and necrotic lesions.

Chlorophyll content

Chlorophyll content of leaves was estimated at the end ofthe treatment regime from stressed plants and controls asdescribed earlier (Puckette et al. 2007).

Leaf water potential and relative water content

Leaf water potential in control, drought, ozone and com-bined stress-treated plants were measured using leaf psy-chrometers on days 57 and 60 (3 and 6 d after treatmentinitiation, respectively). All the psychrometers were cali-brated prior to the experiment. Five replicate samples wereassayed for this experiment. Values of leaf water potentialwere corrected to 27.5 °C and also with the calibrationfactor for each psychrometer. The average values and stan-dard deviations were calculated in Excel.

Relative water content (RWC) in drought-treated plantswas determined by taking fresh weight (FW), turgid weight(TW) and dry weights (DW) of leaves. Four replicates of 10randomly picked leaves in each replicate were used for thisassay. RWC was determined using formula: ((FW – DW)/(TW – DW)) * 100.

Stomatal conductance, photosynthesis andtranspiration rate

Stomatal conductance, photosynthesis and transpirationrates were measured 1 d after the initiation of the stresstreatments and at the end of the 6 d stress treatments.Leaflets of treated and control plants were sealed in a gasexchange cuvette of Li-Cor 6400 Portable Photosynthesissystem (Li-Cor, Lincoln, NE, USA). An Arabidopsiscuvette was used to snugly fit the small leaflet of Medi-cago. Measurements were taken under the followingconditions: leaf temperature: 24 °C, CO2 concentration:380 mL L-1, airflow: 0.1 L min-1, white light illumination:200 mmol m-2 s-1 and relative humidity: 35%. Measure-ments were taken 5 min after the leaves were sealed incuvette under conditions described earlier. Fifteen leafletssampled from different plants were tested for each of thetreatments.

ROS, NO and antioxidant assays

ROS, NO and antioxidants were measured 1 d after treat-ment initiation and at the end of 6 d treatment. ROS levelswere measured in control, drought, ozone and combined

drought- and ozone stress-treated leaves using a Versa-Fluor Fluorometer (Bio-Rad, Hercules, CA, USA) asdescribed earlier (Mahalingam et al. 2006). The total ROSlevels in each sample were plotted as relative fluorescenceunits (RFU) per milligram of protein. NO levels weremeasured using a spectrophotometer as described earlier(Mahalingam et al. 2006).

AsA dehydroascorbate (DHA), GSH, oxidized glu-tathione (GSSG) – were measured in the stress-treated andcontrol plants as described earlier (Mahalingam et al. 2006).Each assay was conducted thrice using pooled leaf tissuesamples from three biological replicates.

Statistical analyses

R-studio was to conduct two-way analysis of variance todetermine treatment effects (drought, ozone, combinedozone and drought), at the beginning (day 1) and at the endof treatment (day 6) and treatment ¥ day interactions. Thevarious physiological and biochemical measurements wereinput as variable factor. Tukey’s honestly significant differ-ences between control and treatments were calculated inR-studio and adjusted P-value of <0.05 was used as a cut-offfor identifying traits showing significant effect in responseto stress treatments.

RNA isolations and microarray hybridizations

Leaf tissues from control and treated plants were harvestedat the end of stress treatments (10 d of drought stress, 6 d ofozone treatment and combined drought and ozone) snapfrozen in liquid nitrogen. Total RNA isolations were doneusing Trizol reagent (Invitrogen, Carlsbad, CA, USA), fol-lowed by clean-up using the RNeasy kit (Qiagen, Valencia,CA, USA). Quality of RNA was analysed on a Bioanalyzer(Agilent, Palo Alto, CA, USA). About 10 mg of purifiedtotal RNA was used as template for hybridization. cDNAsynthesis, amplification, probe labelling and hybridizationswere conducted as described in the manufacturer’s instruc-tions (Affymetrix, Santa Clara, CA, USA). Three biologicalreplicates were used for hybridizations with GenechipMedicago genome arrays (Affymetrix).

Microarray data analyses

Data extraction, normalization and identification of differ-entially expressed genes were conducted as describedearlier (Benedito et al. 2008). Changes in RNA abundancein response to stress treatment were obtained by calcula-tion of the signal log2 ratio of each gene signal in thecontrol RNA samples relative to the stress treatment RNAsamples, with values from the latter in the numerator. Probesets that showed twofold or greater differences in transcriptlevels in two or more replicates were selected. Furthermore,in order to minimize family-wide error rates in multiplecomparisons, only probe sets with Bonferroni correctedP-value <9.8e-07 were selected for further analysis. All theraw data related to the microarray experiments in this study

708 N. J. Iyer et al.


have been submitted to MIAMEXPRESS database (http://www.ebi.ac.uk/miamexpress/) under the accession numberE-MEXP-3657.

Enriched gene ontologies

Differentially expressed genes from each treatment wereanalysed to determine the overlap between the genesinduced or repressed in response to drought or ozone andcombined drought and ozone treatments. The MedicagoAffymetrix identifiers for each category were input for sin-gular enrichment analysis in the agriGO toolkit (http://bioinfo.cau.edu.cn/agriGO/index.php) (Du et al. 2010).Medicago Affymetrix genome array was selected asbackground. Fisher’s statistical test and Yekutieli [false dis-covery rate (FDR) under dependency] for multi-test adjust-ment method were selected to identify enriched geneontology (GO) categories with five or more mappingentries and significance level of 0.05. Graphical outputs ofthe significant GO terms were generated by agriGO.

RESULTS

Phenotypic responses to ozone, drought andcombined ozone and drought stress

Short-term ozone stress for 6 d led to chlorosis and smallnecrotic lesions (8% of the leaves) in Jemalong. Droughtstress in these plants led to chlorosis, wilting and, some-times, collapse of entire trifoliates (10% of the leaves).Interestingly, in plants subjected to combined drought andozone stress, the only symptom was mild chlorosis (4% ofthe leaves) (Fig. 1).

Physiological responses to drought and ozone

Chlorophyll contentConsistent with the phenotypes observed, leaf chlorophyllcontent showed a significant reduction in response todrought or ozone stress (Fig. 1; Table 1).

Leaf water potentialLeaf water potential of well-watered plants under ambientozone conditions did not fluctuate much (–1.7 MPa). Leaf

Table 1. Summary of significance analysis of drought, ozone and combined ozone and drought stress experiments in Medicago truncatula

Ambient Ozone

Measurements Significance Day Watered Drought Watered Drought

Chlorophyll (mg per gprotein)

T 6 0.31 � 0.04 0.15 � 0.01 (0.00009) 0.17 � 0.02 (0.00032) 0.26 � 0.03

Leaf water potential (MPa) T, D, TxD 6 -1.85 � 0.10 -2.65 � 0.18 (0.00000) -2.06 � 0.07 -2.0 � 0.05Stomatal conductance

(mol m-2 s-1)T, D, TxD 1 0.15 � 0.03 0.06 � 0.02 (0.00119) 0.08 � 0.03 (0.01327) 0.05 � 0.02 (0.00021)

6 0.14 � 0.02 0.05 � 0.01 (0.00054) 0.16 � 0.02 0.07 � 0.01 (0.00080)Photosynthesis (mmol m-2 s-1) T, D 1 7.5 � 0.7 5 � 0.5 (0.00595) 5.2 � 0.8 (0.01367) 5.7 � 0.9

