Photosynthetic Responses of Canola and Wheat to Elevated Levels of CO2, O3 and Water Deficit in Open-Top Chambers

The effects of elevated CO2 (700 ppm) and O3 (80 ppb) alone and in combination on the photosynthetic efficiency of canola and wheat plants were investigated in open-top chambers (OTCs). The plants were fumigated for four weeks under well-watered and water-stressed (water deficit) conditions. The fast chlorophyll a fluorescence transients were measured after 2 and 4 weeks of fumigation, as well as in control plants, and analyzed by the JIP-test, which is a non-destructive, non-invasive, informative, very fast and inexpensive technique used to evaluate the changes in photosynthetic efficiency. Biomass measurements were taken only after 4 weeks of fumigation. The performance index (PItotal), an overall parameter calculated from the JIP-test formulae, was reduced by elevated CO2 and O3 under well-watered conditions. In the absence of any other treatment, water stress caused a decrease of the PItotal, and it was partly eliminated by fumigation with elevated CO2 and CO2 + O3. This finding was also supported by the biomass results, which revealed a higher biomass under elevated CO2 and CO2 + O3. The decrease in biomass induced by elevated O3 was likely caused by the decline of photosynthetic efficiency. Our findings suggest that elevated CO2 reduces the drought effect both in the absence and presence of O3 in canola and wheat plants. The study also indicates that elevated O3 would pose a threat in future to agricultural crops.


Introduction
The concentrations of carbon dioxide (CO 2 ) and ozone (O 3 ) are increasing at a steady rate in the atmosphere [1,2]. The increasing CO 2 trend is mostly caused by the use of fossil fuels for combustion [2]. It is projected that CO 2 concentration will rise to about 550 ppm by 2050 [3]. Elevated CO 2 stimulates plant growth and development but elevated O 3 often has the opposite effect [4,5]. Ozone causes considerable damage in agricultural crops, which includes visible injury, reduced photosynthetic capacity, modifications to carbon allocation and reduced yield quantity and quality [6,7]. Prolonged exposure to O 3 levels above 40 ppb decreases crop yields due to reduced photosynthesis and disruption of metabolism [8]. These findings suggest that agricultural crops in southern Africa may be at risk because of elevated O 3 levels [9]. The seasonal variation of O 3 indicates the highest O 3 concentrations in spring and winter and the lowest in summer [10]. The maximum O 3 concentrations in this region are between 40-60 ppb and can rise to more than 90 ppb in the spring season [11]. What is of concern is how these changes will interact with one another and influence plant growth, as well as the interaction of these gasses with other factors of climate change, such as droughts.

Photosynthetic Responses of Canola to Elevated Levels of CO 2 and O 3
The averages of the raw fluorescence transients of canola leaves were plotted on a logarithmic time scale from 20 µs to 1 s and the values are expressed as F t /F 0 ( Figure 1). The steps O, J, I and P are indicated. In order to reveal hidden differences, the fluorescence data were normalized between O (20 µs) and K (300 µs) steps, as V OK = (F t − F 0 )/(F K − F 0 ), and plotted as difference kinetics ∆V OK = V OK(treatment) − V OK (control) . Also, fluorescence data were normalized between the steps O and J (2 ms), as V OJ = (F t − F 0 )/(F J − F 0 ), and plotted as difference kinetics ∆V OJ = V OJ (treatment) − V OJ (control) . These allowed the visualization of the positive ∆L (0.15 ms) and ∆K-band (0.3 ms) in the O 3 treatment (Figure 2). The appearance of ∆L and ∆K-band is regarded as a good indicator to detect the physiological disturbances of plants caused by environmental conditions [30].  Figure 2). The appearance of ΔL and ΔK-band is regarded as a good indicator to detect the physiological disturbances of plants caused by environmental conditions [30].       Canola plants fumigated with elevated CO 2 and O 3 caused the decline of the PI total when subjected to well-watered conditions ( Figure 3). However, in water-stressed plants, no significant difference was found when compared with the control. The combination of elevated CO 2 and O 3 had higher PI total values under both water regimes ( Figure 3). Canola plants fumigated with elevated CO2 and O3 caused the decline of the PItotal when subjected to well-watered conditions ( Figure 3). However, in water-stressed plants, no significant difference was found when compared with the control. The combination of elevated CO2 and O3 had higher PItotal values under both water regimes ( Figure 3). Average (of all weeks) PItotal of canola plants exposed to CO2, O3 and CO2 + O3 under wellwatered and water-stressed conditions for four weeks. For the same water treatment, different letters show statistically significant differences (p < 0.05).
