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


R. Mahalingam. Fax: +405-744-7799; e-mail:


Drought and tropospheric ozone are escalating climate change problems that can co-occur. In this study, we observed Medicago truncatula cultivar Jemalong that is sensitive to ozone and drought stress when applied singly, showed tolerance when subjected to a combined application of these stresses. Lowered stomatal conductance may be a vital tolerance mechanism to overcome combined ozone and drought. Sustained increases in both reduced ascorbate and glutathione in response to combined stress may play a role in lowering reactive oxygen species and nitric oxide toxicity. Transcriptome analysis indicated that genes associated with glucan metabolism, responses to temperature and light signalling may play a role in dampening ozone responses due to drought-induced stomatal closure during combined occurrence of these two stresses. Gene ontologies for jasmonic acid signalling and innate immunity were enriched among the 300 differentially expressed genes unique to combined stress. Differential expression of transcription factors associated with redox, defence signalling, jasmonate responses and chromatin modifications may be important for evoking novel gene networks during combined occurrence of drought and ozone. The alterations in redox milieu and distinct transcriptome changes in response to combined stress could aid in tweaking the metabolome and proteome to annul the detrimental effects of ozone and drought in Jemalong.


In field conditions, plants face several distinct environmental extremes either simultaneously or at different times during the growing season (Tester & Bacic 2005). For example, drought and heat stress represent the conditions commonly encountered by plants growing in arid regions (Mittler et al. 2001; Moffat 2002). Analysis of these two stresses in Arabidopsis and tobacco plants showed that combination of heat and drought affected plants differently when compared with drought or heat stress applied individually with reference to photosynthesis, respiration, stomatal conductance and leaf temperature (Rizhsky, Liang & Mittler 2002; Rizhsky et al. 2004). In the wake of global climate change caused by increasing levels of CO2, several studies reported on the interactive effects of CO2 and tropospheric 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 plant biomass (Heagle 1989) by affecting allocation of assimilates and induces senescence process in plants (Pell, Schlagnhaufer & Arteca 1997; Miller, Arteca & Pell 1999). Highest ozone concentrations usually occur around midday and during summer season (Lorenzini, Nali & Panicucci 1994), in conjunction with high light and/or drought. Studies on combined effects of high light and ozone in Phaseolus vulgaris indicated that the former exacerbated the detrimental effects of ozone on photosynthesis (Guidi, Tonini & Soldatini 2000). Interactive effects of ozone and drought have been well studied in tree species. Birch saplings grown under field conditions showed extensive ozone injury, reduced height and lowered stomatal density under restricted water supply conditions (Paakkonen et al. 1998). Field experiments on combined ozone and drought in Norway spruce and beech revealed that ozone affected growth and biomass very differently in two different clones of these trees (Dixon, Le Thiec & Garrec, 1998, Karlsson et al. 1997). In the needles of conifers, the impact of combined ozone and drought seems to vary depending on the species. For example, drought prevents ozone injury in ponderosa pine (Beyers, Riechers & Temple 1992; Temple et al. 1993), while ozone retards the ability of Aleppo pine to tolerate drought (Alonso et al. 2001). Modelling studies on the interactive effects of drought and ozone using yield forecasting predicted a general drought-induced reduction of crop sensitivity to ozone and it varied between crops, regions and years (King 1988).

Since the flux of ozone into the plant is through the stomata, it has been argued that differences in sensitivity of plants to this pollutant are partly due to differences in stomatal conductance (Reich 1987). Based on the ozone uptake models, drought-induced stomatal closure would limit ozone uptake into leaves and hence protect plants from ozone stress (Panek & Goldstein 2001; Panek, Kurpius & Goldstein 2002; Grunehage & Jager 2003). However, this simplistic model was challenged by observations that ozone caused stomatal ‘sluggishness’ that leads to incomplete stomatal closure and hence exacerbated the effects of drought (Grulke et al. 2003, 2005; Karnosky et al. 2005). The presence of night-time ozone in rural locations (McCurdy 1994) could also greatly reduce the biomass of plants (Winner et al. 1989), especially if ozone hindered opening of guard cells after a dark exposure (Torsethaugen, Pell & Assmann 1999). These studies showed that physiological impacts of ozone and water deprivation together on plants are complex and merits attention given that concentrations of ozone in the troposphere will continue to increase in the future (Ashmore 2005; Ainsworth, Rogers & Leakey 2008).