6 6.5 � 1.0 4.5 � 0.5 (0.03497) 5.2 � 0.3 4.7 � 0.5ROS (RFU in %) T, D, TxD 6 100 � 0 201 � 32 (0.01029) 282 � 58 (0.00002) 110 � 10Nitric oxide (mmol per g wt) T, D, TxD 6 6.4 � 0.7 11.4 � 1 (0.00002) 6.8 � 0.5 8 � 1Ascorbate (mmol per g wt) T, D, TxD 6 0.64 � 0.05 0.55 � 0.15 0.86 � 0.05 1.29 � 0.18 (0.0008)Dehydroascorbate (mmol

per g wt)T, D, TxD 1 0.19 � 0.14 0.59 � 0.19 (0.01876) 0.58 � 0.17 (0.02270) 0.6 � 0.1 (0.01652)

6 0.14 � 0.10 0.40 � 0.05 0.75 � 0.10 (0.00040) 0.12 � 0.07Glutathione (nmol per g wt) T 1 251 � 27 450 � 47 459 � 18 550 � 95 (0.01052)

6 287 � 81 362 � 42 605 � 107 (0.00614) 707 � 162GSSG (nmol per g wt) T, TxD 1 66 � 11 124 � 22 (0.01983) 131 � 13 (0.00824) 86 � 25

6 22 � 2.7 169 � 27 (0.00000) 89 � 11 (0.00661) 95 � 19

Statistically significant values or interactions from two-way analysis of variance were designated as D = day, T = treatment andTxD = treatment ¥ day interactions. Each value is a mean (n = 4) followed by standard error. Adjusted P-values (<0.05) from Tukey’shonestly significant difference test (comparisons with ambient watered control plants) are shown in parenthesis.ROS, reactive oxygen species; RFU, relative fluorescent unit; GSSG, oxidized glutathione.

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Figure 1. Phenotypic responses of Medicago truncatula cv.Jemalong to drought, ozone and combined stress. Top panelshows a sample trifoliate exhibiting the symptoms associatedwith drought, ozone or combined stress. Photographs were takenat the end of the stress treatment. Graph depicts the totalchlorophyll content at the end of stress treatments. Error barsrepresent standard deviations (n = 4). aMeasurement that issignificantly different compared to controls.



water potential decreased to -2.0 MPa on day 57,and further withholding of water for 3 more days led tosignificant decrease in the leaf water potential (–2.9 MPaon day 60) compared with corresponding control plants(Fig. 2a). Changes in leaf water potential in response toozone and combined stress was similar in pattern todrought alone, after 3 d of treatment. However, thereduction in water potential was not significant. Interest-ingly, leaf water potential in ozone and combinedstress-treated plants were similar to the control levelsby the end of treatment period. The marked changes inleaf water potential of drought stressed plants were alsoaccompanied by a significant decrease in their leaf watercontent (Supporting Information Fig. S1).

Stomatal conductanceThere were significant differences in the stomatal conduc-tance in response to stress treatments (Table 1, Fig. 2b).These effects were seen clearly within 1 d after imposition ofstress and at the end of the 6 d treatment in response todrought and combined stress treatments. Interestingly, sto-matal conductance at the end of first day of ozone treatmentwas lower than control plants, and by the end of the 6 d

treatment, it was comparable to values observed in controlplants.

Photosynthesis rateRates of photosynthesis were reduced by nearly 40% inJemalong in response to ozone and drought treatments bythe end of day 1 (Table 1, Fig. 2c). This decrease prevailedon the sixth day of drought treatment. Combined stresstreatment showed a trend towards reducing photosynthesis;however, it was not statistically significant.

TranspirationTranspiration rates were comparable to control plants atthe end of ozone treatment (Fig. 2d) and were consistentwith the observations on stomatal conductance (Fig. 2b).Transpiration rates at the end of 6 d drought and combinedstress treatments were only slightly lowered when com-pared to control plants (Table 1, Fig. 2d).

Biochemical responses to drought and ozone

Total ROSTotal ROS levels showed significant increases in responseto single stresses at the end of treatment regime (Table 1,

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Figure 2. Physiological responses of Medicago truncatula cv. Jemalong subjected to drought and ozone stress. Measurements of leafwater potential, stomatal conductance, photosynthesis rates and transpiration rates were conducted 1 and 6 d after the stress treatmentinitiation. (a). Leaf water potential; (b) stomatal conductance; (c) photosynthesis rate; (d) Transpiration rate. Error bars representstandard deviations (n = 4). aMeasurement that is significantly different compared with corresponding control samples (P � 0.05).



Fig. 3a). ROS levels nearly doubled in response to drought,while nearly threefold increase in ROS was recorded inresponse to ozone stress. Interestingly, ROS levels inresponse to combined stress were comparable to levelsin control plants (Table 1).

NOHighest NO levels were observed at the end of droughttreatment (Fig. 3b). Levels of NO in response to ozone atearly time point were lower than in control plants, andsignificantly lower than the levels in drought stressed plantsat both early and late time points. NO levels at the end ofthe combined stress treatment were similar to controlplants.

AsA and DHALevels of reduced AsA were significantly increased bycombined ozone and drought stress treatment (Fig. 3c).Drought stress did not alter the AsA levels, while ozonetreatment did lead to an increase in this important antioxi-dant. DHA levels showed an increase in response to singleand combined stress treatments within 1 d after treatment(Fig. 3d). Further increase in DHA levels were observed bythe end of 6 d ozone treatment. Levels of DHA at the endof the combined stress treatment returned to control levels.

GSH and GSSGInitial increases in GSH levels were consistently seen inboth single and combined stress treatments (Fig. 3e).

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Figure 3. Biochemical responses of Medicago truncatula cv. Jemalong subjected to drought and ozone stress. Measurements wereconducted 1 and 6 d after the stress treatment initiation. (a) Total reactive oxygen species (ROS); (b) nitric oxide (NO); (c) reducedascorbate (AsA); (d) dehydroascorbate (DHA); (e) reduced glutathione (GSH); (f) oxidized glutathione (GSSG). Measurements wereconducted from pooled tissues of three biological replicates and each measurement is the average of three technical replicates.aMeasurement that is significantly different compared with corresponding control (P � 0.05).RFU, relative fluorescent unit.



However, by the end of stress treatment, increase in GSHlevels in response to combined stress was 3–4-fold highercompared to control plants, and nearly twofold more thanin drought stress plants. Nearly 4–8-fold increase in GSSGlevels was recorded in response to drought, while ozone andcombined stress treatments showed a 2–3-fold increasecompared to corresponding controls (Fig. 3f).

Differential gene expression in response todrought and ozone

Changes in steady-state levels of transcripts in M. trunca-tula leaves subjected to ozone, drought or their combinationwere analysed using Affymetrix gene chips. Correlationsbetween the three biological replicates were between 0.87and 0.95, indicating high reproducibility of the stress treat-ments and the molecular techniques related to Affymetrixgene chip hybridizations. Reproducible differential expres-sion of 5390 probe sets was observed in drought or ozone orcombined ozone and drought stress treatments (SupportingInformation Tables S1 and S2). The largest number of dif-ferentially expressed genes was observed in response toozone (Fig. 4). Although least number of differentiallyexpressed probe sets was recorded in response to drought(Fig. 4), it had the largest number of uniquely up- or down-regulated probe sets among the three stressors tested.

Combined application of drought and ozone showed a sig-nificant overlap with ozone stress treatment on the basis ofthe number of commonly differentially expressed probesets. More than 1100 probe sets were commonly differen-tially expressed in all stress treatments (Fig. 4).

In the next stage of the analysis, we set out to identifyenriched GOs associated with each subgroup in the Venndiagram (Fig. 4). The Affymetrix probe set identifiers foreach of the subsets (Fig. 4) was loaded into agriGO and thedi-acyclic graphs showing over-represented GOs wereretrieved for categories with at least five mapping entries(Fig. 5, Supporting Information Fig. S2–S6). Increasingthe number of entries to more than five yielded veryfew enriched GOs, while lowering the number below fiveresulted in a large number of enriched GOs containing only3–4 probe sets representing only 1–2 genes.