The PItotal is an overall parameter calculated from the JIP-test that combines biophysical parameters. The parameters are the density of reaction centers (RC/ABS); the parameter (φPo /(1 − φPo), where φPo represents the maximum quantum yield of primary photochemistry; the parameter (ψEo /(1 − ψEo), where ψEo represents the efficiency with which an electron moves into the electron transport chain further than QA -; the parameter (δRo /(1 − δRo), where δRo represents the efficiency with which an electron from the intersystem electron carriers is transferred to reduce end electron acceptors at the PSI (Photosystem I) acceptor side. The statistical analysis of the effect of elevated CO2, O3 and CO2 + O3 on the components of the PItotal in canola leaves is shown in Table 1. Plants in the O3 treatment enhanced the parameter ψEo/(1 − ψEo) under both water regimes, while the parameter δRo /(1 − δRo) was reduced. The combination of CO2 and O3 improved the parameter δRo /(1 − δRo) when subjected to well-watered and water-stressed conditions ( Table 1).
Elevated CO2 and the combination of CO2 and O3 had no significant effect on the above ground biomass of well-watered plants. However, canola plants exposed to O3 (80 ppb) caused a significant reduction in biomass under well-watered conditions ( Figure 4). In water-stressed plants, none of the treatments had a significant effect on biomass production, but based on average values, elevated CO2 and the combination of CO2 and O3 led to a higher biomass than the control (Figure 4). The PI total is an overall parameter calculated from the JIP-test that combines biophysical parameters. The parameters are the density of reaction centers (RC/ABS); the parameter (ϕ Po /(1 − ϕ Po ), where ϕ Po represents the maximum quantum yield of primary photochemistry; the parameter (ψ Eo /(1 − ψ Eo ), where ψ Eo represents the efficiency with which an electron moves into the electron transport chain further than Q A -; the parameter (δ Ro /(1 − δ Ro ), where δ Ro represents the efficiency with which an electron from the intersystem electron carriers is transferred to reduce end electron acceptors at the PSI (Photosystem I) acceptor side. The statistical analysis of the effect of elevated CO 2 , O 3 and CO 2 + O 3 on the components of the PI total in canola leaves is shown in Table 1. Plants in the O 3 treatment enhanced the parameter ψ Eo /(1 − ψ Eo ) under both water regimes, while the parameter δ Ro /(1 − δ Ro ) was reduced. The combination of CO 2 and O 3 improved the parameter δ Ro /(1 − δ Ro ) when subjected to well-watered and water-stressed conditions ( Table 1).
Elevated CO 2 and the combination of CO 2 and O 3 had no significant effect on the above ground biomass of well-watered plants. However, canola plants exposed to O 3 (80 ppb) caused a significant reduction in biomass under well-watered conditions ( Figure 4). In water-stressed plants, none of the treatments had a significant effect on biomass production, but based on average values, elevated CO 2 and the combination of CO 2 and O 3 led to a higher biomass than the control ( Figure 4). The radar plot indicates that the decline of PItotal in well-watered canola plants exposed to elevated O3 was caused by the parameter δRo /(1 − δRo) ( Figure 5). This drop was linked to the decrease of biomass accumulation. The higher PItotal in canola plants fumigated with a combination of CO2 and O3 was sustained by the parameter, δRo /(1 − δRo).  The radar plot indicates that the decline of PI total in well-watered canola plants exposed to elevated O 3 was caused by the parameter δ Ro /(1 − δ Ro ) ( Figure 5). This drop was linked to the decrease of biomass accumulation. The higher PI total in canola plants fumigated with a combination of CO 2 and O 3 was sustained by the parameter, δ Ro /(1 − δ Ro ). The radar plot indicates that the decline of PItotal in well-watered canola plants exposed to elevated O3 was caused by the parameter δRo /(1 − δRo) ( Figure 5). This drop was linked to the decrease of biomass accumulation. The higher PItotal in canola plants fumigated with a combination of CO2 and O3 was sustained by the parameter, δRo /(1 − δRo).