A common biochemical response in plants to stresses including ozone and drought is the production of reactive oxygen species (ROS) (Mittler 2002; Mahalingam & Fedoroff 2003). In fact, ozone toxicity in plants is related to the formation 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 production of free radicals (Quartacci & Navariizzo 1992; Biehler & Fock 1996). Changes in ROS levels in turn perturb the redox homeostasis via changes in ascorbate (AsA) and glutathione (GSH) (Noctor & Foyer 1998). Apart from ROS scavenging, both AsA and GSH play a role in development via regulating cell cycle and serving as cofactors for other proteins (Potters et al. 2000; Arrigoni & De Tullio 2002; Rouhier, Lemaire & Jacquot 2008; Vivancos et al. 2010). Thus, changes in redox balance can have a significant impact on the proteome both during development and stress (Foyer & Noctor 2009).

ROS and NO bursts induced by stressors like ozone in turn affect interactions between phytohormones jasmonic acid (JA), salicylic acid (SA), ethylene and abscisic acid (ABA) (Rao, Koch & Davis 2000; Rao & Davis 2001; Overmyer, Brosche & Kangasjarvi 2003). Increase in ozone-induced ethylene biosynthesis that is dependent on a functional SA signalling pathway leads to increase in ABA biosynthesis which regulates stomatal conductance (Rao, Lee & Davis 2002; Ahlfors et al. 2004). Interestingly, ozone has been shown to suppress ABA-induced stomatal closure via an ethylene-dependent mechanism (Wilkinson & Davies 2009, 2010). Thus, changes in the biochemical milieu in responses to single stresses and combined stresses synergize or antagonize different hormonal signalling pathways that ultimately trigger distinct physiological responses.

Since physiological and biochemical changes are mostly regulated by proteins, efforts were focused on isolation and characterization of protein coding genes such as phosphoenolpyruvate caraboxylase and ribulose 1·5-bisphosphate carboxylase/oxygenase activase, commonly induced by combined stresses (Fontaine, Cabane & Dizengremel 2003). Recently, genomics approaches using microarrays to analyse transcriptional changes to combined stresses clearly showed that responses of plants to combined stresses were markedly different when compared with response to individual 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 gene expression in response to combined ozone and drought stress in Medicago truncatula cultivar Jemalong that was previously identified as an ozone-sensitive line (Puckette, Weng & Mahalingam 2007). Changes in gene expression were juxtaposed with measurements of physiological and biochemical traits following single and combined ozone and drought stresses. This integrative analysis led to the identification of several novel mechanisms that may be useful for developing resistance to combined occurrences of ozone and drought stress in plants.


Plant material and growing conditions

M. truncatula cultivar Jemalong was selected for this study based on their sensitive responses to ozone and drought (Puckette et al. 2007). Plants were grown in 10″ × 10″ trays filled with 750 g of MetroMix 200 (Scotts-Sierra Horticultural Products Company, Marysville, OH, USA). Excess seedlings 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 µmol s−1 m−2, 10 h/14 h day and night conditions. Each tray was irrigated with 600 mL of water. Watering was done every 3 d, maintaining the moisture levels in each tray as closely as possible. Trays were randomly rearranged every 3 d to minimize variation in microenvironment among trays in different shelves and in the two different growth chambers. All the plants were maintained 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, ozone and combined drought and ozone stress.

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

Ozone stress was imposed on 54-day-old plants using an earlier described ozone generation setup (Puckette et al. 2007) by exposing plants to 1.75 times of the ambient ozone levels (70 nmol mol−1) for 6 h per day for 6 consecutive days. The AOT40 value for this short-term ozone exposure treatment was 140 ppbh and was calculated using the formula:


where Ci is the average ozone concentration used for the treatment, T is the threshold concentration, h is the total possible number of hours over the investigation period and h0 is the number of measured hourly values (Mauzerall & Wang 2001).

Water levels in ozone-treated plants were maintained to same levels as in control plants. The stress treatments were repeated three times. Symptoms on the plants were evaluated visually at the end of stress treatments. The total number of leaves and number of symptomatic leaves on each plant at the end of stress treatment were recorded and used for calculating the percentage of leaves showing symptoms such as chlorosis and necrotic lesions.

Chlorophyll content

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

Leaf water potential and relative water content

Leaf water potential in control, drought, ozone and combined stress-treated plants were measured using leaf psychrometers on days 57 and 60 (3 and 6 d after treatment initiation, respectively). All the psychrometers were calibrated prior to the experiment. Five replicate samples were assayed for this experiment. Values of leaf water potential were corrected to 27.5 °C and also with the calibration factor for each psychrometer. The average values and standard deviations were calculated in Excel.