Among the genes unique to combined stress, GOs asso-ciated with response to JA and innate immunity was signifi-cant (Fig. 5a). Glycoprotein catabolism was repressed.Among the genes that were induced in all stress treatments,several GO categories were significant. This included cellwall, amino acid and isoprenoid catabolism, trehaloseand flavonoid biosynthesis, SA and JA-mediated signallingpathways (Supporting Information Fig. S2). Among thegenes repressed in all the stress treatments, GOs for nitro-gen metabolism, peptide transport and inorganic ion trans-port were over-represented (Fig. 5b). Among the genesunique to drought stress, GOs associated with ABA signal-ling, proline biosynthesis, oxidative stress, heat stress andhighlight were significant (Supporting Information Fig. S3).Cell wall biogenesis, catabolism of amino acids, aminogly-cans and protein chromophore linkages were other GOssignificantly enriched in drought stress. Glucan metabolismwas significantly repressed by drought stress (SupportingInformation Fig. S4).

Maximum number of significantly enriched GOs wasobserved in response to ozone stress, consistent with thelargest number of differentially expressed genes. GOs asso-ciated with phenylalanine ammonia-lyase biosynthesis,glucose, sucrose and glucan metabolism were significantlyenriched (Supporting Information Fig. S5). Interestingly,GOs for circadian rhythm, photosynthetic electron trans-port, red or far-red light signalling and responses to tem-perature stimulus and inorganic substances were repressedin response to ozone treatment (Supporting InformationFig. S6).

Transcriptional regulationGO annotations for only about 70% of the M. truncatulaprobe sets were available in agriGO. Hence, we sought toidentify the Arabidopsis homologs for the probe sets on theMedicago Affymetrix array. Use of Arabidopsis identifiersresulted in the same set of enriched GOs as describedearlier. Only new GO that was identified using thisapproach was the transcription regulation category. Ofthe 51 probe sets that were responsive to both single andcombined stress, 23 were up-regulated and 28 were

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Figure 4. Venn diagram representation of the differentiallyexpressed genes in M. truncatula cultivar Jemalong in response todrought, ozone and combined stress. Numbers in regular fontrepresent induced genes while the numbers in italics representrepressed genes.



down-regulated. WRKY family members were predomi-nant among the transcription factors (TFs) common tosingle and combined stresses. Of the 22 probe sets uniquelydifferentially expressed in response to combined stress, 13were up-regulated and nine were down-regulated (Table 2).Several TFs such as MYC3 and WRKY50 unique to com-bined stress are associated with JA signalling. Fifteen probesets were unique to single stresses but were not differen-tially expressed in response to combined stress. Severalgenes annotated as ethylene response factors were identi-fied in response to ozone, while single AP2 domain contain-ing TFs such as DREB and RAPs were common to droughtstress.

DISCUSSION

In nature, plants are exposed to multiple stresses simulta-neously. Several studies have shown that the responses tocombined stresses are unique and cannot be extrapolated tothe observed responses to stresses when applied singly(Rizhsky et al. 2002, 2004; Mittler 2006; Jambunathan,

Puckette & Mahalingam 2010; Mittler & Blumwald 2010;Biswas & Jiang 2011). In this study, we report that simulta-neous application of drought and ozone evokes an attenu-ated phenotypic response in M. truncatula cv. Jemalong, incontrast to their extreme susceptibility to each of thesestressors singly (Fig. 1). Based on the changes in the physi-ological and biochemical parameters examined, it appearsthat drought stress was the most severe, followed by ozone,while the combination of ozone and drought was the leastdamaging (Table 1).

In pine, drought alone or combined drought and ozonestress lowered the needle water potential (Alonso et al.2001), which is in contrast to our studies (Fig. 2a). In theAlonso et al. (2001) study, pine seedlings were first sub-jected to ozone stress followed by drought, while in ourstudies, a mild drought was prevailing when the ozonestress was applied. The differences in the order in whichthe stressors were applied could also be a reason for theobserved differences between pine and Medicago. Thecontrasting observations could be simply due to differ-ences in the plant species. Net photosynthesis rates were

Figure 5. Gene ontology analysis of drought and ozone stress responsive genes in Medicago truncatula cv. Jemalong using agriGO.(a) Significantly over-represented GOs associated with genes induced in response to combined drought and ozone stress.(b) Significantly over-represented GOs associated with genes repressed in response to single and combined stress. The coloured boxesindicate significantly enriched GOs. The colour scale from yellow to red represents increasing levels of statistical significance as indicatedin the figure.



significantly down when the stresses were applied singly orin combination (Fig. 2c) supporting the notion that carbonfixation processes are compromised in response to envi-ronmental perturbations such as drought and ozone (Maet al. 2012; Wilkinson et al. 2012). Thus, even if the pheno-typic symptoms in response to combined stress were lesssevere compared with the single stresses, the short-termphysiological response, especially photosynthesis rate, wasnegatively affected. This response is again consistent withthe lower stomatal conductance that was observed inresponse to drought and combined stress (Fig. 2b). Severalstudies have shown that water stress may lower the effectsof ozone by reducing stomatal conductance and in turnlowering ozone uptake (Temple, Taylor & Benoit 1985;Tingey & Hogsett 1985; Pearson & Mansfield 1993;Reichenauer & Bolhar-Nordenkampf 1999). The higherstomatal conductance observed in response to ozone inJemalong may be due to stomatal sluggishness and couldbe caused by damage to ion channels (Torsethaugen et al.1999; Vahisalu et al. 2008). The changes in transpirationrates were in line with the stomatal conductance responsesto single and combined stresses. Thus, considering only thephysiological traits, it appears that lowering stomatal con-ductance may be an important stress avoidance strategy to

combined ozone and drought stress in Jemalong. Long-term exposure to drought and ozone stresses during cropseason will help assess the impact of lowered stomatalconductance on economically important traits such asyield and biomass.

ROS are key signalling molecules during environmentalperturbations and normal development (Mahalingam &Fedoroff 2003; Mittler et al. 2011). Numerous studies havedocumented changes in ROS in response to drought orozone (Kangasjarvi et al. 2005; Mahalingam et al. 2005;Puckette, Tang & Mahalingam 2008; Kar 2011; Lee & Park2012). A striking finding of our study is that combinedstress does not alter the ROS or NO levels in Jemalongduring the two tested time points (Fig. 3a,b). This may beattributed to active increases in reduced AsA levels(Fig. 2c) that have been touted as the first line of defenceagainst apoplastic ROS generators such as ozone (Kangas-jarvi et al. 1994; Conklin, Williams & Last 1996), as well asincreases in GSH (Fig 2e). It is also possible that there areother mechanisms apart from the AsA-GSH cycle forkeeping the ROS levels under control during combinedstress. An increase in ROS (as observed in ozone alone)can be accompanied by an increase in NO levels (asobserved in drought alone), and these reactive species

Table 2. Differentially expressedtranscription factor genes unique tocombined ozone and drought stress inMedicago truncatula

TIGR TC TAIR Hit Dr Oz Dr + Oz Brief description

TC126060 AT5G46760 1.28 1.70 2.39 MYC3 activates JA-responsesTC134696 AT4G23810 0.82 1.43 2.01 WRKY53 associated with senescenceTC125948 AT4G34410 1.43 1.50 2.53 Redox responsive subfamily B-3 of