Photosynthetic Responses of Wheat to Elevated Levels of CO 2 and O 3
The average Chl a fluorescence transient of dark-adapted wheat leaves for the four treatments under well-watered ( Figure 6A) and water-stressed ( Figure 6B) conditions were plotted on a logarithmic time scale, 20 µs to 1 s and expressed as F t /F 0 for clarity. The four transients showed a typical OJIP shape, with small differences between them. In order to show the hidden differences, the fluorescence data were normalized between O (20 µs) and K (300 µs) steps, as V OK = (F t − F 0 )/(F K − F 0 ), and plotted as difference kinetics ∆V OK = V OK(treatment) − V OK (control) , which revealed the ∆L-band. A positive ∆L-band indicates lower energetic connectivity while a negative ∆L-band indicates higher energetic connectivity [31]. Furthermore, the fluorescence data were normalized between the steps O and J (2 ms), as V OJ = (F t − F 0 )/(F J − F 0 ), and plotted as difference kinetics ∆V OJ = V OJ(treatment) − V OJ (control) revealing the ∆K-band.

Photosynthetic Responses of Wheat to Elevated Levels of CO2 and O3
The average Chl a fluorescence transient of dark-adapted wheat leaves for the four treatments under well-watered ( Figure 6A) and water-stressed ( Figure 6B) conditions were plotted on a logarithmic time scale, 20 µs to 1 s and expressed as Ft/F0 for clarity. The four transients showed a typical OJIP shape, with small differences between them. In order to show the hidden differences, the fluorescence data were normalized between O (20 µs) and K (300 µs) steps, as VOK = (Ft − F0)/(FK − F0), and plotted as difference kinetics ΔVOK = VOK(treatment) − VOK (control), which revealed the ΔL-band. A positive ΔL-band indicates lower energetic connectivity while a negative ΔL-band indicates higher energetic connectivity [31]. Furthermore, the fluorescence data were normalized between the steps O and J (2 ms), as VOJ = (Ft − F0)/(FJ − F0), and plotted as difference kinetics ΔVOJ = VOJ(treatment) − VOJ (control) revealing the ΔK-band. A positive ΔK-band shows an increased reduction rate of quinone (QA), from QA to QA − , which suggests that the oxygen evolving complex (OEC) may have become leaky and offers access to nonwater electron donors [32]. Positive ΔL-and ΔK-bands in the O3 treatment were revealed clearly under both water regimes ( Figure 7). Elevated CO2 resulted in a significant decrease of the total performance index (PItotal) in wheat plants subjected to well-watered conditions ( Figure 8). However, no significant difference was found in water-stressed plants exposed to elevated CO2. Ozone fumigation led to a significant decline in PItotal under both water regimes. The combination of elevated CO2 and O3 did not affect the photosynthetic performance (based on the PItotal) of well-watered wheat plants. However, in waterstressed wheat plants, the PItotal increased significantly by 9%, compared with the non-fumigated plants ( Figure 8). A positive ∆K-band shows an increased reduction rate of quinone (Q A ), from Q A to Q A − , which suggests that the oxygen evolving complex (OEC) may have become leaky and offers access to non-water electron donors [32]. Positive ∆Land ∆K-bands in the O 3 treatment were revealed clearly under both water regimes ( Figure 7). Elevated CO 2 resulted in a significant decrease of the total performance index (PI total ) in wheat plants subjected to well-watered conditions ( Figure 8). However, no significant difference was found in water-stressed plants exposed to elevated CO 2 . Ozone fumigation led to a significant decline in PI total under both water regimes. The combination of elevated CO 2 and O 3 did not affect the photosynthetic performance (based on the PI total ) of well-watered wheat plants. However, in water-stressed wheat plants, the PI total increased significantly by 9%, compared with the non-fumigated plants (Figure 8).     