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

Stomatal conductance, photosynthesis and transpiration rate

Stomatal conductance, photosynthesis and transpiration rates were measured 1 d after the initiation of the stress treatments and at the end of the 6 d stress treatments. Leaflets of treated and control plants were sealed in a gas exchange cuvette of Li-Cor 6400 Portable Photosynthesis system (Li-Cor, Lincoln, NE, USA). An Arabidopsis cuvette was used to snugly fit the small leaflet of Medicago. Measurements were taken under the following conditions: 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%. Measurements were taken 5 min after the leaves were sealed in cuvette under conditions described earlier. Fifteen leaflets sampled from different plants were tested for each of the treatments.

ROS, NO and antioxidant assays

ROS, NO and antioxidants were measured 1 d after treatment initiation and at the end of 6 d treatment. ROS levels were measured in control, drought, ozone and combined drought- and ozone stress-treated leaves using a Versa-Fluor Fluorometer (Bio-Rad, Hercules, CA, USA) as described earlier (Mahalingam et al. 2006). The total ROS levels in each sample were plotted as relative fluorescence units (RFU) per milligram of protein. NO levels were measured using a spectrophotometer as described earlier (Mahalingam et al. 2006).

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

Statistical analyses

R-studio was to conduct two-way analysis of variance to determine treatment effects (drought, ozone, combined ozone and drought), at the beginning (day 1) and at the end of treatment (day 6) and treatment × day interactions. The various physiological and biochemical measurements were input as variable factor. Tukey's honestly significant differences between control and treatments were calculated in R-studio and adjusted P-value of <0.05 was used as a cut-off for identifying traits showing significant effect in response to stress treatments.

RNA isolations and microarray hybridizations

Leaf tissues from control and treated plants were harvested at the end of stress treatments (10 d of drought stress, 6 d of ozone treatment and combined drought and ozone) snap frozen in liquid nitrogen. Total RNA isolations were done using Trizol reagent (Invitrogen, Carlsbad, CA, USA), followed 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 µg of purified total RNA was used as template for hybridization. cDNA synthesis, amplification, probe labelling and hybridizations were conducted as described in the manufacturer's instructions (Affymetrix, Santa Clara, CA, USA). Three biological replicates were used for hybridizations with Genechip Medicago genome arrays (Affymetrix).

Microarray data analyses

Data extraction, normalization and identification of differentially expressed genes were conducted as described earlier (Benedito et al. 2008). Changes in RNA abundance in response to stress treatment were obtained by calculation of the signal log2 ratio of each gene signal in the control RNA samples relative to the stress treatment RNA samples, with values from the latter in the numerator. Probe sets that showed twofold or greater differences in transcript levels in two or more replicates were selected. Furthermore, in order to minimize family-wide error rates in multiple comparisons, only probe sets with Bonferroni corrected P-value <9.8e-07 were selected for further analysis. All the raw data related to the microarray experiments in this study have been submitted to MIAMEXPRESS database ( under the accession number E-MEXP-3657.

Enriched gene ontologies

Differentially expressed genes from each treatment were analysed to determine the overlap between the genes induced or repressed in response to drought or ozone and combined drought and ozone treatments. The Medicago Affymetrix identifiers for each category were input for singular enrichment analysis in the agriGO toolkit ( (Du et al. 2010). Medicago Affymetrix genome array was selected as background. Fisher's statistical test and Yekutieli [false discovery rate (FDR) under dependency] for multi-test adjustment method were selected to identify enriched gene ontology (GO) categories with five or more mapping entries and significance level of 0.05. Graphical outputs of the significant GO terms were generated by agriGO.


Phenotypic responses to ozone, drought and combined ozone and drought stress

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

Figure 1.

Phenotypic responses of Medicago truncatula cv. Jemalong to drought, ozone and combined stress. Top panel shows a sample trifoliate exhibiting the symptoms associated with drought, ozone or combined stress. Photographs were taken at the end of the stress treatment. Graph depicts the total chlorophyll content at the end of stress treatments. Error bars represent standard deviations (n = 4). aMeasurement that is significantly different compared to controls.

Physiological responses to drought and ozone

Chlorophyll content

Consistent with the phenotypes observed, leaf chlorophyll content showed a significant reduction in response to drought or ozone stress (Fig. 1; Table 1).