ERF/AP2TC129809 AT1G46480 0.70 1.57 2.23 WUSCHEL-related homeobox geneBE940931 AT5G26170 1.55 1.80 2.68 WRKY50 – JA inducible defence

responsesTC112993 AT4G04450 1.38 1.95 2.08 WRKY 42BQ147546 AT1G68320 1.84 1.92 2.06 MYB62 -phosphate starvationTC132070 AT1G50600 1.29 1.62 2.03 scarecrow-like protein (SCL5), GRASTC129528 AT4G17785 1.88 1.87 2.64 Putative transcription factor, MYB39TC119107 AT5G57390 1.25 1.71 2.05 AINTEGUMENTA-LIKE 5TC121790 AT1G69580 1.92 1.55 2.43 Homeodomain-like superfamily proteinTC129232 AT5G46910 0.98 2.00 2.21 Jumonji – C5HC2 type protein, response

to chitinTC126482 AT1G56170 1.75 1.81 2.16 Nuclear factor Y-C2, NF-YC2, ER stressTC115105 AT5G61590 0.55 0.56 0.20 B-3 subfamily of ERF/AP2TC121489 AT1G76880 0.72 0.50 0.45 Homeodomain protein, SANT and Myb

domainTC119841 AT5G58900 0.53 0.54 0.43 Homeodomain-Myb-like, SANT, DNAj,

HSP40TC113329 AT4G34530 0.53 0.56 0.31 Cryptochrome-interacting basic-

helix-loop-helix, CIB1, floral transitionTC122656 AT4G24240 0.66 0.84 0.41 Calmodulin-binding WRKY7-repressorTC122656 AT1G29280 0.51 0.68 0.30 WRKY65TC121586 AT5G08520 0.67 0.82 0.46 Homeodomain- Myb-like, SANT, DNAj,

HSP40

Values in these columns represent average log2-fold change of treatment/control from threereplicate hybridizations. Entries in italicized font represent repressed genes.Dr, drought; Oz, ozone; Dr + Oz, combined drought and ozone stress; TIGR TC, tentativeconsensus identifiers for the Medicago Affymetrix probe sets obtained from TIGR; TAIRhit, best Arabidopsis match for the Medicago sequences retrieved from TAIR site.



could lead to the formation of less reactive peroxynitrite(Delledonne et al. 2001; Vandelle & Delledonne 2011)during combined stress. Using peroxynitrite specific dyessuch as HKGreen-2 (Sun et al. 2009; Gaupels et al. 2011)will be valuable to validate this hypothesis.

This begs the question why AsA levels were not elevatedin response to ozone alone in Jemalong. AsA-GSH cycleplays an important role in recycling DHA (Creissen & Mul-lineaux 2002). Recycling of DHA that accumulates in theapoplast in response to ozone is carried out by DHA reduc-tase enzyme using GSH as a reductant (Yoshida et al. 2006).We were not able to identify any probe sets for DHA reduc-tase in the Medicago Affymetrix array. It should also benoted that several studies in different plant systems haveindicated lack of correlation between transcript levels andprotein activity of AsA-GSH genes (Creissen & Mullineaux2002).

Since a mild drought stress was already in effect when theozone treatments were initiated, it could be argued that thereduced effects of ozone are due to stomatal closing inresponse to drought. We used the gene expression profilingdata to examine the hypothesis that reduced effect of ozonewhen combined with drought stress may be due to pro-cesses up-regulated by ozone that are negatively regulatedby drought but are not perturbed during combined stress.The GO for glucan metabolism was identified amongozone-induced genes and drought-repressed genes. Genesin glucan metabolism belonged to different members ofxyloglucan endotransglycosylase (XET) family andinvertase/pectin methylesterase family. Xyloglucan is ahemicellulose that is important for tightening or looseningcellulose microfibrils which in turn enable the cell to changeshape in growing zones and retain shapes after cell matu-ration (Hayashi & Kaida 2011). Down-regulation of XETgene family members in response to low water potential insoybeans and tobacco decreased cell wall extensibility butincreased wall thickness that could prevent water loss(Herbers et al. 2001; Wu et al. 2005). On the contrary,up-regulation of XETs in response to ozone may promotewall biogenesis and increase in cell or stomatal density asobserved in birch tress (Gunthardt-Goerg et al. 1993;Paakkonen et al. 1995; Frey et al. 1996; Kontunen-Soppelaet al. 2010). This increase in cell number may aid in evenozone distribution within leaf tissues for efficient detoxifi-cation processes (Paakkonen et al. 1995; Hetherington &Woodward 2003). Thus, transgenic approaches modulatingXET gene expression may not deliver the desired impactwhen combined ozone and drought stress are operativesimultaneously.

On the same lines of the above-stated hypothesis, weexamined for GO categories that are up-regulated indrought and down-regulated in response to ozone. GOsfor response to temperature and response to light stimuluswere identified in this analysis. The significant decreasein stomatal conductance to drought in Jemalong couldlead to an increase in the leaf temperature as reportedfor legumes (Reynolds-Henne et al. 2010). We speculatethat the observed increase in stomatal conductance and

transpiration rates in response to ozone treatment couldlead to a cooling effect and supports the observed down-regulation of genes responding to temperature stimulus.Thermal imaging of the leaf responses during drought andozone stress will verify these hypotheses. Closer inspectionof the gene function revealed that several of these genesare pseudo-response regulators essential for temperatureresponsiveness of the circadian clock (Salome & McClung2005). Circadian clocks evolved to enhance plant fitness toa perpetually changing environment and recent studiesusing Arabidopsis clock mutants have established inter-connections between stress, circadian clock and primarymetabolism (Kant et al. 2008; Fukushima et al. 2009; Leg-naioli, Cuevas & Mas 2009; Sanchez, Shin & Davis 2011).How combined occurrence of stresses can impact circa-dian clocks is not known yet and will be an important areaof research in the wake of global climate changes. One ofthe clock genes repressed in response to combined stressis the homolog of GIGANTEA, loss of function of whichin Arabidopsis leads to oxidative stress tolerance (Kurepaet al. 1998; Fowler et al. 1999; Park et al. 1999; Cao, Jiang &Zhang 2006). It is tempting to speculate that the down-regulation of M. truncatula Gigantea may play a similarrole in imparting tolerance to combined drought andozone stress.

One of the GOs unique to combined stress was related toinnate immunity. Innate immune response in plants is trig-gered by R-genes in response to biotic stresses (Felix et al.1999; Mizel et al. 2003; Spoel & Dong 2012). The role ofthese R-proteins in response to abiotic stress is not knowncurrently. In plant–pathogen interactions, R-proteins physi-cally interact with guard proteins such as RIN4 (Mackeyet al. 2002, 2003; Axtell & Staskawicz 2003; Kim et al. 2005b,2005a) that interacts with plasma membrane associatedH+-ATPases to regulate stomatal aperture (Liu et al. 2009).We speculate that some of the R-proteins identified in thisstudy may be associated with RIN4-like proteins that couldbe important in regulating stomatal behaviour duringcombined stress.

The modulation of TFs exclusive to combined stress oronly by drought or ozone alone highlights the plant’s plas-ticity in responding to environmental perturbations.Members of the AP2/ERF family confer tolerance to mul-tiple stresses (Xu et al. 2011) and are key regulators ofredox responsive gene networks (Khandelwal et al. 2008).Subtle changes in GSH levels could also play a crucial rolein the regulation of redox-responsive genes via componentssuch as redox-regulated NPR1 protein (Tada et al. 2008;Brosche & Kangasjarvi 2012). Activation of jasmonateresponsive MYC3 TF along with the JASMONATE ZIMDOMAIN (JAZ) family repressors JAZ1 and JAZ2 (Sup-porting Information Table S1) (Figueroa & Browse 2012;Kazan & Manners 2012) suggests the involvement of JAsignalling during combined stress and is currently underfurther investigation. Several WRKY TFs have been shownto be responsive to JA, biotic and abiotic stresses and stresscombinations (Rizhsky et al. 2002; Qiu & Yu 2009; Gao et al.2011; Peng et al. 2011). Identification of five different



WRKY family members in response to combined ozoneand drought stress suggests that this family of proteinshave a crucial role to play when multiple stressesoperate simultaneously. The Myb TFs such as Myb62 andSCARECROW-like TFs may be important for remodellingroot architecture during stress (Devaiah et al. 2009; Cui,Hao & Kong 2012). The novel Myb-like factors with SANTdomain and JUMNONJI could be involved in chromatinremodelling (Lu et al. 2011; Luo et al. 2012) during com-bined stress.

CONCLUSIONS

The set of biochemical traits analysed in this study showeddistinct signatures for ozone, drought and combined stresssuggesting changes in redox metabolism play a pivotal rolein determining the plant responses to single or combinedstresses. A large-scale metabolite analysis, especially phyto-hormones, will be valuable for understanding the signallingmechanisms invoked during combined stress. Based on asingle time-point transcriptome analysis, there was a con-siderable overlap in the genes expressed in response toozone and a combination of drought and ozone, consistentwith the similar phenotypic manifestations (chlorosis) tothese stresses. Despite short-term duration of stress regimeused in this study, the identification of 300 probe sets thatare unique to combined ozone and drought providescompelling molecular evidence that plants perceiveco-occurring ozone and drought as a new stress state. Sincechanges in gene expression are not necessarily tantamountto changes in protein levels or activity, this transcriptomeanalysis reflects a small proportion of the molecularresponse to combined ozone and drought stress. From theclimate change perspective, this study shows that there aregermplasm resources that can be exploited for breeding orengineering crop plants that can thrive well when droughtand ozone stress occur simultaneously.

ACKNOWLEDGMENTS

The National Research Initiative Competitive Grant no.2007–35100-18276 from the USDA National Institute ofFood and Agriculture, and Oklahoma Agricultural Experi-ment Station supported this project.

REFERENCES

Ahlfors R., Lang S., Overmyer K., et al. (2004) Arabidopsis radical-induced cell death1 belongs to the WWE protein–protein inter-action domain protein family and modulates abscisic acid,ethylene, and methyl jasmonate responses. The Plant Cell 16,1925–1937.

Ahlfors R., Brosche M., Kollist H. & Kangasjarvi J. (2009) Nitricoxide modulates ozone-induced cell death, hormone biosynthe-sis and gene expression in Arabidopsis thaliana. The PlantJournal 58, 1–12.

Ainsworth E.A., Rogers A. & Leakey A.D. (2008) Targets for cropbiotechnology in a future high-CO2 and high-O3 world. PlantPhysiology 147, 13–19.

Alonso R., Elvira S., Castillo F.J. & Gimeno B.S. (2001) Interactiveeffects of ozone and drought stress on pigments and activities ofantioxidative enzymes in Pinus halepensis. Plant, Cell & Envi-ronment 25, 905–916.

Arrigoni O. & De Tullio M.C. (2002) Ascorbic acid: much morethan just an antioxidant. Biochimica et Biophysica Acta 1569,1–9.

Ashmore M.R. (2005) Assessing the future global impacts of ozoneon vegetation. Plant, Cell & Environment 28, 949–964.

Atienza S.G., Faccioli P., Perrotta G., Dalfino G., Zschiesche W.,Humbeck K., Stanca A.M. & Cattivelli L. (2004) Large scaleanalysis of transcript abundance in barley subjected to severalsingle and combined abiotic stress conditions. Plant Science 167,1359–1365.

Axtell M.J. & Staskawicz B.J. (2003) Initiation of RPS2-specifieddisease resistance in Arabidopsis is coupled to the AvrRpt2-directed elimination of RIN4. Cell 112, 369–377.

Baier M., Kandlbinder A., Golldack D. & Dietz K.J. (2005) Oxida-tive stress and ozone: perception, signalling and response. Plant,Cell & Environment 28, 1012–1020.

Benedito V.A., Torres-Jerez I., Murray J.D., et al. (2008) A geneexpression atlas of the model legume Medicago truncatula. ThePlant Journal 55, 504–513.

Beyers J., Riechers G. & Temple P.J. (1992) Effects of long-termozone exposure and drought on the photosynthetic capacity ofponderosa pine (Pinus ponderosa Laws.). New Phytologist 122,81–90.

Biehler K. & Fock H. (1996) Evidence for the contribution of theMehler-peroxidase reaction in dissipating excess electrons indrought-stressed wheat. Plant Physiology 112, 265–272.

Bilgin D.D., Aldea M., O’Neill B.F., Benitez M., Li M., Clough S.J.& DeLucia E.H. (2008) Elevated ozone alters soybean–virusinteraction. Molecular Plant-Microbe Interactions 21, 1297–1308.

Biswas D.K. & Jiang G.M. (2011) Differential drought-inducedmodulation of ozone tolerance in winter wheat species. Journalof Experimental Botany 62, 4153–4162.

Brosche M. & Kangasjarvi J. (2012) Low antioxidant concentra-tions impact on multiple signalling pathways in Arabidopsisthaliana partly through NPR1. Journal of Experimental Botany63, 1849–1861.

Burkey K.O., Booker F.L., Pursley W.A. & Heagle A.S. (2007)Elevated carbon dioxide and ozone effects on peanut: II. Seedyield and quality. Crop Science 47, 1488–1497.

Cao S., Jiang S. & Zhang R. (2006) The role of GIGANTEA genein mediating the oxidative stress response and in Arabidopsis.Plant Growth Regulation 48, 261–270.

Casteel C.L., O’Neill B.F., Zavala J.A., Bilgin D.D., BerenbaumM.R. & Delucia E.H. (2008) Transcriptional profiling revealselevated CO2 and elevated O3 alter resistance of soybean(Glycine max) to Japanese beetles (Popillia japonica). Plant, Cell& Environment 31, 419–434.

Conklin P.L., Williams E.H. & Last R.L. (1996) Environmentalstress sensitivity of an ascorbic acid-deficient Arabidopsismutant. Proceedings of the National Academy of Sciences of theUnited States of America 93, 9970–9974.

Creissen G.P. & Mullineaux P. (2002) The Molecular Biology of theAscorbate-Glutathione Cycle in Higher Plants. Taylor andFrancis, London.

Cui H., Hao Y. & Kong D. (2012) SCARECROW has a SHORT-ROOT-independent role in modulating the sugar response. PlantPhysiology 158, 1769–1778.

Delledonne M., Zeier J., Marocco A. & Lamb C. (2001) Signalinteractions between nitric oxide and reactive oxygen interme-diates in the plant hypersensitive disease resistance response.Proceedings of the National Academy of Sciences of the UnitedStates of America 98, 13454–13459.



Devaiah B.N., Madhuvanthi R., Karthikeyan A.S. & RaghothamaK.G. (2009) Phosphate starvation responses and gibberellic acidbiosynthesis are regulated by the MYB62 transcription factor inArabidopsis. Molecular Plant 2, 43–58.

Dixon M., Le Thiec D. & Garrec J.P. (1998) Reactions of Norwaysprice and beech trees to 2 years of ozone and episodic drought.Environmental and Experimental Botany 40, 77–91.

Du Z., Zhou X., Ling Y., Zhang Z. & Su Z. (2010) agriGO: a GOanalysis toolkit for the agricultural community. Nucleic AcidsResearch 38, W64–W70.