Table 1. Summary of significance analysis of drought, ozone and combined ozone and drought stress experiments in Medicago truncatula
  1. Statistically significant values or interactions from two-way analysis of variance were designated as D = day, T = treatment and TxD = treatment × day interactions. Each value is a mean (n = 4) followed by standard error. Adjusted P-values (<0.05) from Tukey's honestly significant difference test (comparisons with ambient watered control plants) are shown in parenthesis.

  2. ROS, reactive oxygen species; RFU, relative fluorescent unit; GSSG, oxidized glutathione.

Chlorophyll (µg per g protein)T60.31 ± 0.040.15 ± 0.01 (0.00009)0.17 ± 0.02 (0.00032)0.26 ± 0.03
Leaf water potential (MPa)T, D, TxD6−1.85 ± 0.10−2.65 ± 0.18 (0.00000)−2.06 ± 0.07−2.0 ± 0.05
Stomatal conductance (mol m−2 s−1)T, D, TxD10.15 ± 0.030.06 ± 0.02 (0.00119)0.08 ± 0.03 (0.01327)0.05 ± 0.02 (0.00021)
60.14 ± 0.020.05 ± 0.01 (0.00054)0.16 ± 0.020.07 ± 0.01 (0.00080)
Photosynthesis (µmol m−2 s−1)T, D17.5 ± 0.75 ± 0.5 (0.00595)5.2 ± 0.8 (0.01367)5.7 ± 0.9
66.5 ± 1.04.5 ± 0.5 (0.03497)5.2 ± 0.34.7 ± 0.5
ROS (RFU in %)T, D, TxD6100 ± 0201 ± 32 (0.01029)282 ± 58 (0.00002)110 ± 10
Nitric oxide (µmol per g wt)T, D, TxD66.4 ± 0.711.4 ± 1 (0.00002)6.8 ± 0.58 ± 1
Ascorbate (µmol per g wt)T, D, TxD60.64 ± 0.050.55 ± 0.150.86 ± 0.051.29 ± 0.18 (0.0008)
Dehydroascorbate (µmol per g wt)T, D, TxD10.19 ± 0.140.59 ± 0.19 (0.01876)0.58 ± 0.17 (0.02270)0.6 ± 0.1 (0.01652)
60.14 ± 0.100.40 ± 0.050.75 ± 0.10 (0.00040)0.12 ± 0.07
Glutathione (nmol per g wt)T1251 ± 27450 ± 47459 ± 18550 ± 95 (0.01052)
6287 ± 81362 ± 42605 ± 107 (0.00614)707 ± 162
GSSG (nmol per g wt)T, TxD166 ± 11124 ± 22 (0.01983)131 ± 13 (0.00824)86 ± 25
622 ± 2.7169 ± 27 (0.00000)89 ± 11 (0.00661)95 ± 19

Leaf water potential

Leaf water potential of well-watered plants under ambient ozone conditions did not fluctuate much (–1.7 MPa). Leaf water potential decreased to −2.0 MPa on day 57, and further withholding of water for 3 more days led to significant decrease in the leaf water potential (–2.9 MPa on day 60) compared with corresponding control plants (Fig. 2a). Changes in leaf water potential in response to ozone and combined stress was similar in pattern to drought alone, after 3 d of treatment. However, the reduction in water potential was not significant. Interestingly, leaf water potential in ozone and combined stress-treated plants were similar to the control levels by the end of treatment period. The marked changes in leaf water potential of drought stressed plants were also accompanied by a significant decrease in their leaf water content (Supporting Information Fig. S1).

Figure 2.

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

Stomatal conductance

There were significant differences in the stomatal conductance in response to stress treatments (Table 1, Fig. 2b). These effects were seen clearly within 1 d after imposition of stress and at the end of the 6 d treatment in response to drought and combined stress treatments. Interestingly, stomatal conductance at the end of first day of ozone treatment was lower than control plants, and by the end of the 6 d treatment, it was comparable to values observed in control plants.

Photosynthesis rate

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


Transpiration rates were comparable to control plants at the end of ozone treatment (Fig. 2d) and were consistent with the observations on stomatal conductance (Fig. 2b). Transpiration rates at the end of 6 d drought and combined stress treatments were only slightly lowered when compared to control plants (Table 1, Fig. 2d).