Felix G., Duran J.D., Volko S. & Boller T. (1999) Plants have asensitive perception system for the most conserved domain ofbacterial flagellin. The Plant Journal 18, 265–276.

Figueroa P. & Browse J. (2012) The Arabidopsis JAZ2 promotercontains a G-Box and thymidine-rich module that are necessaryand sufficient for jasmonate-dependent activation by MYC tran-scription factors and repression by JAZ proteins. Plant and CellPhysiology 53, 330–343.

Fontaine V., Cabane M. & Dizengremel P. (2003) Regulation ofphosphoenolpyruvate carboxylase in Pinus halepensis needlessubmitted to ozone and water stress. Physiologia Plantarum 117,445–452.

Fowler S., Lee K., Onouchi H., Samach A., Richardson K., MorrisB., Coupland G. & Putterill J. (1999) GIGANTEA: a circadianclock-controlled gene that regulates photoperiodic flowering inArabidopsis and encodes a protein with several possiblemembrane-spanning domains. The EMBO Journal 18, 4679–4688.

Foyer C.H. & Noctor G. (2009) Redox regulation in photosyntheticorganisms: signaling, acclimation, and practical implications.Antioxidants & Redox Signaling 11, 861–905.

Frey B., Scheidegger C., Gunthardt-Goerg M.S. & Matyssek R.(1996) The effects of ozone and nutrient supply on stomatalresponse in birch (Betula pendula) leaves as determined bydigital image-analysis and x-ray microanalysis. New Phytologist32, 221–235.

Fukushima A., Kusano M., Nakamichi N., Kobayashi M., HayashiN., Sakakibara H., Mizuno T. & Saito K. (2009) Impact of clock-associated Arabidopsis pseudo-response regulators in metaboliccoordination. Proceedings of the National Academy of Sciencesof the United States of America 106, 7251–7256.

Gao Q.M., Venugopal S., Navarre D. & Kachroo A. (2011) Lowoleic acid-derived repression of jasmonic acid-inducible defenseresponses requires the WRKY50 and WRKY51 proteins. PlantPhysiology 155, 464–476.

Gaupels F., Spiazzi-Vandelle E., Yang D. & Delledonne M. (2011)Detection of peroxynitrite accumulation in Arabidopsis thalianaduring the hypersensitive defense response. Nitric Oxide 25,222–228.

Gillespie K.M., Xu F., Richter K.T., McGrath J.M., Markelz R.J.,Ort D.R., Leakey A.D. & Ainsworth E.A. (2012) Greater anti-oxidant and respiratory metabolism in field-grown soybeanexposed to elevated O3 under both ambient and elevated CO2.Plant, Cell & Environment 35, 169–184.

Grulke N.E., Johnson R., Monschein S., Nikolova P. & Tausz M.(2003) Variation in morphological and biochemical O3 injuryattributes of mature Jeffrey pine within canopies and betweenmicrosites. Tree Physiology 23, 923–929.

Grulke N.E., Dobrowolski W., Mingus P. & Fenn M.E. (2005) Cali-fornia black oak response to nitrogen amendment at a high O3,nitrogen-saturated site. Environmental Pollution 137, 536–545.

Grunehage L. & Jager H.-J. (2003) From critical levels to criticalloads of ozone: a discussion of a new experimental and modelingapproach for establishing flux–response relationships for agricul-tural crops and native plant species. Environmental Pollution125, 99–110.

Guidi L., Tonini M. & Soldatini G.F. (2000) Effects of high light andozone fumigation on photosynthesis in Phaseolus vulgaris. PlantPhysiology and Biochemistry 38, 717–725.

Gunthardt-Goerg M.S., Matyssek R., Scheidegger C. & Keller T.(1993) Differentiation and structural decline in the leaves andbark of birch (Betula pendula) under low ozone conentrations.Trees (Berlin) 7, 104–114.

Gupta P., Duplessis S., White H., Karnosky D.F., Martin F.& Podila G.K. (2005) Gene expression patterns of trembl-ing aspen trees following long-term exposure to interactingelevated CO2 and tropospheric O3. New Phytologist 167, 129–141.

Hayashi T. & Kaida R. (2011) Functions of xyloglucans in plants.Molecular Plant 4, 17–24.

Heagle A.S. (1989) Ozone and crop yield. Annual Review of Phy-topathology 27, 397–412.

Herbers K., Lorences E.P., Barrachina C. & Sonnewald U. (2001)Functional characterisation of Nicotiana tabacum xyloglucanendotransglycosylase (NtXET-1): generation of transgenictobacco plants and changes in cell wall xyloglucan. Planta 212,279–287.

Hetherington A.M. & Woodward F.I. (2003) The role of stomatain sensing and driving environmental change. Nature 424, 901–908.

Jambunathan N., Puckette M. & Mahalingam R. (2010) Transcrip-tomic analysis of combined stresses in plants. In Molecular Tech-niques for Crop Improvement (eds M. Jain & D.S. Brar) pp.511–524. Springer, New York, NY.

Kangasjarvi J., Talvinen J., Utriainen M. & Karjalainen R. (1994)Plant defense systems induced by ozone. Plant, Cell & Environ-ment 17, 783–794.

Kangasjarvi J., Jaspers P. & Kollist H. (2005) Signalling and celldeath in ozone-exposed plants. Plant, Cell & Environment 28,1021–1036.

Kant P., Gordon M., Kant S., Zolla G., Davydov O., Heimer Y.M.,Chalifa-Caspi V., Shaked R. & Barak S. (2008) Functional-genomics-based identification of genes that regulate Arabidopsisresponses to multiple abiotic stresses. Plant, Cell & Environment31, 697–714.

Kar R.K. (2011) Plant responses to water stress: role of reactiveoxygen species. Plant Signal Behav 6, 1741–1745.

Karlsson P.E., Medin E.L., Wallin G., Sellden G. & Skarby L. (1997)Effects of ozone and drought stress on the physiology andgrowth of two clones of Norway spruce (Picea abies). New Phy-tologist 136, 265–275.

Karnosky D.F., Pregitzer K.S., Zak D.R., Kubiske M.E., HendreyG.R., Weinstein D., Nosal M. & Percy K.E. (2005) Scaling ozoneresponses of forest trees to the ecosystem level in a changingclimate. Plant, Cell & Environment 28, 965–981.

Kazan K. & Manners J.M. (2012) JAZ repressors and the orches-tration of phytohormone crosstalk. Trends in Plant Science 17,22–31.

Khandelwal A., Elvitigala T., Ghosh B. & Quatrano R.S. (2008)Arabidopsis transcriptome reveals control circuits regulatingredox homeostasis and the role of an AP2 transcription factor.Plant Physiology 148, 2050–2058.

Kim H.S., Desveaux D., Singer A.U., Patel P., Sondek J. & DanglJ.L. (2005b) The Pseudomonas syringae effector AvrRpt2cleaves its C-terminally acylated target, RIN4, from Arabidopsismembranes to block RPM1 activation. Proceedings of theNational Academy of Sciences of the United States of America102, 6496–6501.

Kim M.G., Da Cunha L., McFall A.J., Belkhadir Y., DebRoy S.,Dangl J.L. & Mackey D. (2005a) Two Pseudomonas syringaetype III effectors inhibit RIN4-regulated basal defense inArabidopsis. Cell 121, 749–759.



King D.A. (1988) Modeling the impact of ozone ¥ drought interac-tions on regional crop yields. Environmental Pollution 53, 351–364.

Kontunen-Soppela S., Riikonen J., Ruhanen H., et al. (2010) Dif-ferential gene expression in senescing leaves of two silver birchgenotypes in response to elevated CO2 and tropospheric ozone.Plant, Cell & Environment 33, 1016–1028.

Kurepa J., Smalle J., Van Montagu M. & Inze D. (1998) Oxidativestress tolerance and longevity in Arabidopsis: the late-floweringmutant gigantea is tolerant to paraquat. The Plant Journal 14,759–764.

Lee S. & Park C.M. (2012) Regulation of reactive oxygen speciesgeneration under drought conditions in Arabidopsis. PlantSignal Behavior 7, 599–601.

Legnaioli T., Cuevas J. & Mas P. (2009) TOC1 functions as amolecular switch connecting the circadian clock with plantresponses to drought. The EMBO Journal 28, 3745–3757.

Liu J., Elmore J.M., Fuglsang A.T., Palmgren M.G., Staskawicz B.J.& Coaker G. (2009) RIN4 functions with plasma membraneH+-ATPases to regulate stomatal apertures during pathogenattack. PLoS Biology 7, e1000139.

Lorenzini G., Nali C. & Panicucci A. (1994) Surface ozone in Pisa(Italy) – a 6-year study. Atmospheric Environment 28, 3155–3164.

Lu S.X., Knowles S.M., Webb C.J., Celaya R.B., Cha C., Siu J.P. &Tobin E.M. (2011) The Jumonji C domain-containing proteinJMJ30 regulates period length in the Arabidopsis circadianclock. Plant Physiology 155, 906–915.

Luo M., Liu X., Singh P., Cui Y., Zimmerli L. & Wu K. (2012)Chromatin modifications and remodeling in plant abiotic stressresponses. Biochimica et Biophysica Acta 1819, 129–136.

Ma Z., Peng C., Zhu Q., Chen H., Yu G., Li W., Zhou X., Wang W.& Zhang W. (2012) Regional drought-induced reduction in thebiomass carbon sink of Canada’s boreal forests. Proceedings ofthe National Academy of Sciences of the United States of America109, 2423–2427.

McCurdy T.R. (1994) Tropospheric Ozone (ed. D.J. McKee), pp.19–37. Lewis Publishers, Boca Raton, FL, USA.

Mackey D., Holt B.F. 3rd, Wiig A. & Dangl J.L. (2002) RIN4interacts with Pseudomonas syringae type III effector moleculesand is required for RPM1-mediated resistance in Arabidopsis.Cell 108, 743–754.

Mackey D., Belkhadir Y., Alonso J.M., Ecker J.R. & Dangl J.L.(2003) Arabidopsis RIN4 is a target of the type III virulenceeffector AvrRpt2 and modulates RPS2-mediated resistance. Cell112, 379–389.

Mahalingam R. & Fedoroff N. (2003) Stress response, cell deathand signalling: the many faces of reactive oxygen species. Physi-ologia Plantarum 119, 56–68.

Mahalingam R., Shah N., Scrymgeour A. & Fedoroff N. (2005)Temporal evolution of the Arabidopsis oxidative stress response.Plant Molecular Biology 57, 709–730.

Mahalingam R., Jambunathan N., Gunjan S.K., Faustin E., Weng H.& Ayoubi P. (2006) Analysis of oxidative signalling induced byozone in Arabidopsis thaliana. Plant, Cell & Environment 29,1357–1371.

Mauzerall D.L. & Wang X. (2001) Protecting agricultural cropsfrom the effects of tropospheric ozone exposure: reconcilingscience and standard setting in the United States, Europe andAsia. Annual Review of Energy and Environment 26, 237–268.

Miller J.D., Arteca R.N. & Pell E.J. (1999) Senescence-associatedgene expression during ozone-induced leaf senescence in Arabi-dopsis. Plant Physiology 120, 1015–1023.

Mittler R. (2002) Oxidative stress, antioxidants and stress toler-ance. Trends in Plant Science 7, 405–410.

Mittler R. (2006) Abiotic stress, the field environment and stresscombination. Trends in Plant Science 11, 15–19.

Mittler R. & Blumwald E. (2010) Genetic engineering for modernagriculture: challenges and perspectives. Annual Review of PlantBiology 61, 443–462.

Mittler R., Merquiol E., Hallak-Herr E., Rachmilevitch S., KaplanA. & Cohen M. (2001) Living under a ‘dormant’ canopy: amolecular acclimation mechanism of the desert plant Retamaraetam. The Plant Journal 25, 407–416.

Mittler R., Vanderauwera S., Suzuki N., Miller G., Tognetti V.B.,Vandepoele K., Gollery M., Shulaev V. & Van Breusegem F.(2011) ROS signaling: the new wave? Trends in Plant Science 16,300–309.

Mizel S.B., Honko A.N., Moors M.A., Smith P.S. & West A.P. (2003)Induction of macrophage nitric oxide production by Gram-negative flagellin involves signaling via heteromeric toll-likereceptor 5/toll-like receptor 4 complexes. Journal of Immunol-ogy 170, 6217–6223.

Moffat A.S. (2002) Plant genetics – finding new ways to protectdrought-stricken plants. Science 296, 1226–1229.

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

Overmyer K., Brosche M. & Kangasjarvi J. (2003) Reactive oxygenspecies and hormonal control of cell death. Trends in PlantScience 8, 335–342.

Paakkonen E., Metsarinne S., Holopainen T. & Karenlampi L.(1995) The ozone sensitivity of birch (Betula pendula Roth.) inrelation to the developmental stage of leaves. New Phytologist132, 145–154.

Paakkonen E., Vahala J., Pohjola M., Holopainen T. & KarenlampiL. (1998) Physiological, stomatal and ultrastructural ozoneresponses in birch (Betula pendula Roth) are modified by waterstress. Plant, Cell & Environment 21, 671–684.

Panek J.A. & Goldstein A.H. (2001) Response of stomatal conduc-tance to drought in ponderosa pine: implications for carbon andozone uptake. Tree Physiology 21, 337–344.

Panek J.A., Kurpius M.R. & Goldstein A.H. (2002) An evaluationof ozone exposure metrics for a seasonally drought-stressed pon-derosa pine ecosystem. Environmental Pollution 117, 93–100.

Park D.H., Somers D.E., Kim Y.S., Choy Y.H., Lim H.K., Soh M.S.,Kim H.J., Kay S.A. & Nam H.G. (1999) Control of circadianrhythms and photoperiodic flowering by the ArabidopsisGIGANTEA gene. Science 285, 1579–1582.

Pearson M. & Mansfield T.A. (1993) Interacting effects of ozoneand water stress on the stomatal resistance of beech (Fagussylvatica L.). New Phytologist 1123, 351–358.

Pell E.J., Schlagnhaufer C.D. & Arteca R.N. (1997) Ozone-inducedoxidative stress: mechanisms of action and reaction. PhysiologiaPlantarum 100, 264–273.

Peng X.X., Tang X.K., Zhou P.L., Hu Y.J., Deng X.B., He Y. &Wang H.H. (2011) Isolation and expression patterns of riceWRKY82 transcription factor gene responsive to both biotic andabiotic stresses. Agricultural Sciences in China 10, 893–901.

Potters G., Horemans N., Caubergs R.J. & Asard H. (2000) Ascor-bate and dehydroascorbate influence cell cycle progression in atobacco cell suspension. Plant Physiology 124, 17–20.

Puckette M.C., Weng H. & Mahalingam R. (2007) Physiologicaland biochemical responses to acute ozone-induced oxidativestress in Medicago truncatula. Plant Physiology and Biochemistry45, 70–79.

Puckette M.C., Tang Y. & Mahalingam R. (2008) Transcriptomicchanges induced by acute ozone in resistant and sensitive Medi-cago truncatula accessions. BMC Plant Biology 8, 46.