Biochemical responses to drought and ozone

Total ROS

Total ROS levels showed significant increases in response to single stresses at the end of treatment regime (Table 1, Fig. 3a). ROS levels nearly doubled in response to drought, while nearly threefold increase in ROS was recorded in response to ozone stress. Interestingly, ROS levels in response to combined stress were comparable to levels in control plants (Table 1).

Figure 3.

Biochemical responses of Medicago truncatula cv. Jemalong subjected to drought and ozone stress. Measurements were conducted 1 and 6 d after the stress treatment initiation. (a) Total reactive oxygen species (ROS); (b) nitric oxide (NO); (c) reduced ascorbate (AsA); (d) dehydroascorbate (DHA); (e) reduced glutathione (GSH); (f) oxidized glutathione (GSSG). Measurements were conducted 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.


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

AsA and DHA

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


Initial increases in GSH levels were consistently seen in both single and combined stress treatments (Fig. 3e). However, by the end of stress treatment, increase in GSH levels in response to combined stress was 3–4-fold higher compared to control plants, and nearly twofold more than in drought stress plants. Nearly 4–8-fold increase in GSSG levels was recorded in response to drought, while ozone and combined stress treatments showed a 2–3-fold increase compared to corresponding controls (Fig. 3f).

Differential gene expression in response to drought and ozone

Changes in steady-state levels of transcripts in M. truncatula leaves subjected to ozone, drought or their combination were analysed using Affymetrix gene chips. Correlations between the three biological replicates were between 0.87 and 0.95, indicating high reproducibility of the stress treatments and the molecular techniques related to Affymetrix gene chip hybridizations. Reproducible differential expression of 5390 probe sets was observed in drought or ozone or combined ozone and drought stress treatments (Supporting Information Tables S1 and S2). The largest number of differentially expressed genes was observed in response to ozone (Fig. 4). Although least number of differentially expressed 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 significant overlap with ozone stress treatment on the basis of the number of commonly differentially expressed probe sets. More than 1100 probe sets were commonly differentially expressed in all stress treatments (Fig. 4).

Figure 4.

Venn diagram representation of the differentially expressed genes in M. truncatula cultivar Jemalong in response to drought, ozone and combined stress. Numbers in regular font represent induced genes while the numbers in italics represent repressed genes.

In the next stage of the analysis, we set out to identify enriched GOs associated with each subgroup in the Venn diagram (Fig. 4). The Affymetrix probe set identifiers for each of the subsets (Fig. 4) was loaded into agriGO and the di-acyclic graphs showing over-represented GOs were retrieved for categories with at least five mapping entries (Fig. 5, Supporting Information Fig. S2–S6). Increasing the number of entries to more than five yielded very few enriched GOs, while lowering the number below five resulted in a large number of enriched GOs containing only 3–4 probe sets representing only 1–2 genes.

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 boxes indicate significantly enriched GOs. The colour scale from yellow to red represents increasing levels of statistical significance as indicated in the figure.

Among the genes unique to combined stress, GOs associated with response to JA and innate immunity was significant (Fig. 5a). Glycoprotein catabolism was repressed. Among the genes that were induced in all stress treatments, several GO categories were significant. This included cell wall, amino acid and isoprenoid catabolism, trehalose and flavonoid biosynthesis, SA and JA-mediated signalling pathways (Supporting Information Fig. S2). Among the genes repressed in all the stress treatments, GOs for nitrogen metabolism, peptide transport and inorganic ion transport were over-represented (Fig. 5b). Among the genes unique to drought stress, GOs associated with ABA signalling, proline biosynthesis, oxidative stress, heat stress and highlight were significant (Supporting Information Fig. S3). Cell wall biogenesis, catabolism of amino acids, aminoglycans and protein chromophore linkages were other GOs significantly enriched in drought stress. Glucan metabolism was significantly repressed by drought stress (Supporting Information Fig. S4).

Maximum number of significantly enriched GOs was observed in response to ozone stress, consistent with the largest number of differentially expressed genes. GOs associated with phenylalanine ammonia-lyase biosynthesis, glucose, sucrose and glucan metabolism were significantly enriched (Supporting Information Fig. S5). Interestingly, GOs for circadian rhythm, photosynthetic electron transport, red or far-red light signalling and responses to temperature stimulus and inorganic substances were repressed in response to ozone treatment (Supporting Information Fig. S6).

Transcriptional regulation

GO annotations for only about 70% of the M. truncatula probe sets were available in agriGO. Hence, we sought to identify the Arabidopsis homologs for the probe sets on the Medicago Affymetrix array. Use of Arabidopsis identifiers resulted in the same set of enriched GOs as described earlier. Only new GO that was identified using this approach was the transcription regulation category. Of the 51 probe sets that were responsive to both single and combined stress, 23 were up-regulated and 28 were down-regulated. WRKY family members were predominant among the transcription factors (TFs) common to single and combined stresses. Of the 22 probe sets uniquely differentially expressed in response to combined stress, 13 were up-regulated and nine were down-regulated (Table 2). Several TFs such as MYC3 and WRKY50 unique to combined stress are associated with JA signalling. Fifteen probe sets were unique to single stresses but were not differentially expressed in response to combined stress. Several genes annotated as ethylene response factors were identified in response to ozone, while single AP2 domain containing TFs such as DREB and RAPs were common to drought stress.

Table 2. Differentially expressed transcription factor genes unique to combined ozone and drought stress in Medicago truncatula
TIGR TCTAIR HitDrOzDr + OzBrief description
  1. Values in these columns represent average log2-fold change of treatment/control from three replicate hybridizations. Entries in italicized font represent repressed genes.

  2. Dr, drought; Oz, ozone; Dr + Oz, combined drought and ozone stress; TIGR TC, tentative consensus identifiers for the Medicago Affymetrix probe sets obtained from TIGR; TAIR hit, best Arabidopsis match for the Medicago sequences retrieved from TAIR site.

TC126060AT5G467601.281.702.39MYC3 activates JA-responses
TC134696AT4G238100.821.432.01WRKY53 associated with senescence
TC125948AT4G344101.431.502.53Redox responsive subfamily B-3 of ERF/AP2
TC129809AT1G464800.701.572.23WUSCHEL-related homeobox gene
BE940931AT5G261701.551.802.68WRKY50 – JA inducible defence responses
TC112993AT4G044501.381.952.08WRKY 42
BQ147546AT1G683201.841.922.06MYB62 -phosphate starvation
TC132070AT1G506001.291.622.03scarecrow-like protein (SCL5), GRAS
TC129528AT4G177851.881.872.64Putative transcription factor, MYB39
TC119107AT5G573901.251.712.05AINTEGUMENTA-LIKE 5
TC121790AT1G695801.921.552.43Homeodomain-like superfamily protein
TC129232AT5G469100.982.002.21Jumonji – C5HC2 type protein, response to chitin
TC126482AT1G561701.751.812.16Nuclear factor Y-C2, NF-YC2, ER stress
TC115105 AT5G61590 0.55 0.56 0.20 B-3 subfamily of ERF/AP2
TC121489 AT1G76880 0.72 0.50 0.45 Homeodomain protein, SANT and Myb domain
TC119841 AT5G58900 0.53 0.54 0.43 Homeodomain-Myb-like, SANT, DNAj, HSP40
TC113329 AT4G34530 0.53 0.56 0.31 Cryptochrome-interacting basic-helix-loop-helix, CIB1, floral transition
TC122656 AT4G24240 0.66 0.84 0.41 Calmodulin-binding WRKY7-repressor
TC122656 AT1G29280 0.51 0.68 0.30 WRKY65
TC121586 AT5G08520 0.67 0.82 0.46 Homeodomain- Myb-like, SANT, DNAj, HSP40


In nature, plants are exposed to multiple stresses simultaneously. Several studies have shown that the responses to combined stresses are unique and cannot be extrapolated to the 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 simultaneous application of drought and ozone evokes an attenuated phenotypic response in M. truncatula cv. Jemalong, in contrast to their extreme susceptibility to each of these stressors singly (Fig. 1). Based on the changes in the physiological and biochemical parameters examined, it appears that drought stress was the most severe, followed by ozone, while the combination of ozone and drought was the least damaging (Table 1).