Qiu Y.P. & Yu D.Q. (2009) Over-expression of the stress-inducedOSWRKY45 enhances disease resistance and drought tolerance



in Arabidopsis. Environmental and Experimental Botany 65,35–47.

Quartacci M.F. & Navariizzo F. (1992) Water-stress and free-radical mediated changes in sunflower seedlings. Journal of PlantPhysiology 139, 621–625.

Rao M.V. & Davis K.R. (2001) The physiology of ozone inducedcell death. Planta 213, 682–690.

Rao M.V., Koch J.R. & Davis K.R. (2000) Ozone: a tool for probingprogrammed cell death in plants. Plant Molecular Biology 44,345–358.

Rao M.V., Lee H. & Davis K.R. (2002) Ozone-induced ethyleneproduction is dependent on salicylic acid, and both salicylic acidand ethylene act in concert to regulate ozone-induced cell death.The Plant Journal 32, 447–456.

Reich P.B. (1987) Quantifying plant response to ozone: a unifyingtheory. Tree Physiology 3, 63–91.

Reichenauer T.G. & Bolhar-Nordenkampf H.R. (1999) Doesozone exposure protect from photoinhibition? Free RadicalResearch 31, S193–S198.

Reynolds-Henne C.E., Langenegger A., Mani J., Schenk N.,Zumsteg A. & Feller U. (2010) nteractions between tempera-ture, drought and stomatal opening in legumes. Environmentaland Experimental Botany 68, 37–43.

Rizhsky L., Liang H.J. & Mittler R. (2002) The combined effect ofdrought stress and heat shock on gene expression in tobacco.Plant Physiology 130, 1143–1151.

Rizhsky L., Liang H.J., Shuman J., Shulaev V., Davletova S. &Mittler R. (2004) When Defense pathways collide. The responseof Arabidopsis to a combination of drought and heat stress. PlantPhysiology 134, 1683–1696.

Rouhier N., Lemaire S.D. & Jacquot J.P. (2008) The role of glu-tathione in photosynthetic organisms: emerging functions forglutaredoxins and glutathionylation. Annual Review of PlantBiology 59, 143–166.

Salome P.A. & McClung C.R. (2005) PSEUDO-RESPONSEREGULATOR 7 and 9 are partially redundant genes essentialfor the temperature responsiveness of the Arabidopsis circadianclock. The Plant Cell 17, 791–803.

Sanchez A., Shin J. & Davis S.J. (2011) Abiotic stress and the plantcircadian clock. Plant Signal Behavior 6, 223–231.

Spoel S.H. & Dong X. (2012) How do plants achieve immunity?Defence without specialized immune cells. Nature ReviewsImmunology 12, 89–100.

Sun Z.N., Wang H.L., Liu F.Q., Chen Y., Tam P.K. & Yang D.(2009) BODIPY-based fluorescent probe for peroxynitritedetection and imaging in living cells. Organic Letters 11, 1887–1890.

Tada Y., Spoel S.H., Pajerowska-Mukhtar K., Mou Z., Song J., WangC., Zuo J. & Dong X. (2008) Plant immunity requires conforma-tional changes [corrected] of NPR1 via S-nitrosylation andthioredoxins. Science 321, 952–956.

Temple P.J., Taylor O.C. & Benoit L.F. (1985) Cotton yieldresponses to ozone as mediated by soil moisture and evapotrans-piration. Journal of Environmental Quality 14, 55–60.

Temple P.J., Riechers G., Miller P.R. & Lennox R.W. (1993) Growthresponses of ponderosa pine to long-term exposure to ozone, wetand dry acidic deposition and drought. Candian Journal of ForestResearch 23, 59–66.

Tester M. & Bacic A. (2005) Abiotic stress tolerance in grasses.From model plants to crop plants. Plant Physiology 137, 791–793.

Tingey D.T. & Hogsett W.E. (1985) Water-stress reducesozone injury via a stomatal mechanism. Plant Physiology 77,944–947.

Torsethaugen G., Pell E.J. & Assmann S.M. (1999) Ozone inhibitsguard cell K+ channels implicated in stomatal opening.

Proceedings of the National Academy of Sciences of the UnitedStates of America 96, 13577–13582.

Vahisalu T., Kollist H., Wang Y.F., et al. (2008) SLAC1 is requiredfor plant guard cell S-type anion channel function in stomatalsignalling. Nature 452, 487–U415.

Vandelle E. & Delledonne M. (2011) Peroxynitrite formation andfunction in plants. Plant Science 181, 534–539.

Vivancos P.D., Dong Y., Ziegler K., Markovic J., Pallardo F.V.,Pellny T.K., Verrier P.J. & Foyer C.H. (2010) Recruitment ofglutathione into the nucleus during cell proliferation adjustswhole-cell redox homeostasis in Arabidopsis thaliana andlowers the oxidative defence shield. The Plant Journal 64, 825–838.

Wilkinson S. & Davies W.J. (2009) Ozone suppresses soil drying-and abscisic acid (ABA)-induced stomatal closure via anethylene-dependent mechanism. Plant, Cell & Environment 32,949–959.

Wilkinson S. & Davies W.J. (2010) Drought, ozone, ABA and eth-ylene: new insights from cell to plant to community. Plant, Cell &Environment 33, 510–525.

Wilkinson S., Mills G., Illidge R. & Davies W.J. (2012) How is ozonepollution reducing our food supply? Journal of ExperimentalBotany 63, 527–536.

Winner W.E., Lefohn S., Cotter I.S., Greitner C.S., Nellessen J.,Mcevoy L.R., Olson R.L., Atkinson C.J. & Moore L.D. (1989)Plant-responses to elevational gradients of O3 exposures in Vir-ginia. Proceedings of the National Academy of Sciences of theUnited States of America 86, 8828–8832.

Wu Y., Jeong B.R., Fry S.C. & Boyer J.S. (2005) Change inXET activities, cell wall extensibility and hypocotyl elongationof soybean seedlings at low water potential. Planta 220, 593–601.

Xu Z.S., Chen M., Li L.C. & Ma Y.Z. (2011) Functions andapplication of the AP2/ERF transcription factor family in cropimprovement. Journal of Integrative Plant Biology 53, 570–585.

Yoshida S., Tamaoki M., Shikano T., et al. (2006) Cytosolic dehy-droascorbate reductase is important for ozone tolerance in Ara-bidopsis thaliana. Plant and Cell Physiology 47, 304–308.

Received 8 May 2012; received in revised form 20 August 2012;accepted for publication 22 August 2012

SUPPORTING INFORMATION

Additional Supporting Information may be found in theonline version of this article:

Figure S1. Relative water content of control plants andplants under drought stress for 6 d. Four replicates from 10randomly picked leaves were used for assaying the relativewater content of the leaves. Error bars represent standarddeviations.Figure S2. Output from agriGO analysis showing signifi-cantly over-represented gene ontologies induced inresponse to single and combined drought and ozone stress.Figure S3. Output from agriGO analysis showing signifi-cantly over-represented gene ontologies induced inresponse to drought stress.Figure S4. Output from agriGO analysis showing signifi-cantly over-represented gene ontologies repressed inresponse to drought stress.



Figure S5. Output from agriGO analysis showing signifi-cantly over-represented gene ontologies induced inresponse to ozone stress.Figure S6. Output from agriGO analysis showing signifi-cantly over-represented gene ontologies repressed inresponse to ozone stress.

Table S1. List of genes up-regulated in response to singleand/or combined drought and ozone stress.Table S2. List of genes down-regulated in response tosingle and/or combined drought and ozone stress.



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Physiological, biochemical and molecular responses to a combination of drought and ozone in Medicago truncatula