In pine, drought alone or combined drought and ozone stress lowered the needle water potential (Alonso et al. 2001), which is in contrast to our studies (Fig. 2a). In the Alonso et al. (2001) study, pine seedlings were first subjected to ozone stress followed by drought, while in our studies, a mild drought was prevailing when the ozone stress was applied. The differences in the order in which the stressors were applied could also be a reason for the observed differences between pine and Medicago. The contrasting observations could be simply due to differences in the plant species. Net photosynthesis rates were significantly down when the stresses were applied singly or in combination (Fig. 2c) supporting the notion that carbon fixation processes are compromised in response to environmental perturbations such as drought and ozone (Ma et al. 2012; Wilkinson et al. 2012). Thus, even if the phenotypic symptoms in response to combined stress were less severe compared with the single stresses, the short-term physiological response, especially photosynthesis rate, was negatively affected. This response is again consistent with the lower stomatal conductance that was observed in response to drought and combined stress (Fig. 2b). Several studies have shown that water stress may lower the effects of ozone by reducing stomatal conductance and in turn lowering ozone uptake (Temple, Taylor & Benoit 1985; Tingey & Hogsett 1985; Pearson & Mansfield 1993; Reichenauer & Bolhar-Nordenkampf 1999). The higher stomatal conductance observed in response to ozone in Jemalong may be due to stomatal sluggishness and could be caused by damage to ion channels (Torsethaugen et al. 1999; Vahisalu et al. 2008). The changes in transpiration rates were in line with the stomatal conductance responses to single and combined stresses. Thus, considering only the physiological traits, it appears that lowering stomatal conductance may be an important stress avoidance strategy to combined ozone and drought stress in Jemalong. Long-term exposure to drought and ozone stresses during crop season will help assess the impact of lowered stomatal conductance on economically important traits such as yield and biomass.

ROS are key signalling molecules during environmental perturbations and normal development (Mahalingam & Fedoroff 2003; Mittler et al. 2011). Numerous studies have documented changes in ROS in response to drought or ozone (Kangasjarvi et al. 2005; Mahalingam et al. 2005; Puckette, Tang & Mahalingam 2008; Kar 2011; Lee & Park 2012). A striking finding of our study is that combined stress does not alter the ROS or NO levels in Jemalong during the two tested time points (Fig. 3a,b). This may be attributed to active increases in reduced AsA levels (Fig. 2c) that have been touted as the first line of defence against apoplastic ROS generators such as ozone (Kangasjarvi et al. 1994; Conklin, Williams & Last 1996), as well as increases in GSH (Fig 2e). It is also possible that there are other mechanisms apart from the AsA-GSH cycle for keeping the ROS levels under control during combined stress. An increase in ROS (as observed in ozone alone) can be accompanied by an increase in NO levels (as observed in drought alone), and these reactive species could lead to the formation of less reactive peroxynitrite (Delledonne et al. 2001; Vandelle & Delledonne 2011) during combined stress. Using peroxynitrite specific dyes such 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 elevated in response to ozone alone in Jemalong. AsA-GSH cycle plays an important role in recycling DHA (Creissen & Mullineaux 2002). Recycling of DHA that accumulates in the apoplast in response to ozone is carried out by DHA reductase enzyme using GSH as a reductant (Yoshida et al. 2006). We were not able to identify any probe sets for DHA reductase in the Medicago Affymetrix array. It should also be noted that several studies in different plant systems have indicated lack of correlation between transcript levels and protein activity of AsA-GSH genes (Creissen & Mullineaux 2002).

Since a mild drought stress was already in effect when the ozone treatments were initiated, it could be argued that the reduced effects of ozone are due to stomatal closing in response to drought. We used the gene expression profiling data to examine the hypothesis that reduced effect of ozone when combined with drought stress may be due to processes up-regulated by ozone that are negatively regulated by drought but are not perturbed during combined stress. The GO for glucan metabolism was identified among ozone-induced genes and drought-repressed genes. Genes in glucan metabolism belonged to different members of xyloglucan endotransglycosylase (XET) family and invertase/pectin methylesterase family. Xyloglucan is a hemicellulose that is important for tightening or loosening cellulose microfibrils which in turn enable the cell to change shape in growing zones and retain shapes after cell maturation (Hayashi & Kaida 2011). Down-regulation of XET gene family members in response to low water potential in soybeans and tobacco decreased cell wall extensibility but increased 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 promote wall biogenesis and increase in cell or stomatal density as observed in birch tress (Gunthardt-Goerg et al. 1993; Paakkonen et al. 1995; Frey et al. 1996; Kontunen-Soppela et al. 2010). This increase in cell number may aid in even ozone distribution within leaf tissues for efficient detoxification processes (Paakkonen et al. 1995; Hetherington & Woodward 2003). Thus, transgenic approaches modulating XET gene expression may not deliver the desired impact when combined ozone and drought stress are operative simultaneously.

On the same lines of the above-stated hypothesis, we examined for GO categories that are up-regulated in drought and down-regulated in response to ozone. GOs for response to temperature and response to light stimulus were identified in this analysis. The significant decrease in stomatal conductance to drought in Jemalong could lead to an increase in the leaf temperature as reported for legumes (Reynolds-Henne et al. 2010). We speculate that the observed increase in stomatal conductance and transpiration rates in response to ozone treatment could lead to a cooling effect and supports the observed down-regulation of genes responding to temperature stimulus. Thermal imaging of the leaf responses during drought and ozone stress will verify these hypotheses. Closer inspection of the gene function revealed that several of these genes are pseudo-response regulators essential for temperature responsiveness of the circadian clock (Salome & McClung 2005). Circadian clocks evolved to enhance plant fitness to a perpetually changing environment and recent studies using Arabidopsis clock mutants have established interconnections between stress, circadian clock and primary metabolism (Kant et al. 2008; Fukushima et al. 2009; Legnaioli, Cuevas & Mas 2009; Sanchez, Shin & Davis 2011). How combined occurrence of stresses can impact circadian clocks is not known yet and will be an important area of research in the wake of global climate changes. One of the clock genes repressed in response to combined stress is the homolog of GIGANTEA, loss of function of which in Arabidopsis leads to oxidative stress tolerance (Kurepa et 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 similar role in imparting tolerance to combined drought and ozone stress.

One of the GOs unique to combined stress was related to innate immunity. Innate immune response in plants is triggered by R-genes in response to biotic stresses (Felix et al. 1999; Mizel et al. 2003; Spoel & Dong 2012). The role of these R-proteins in response to abiotic stress is not known currently. In plant–pathogen interactions, R-proteins physically interact with guard proteins such as RIN4 (Mackey et al. 2002, 2003; Axtell & Staskawicz 2003; Kim et al. 2005b, 2005a) that interacts with plasma membrane associated H+-ATPases to regulate stomatal aperture (Liu et al. 2009). We speculate that some of the R-proteins identified in this study may be associated with RIN4-like proteins that could be important in regulating stomatal behaviour during combined stress.

The modulation of TFs exclusive to combined stress or only by drought or ozone alone highlights the plant's plasticity in responding to environmental perturbations. Members of the AP2/ERF family confer tolerance to multiple stresses (Xu et al. 2011) and are key regulators of redox responsive gene networks (Khandelwal et al. 2008). Subtle changes in GSH levels could also play a crucial role in the regulation of redox-responsive genes via components such as redox-regulated NPR1 protein (Tada et al. 2008; Brosche & Kangasjarvi 2012). Activation of jasmonate responsive MYC3 TF along with the JASMONATE ZIM DOMAIN (JAZ) family repressors JAZ1 and JAZ2 (Supporting Information Table S1) (Figueroa & Browse 2012; Kazan & Manners 2012) suggests the involvement of JA signalling during combined stress and is currently under further investigation. Several WRKY TFs have been shown to be responsive to JA, biotic and abiotic stresses and stress combinations (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 ozone and drought stress suggests that this family of proteins have a crucial role to play when multiple stresses operate simultaneously. The Myb TFs such as Myb62 and SCARECROW-like TFs may be important for remodelling root architecture during stress (Devaiah et al. 2009; Cui, Hao & Kong 2012). The novel Myb-like factors with SANT domain and JUMNONJI could be involved in chromatin remodelling (Lu et al. 2011; Luo et al. 2012) during combined stress.


The set of biochemical traits analysed in this study showed distinct signatures for ozone, drought and combined stress suggesting changes in redox metabolism play a pivotal role in determining the plant responses to single or combined stresses. A large-scale metabolite analysis, especially phytohormones, will be valuable for understanding the signalling mechanisms invoked during combined stress. Based on a single time-point transcriptome analysis, there was a considerable overlap in the genes expressed in response to ozone and a combination of drought and ozone, consistent with the similar phenotypic manifestations (chlorosis) to these stresses. Despite short-term duration of stress regime used in this study, the identification of 300 probe sets that are unique to combined ozone and drought provides compelling molecular evidence that plants perceive co-occurring ozone and drought as a new stress state. Since changes in gene expression are not necessarily tantamount to changes in protein levels or activity, this transcriptome analysis reflects a small proportion of the molecular response to combined ozone and drought stress. From the climate change perspective, this study shows that there are germplasm resources that can be exploited for breeding or engineering crop plants that can thrive well when drought and ozone stress occur simultaneously.


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