The statistical analysis of the effect of elevated CO 2 , O 3 and CO 2 + O 3 on these parameters under well-watered and water-stressed conditions is presented in Table 2. We note that fumigation on wheat plants had a significant effect on the components of the PI total with the exception of the parameter (ψ Eo /(1 − ψ Eo ) in well-watered plants. Elevated CO 2 and O 3 caused a significant decline in the density of reaction centers and the parameter δ Ro /(1 − δ Ro ) ( Table 2). The combination of CO 2 and O 3 enhanced the density of reaction centers under both water regimes. Ozone fumigation caused a reduction in biomass of wheat plants when subjected to well-watered and water-stressed conditions. Compared with the control, biomass was reduced by about 40% under well-watered and 22% under water-stressed conditions ( Figure 9). In water-stressed plants, fumigation did not have any significant effect on biomass. However, based on average values the plants exposed to elevated CO 2 and CO 2 + O 3 , treatments had a higher biomass compared with the control (Figure 9). The statistical analysis of the effect of elevated CO2, O3 and CO2 + O3 on these parameters under well-watered and water-stressed conditions is presented in Table 2. We note that fumigation on wheat plants had a significant effect on the components of the PItotal with the exception of the parameter (ψEo /(1 − ψEo) in well-watered plants. Elevated CO2 and O3 caused a significant decline in the density of reaction centers and the parameter δRo /(1 − δRo) ( Table 2). The combination of CO2 and O3 enhanced the density of reaction centers under both water regimes. Ozone fumigation caused a reduction in biomass of wheat plants when subjected to wellwatered and water-stressed conditions. Compared with the control, biomass was reduced by about 40% under well-watered and 22% under water-stressed conditions ( Figure 9). In water-stressed plants, fumigation did not have any significant effect on biomass. However, based on average values the plants exposed to elevated CO2 and CO2 + O3, treatments had a higher biomass compared with the control (Figure 9). . Biomass of wheat exposed to CO2, O3 and CO2+O3 under well-watered and water-stressed conditions after four weeks. For the same water treatment, the different letters show statistically significant differences (p < 0.05).
The radar plot of the PItotal and its components and biomass is presented in Figure 10. As shown, the reduction in biomass of ozone-treated plants is related to the decrease of the PItotal. On the other hand, the biomass enhancement in water-stressed wheat plants exposed to the combined effect of elevated CO2 and O3 is well associated with the increase in PItotal. We noted that the PItotal was generally influenced by the density of reaction centers and efficiency, with which an electron from the intersystem electron carriers is transferred to reduce end electron acceptors at the PSI acceptor side (Figure 10). . Biomass of wheat exposed to CO 2 , O 3 and CO 2 +O 3 under well-watered and water-stressed conditions after four weeks. For the same water treatment, the different letters show statistically significant differences (p < 0.05).
The radar plot of the PI total and its components and biomass is presented in Figure 10. As shown, the reduction in biomass of ozone-treated plants is related to the decrease of the PI total. On the other hand, the biomass enhancement in water-stressed wheat plants exposed to the combined effect of elevated CO 2 and O 3 is well associated with the increase in PI total . We noted that the PI total was generally influenced by the density of reaction centers and efficiency, with which an electron from the intersystem electron carriers is transferred to reduce end electron acceptors at the PSI acceptor side ( Figure 10) .

Discussion
Elevated CO2 increases the photosynthetic performance of plants, which in turn results in higher biomass production [33,34], but often declines with time when plants are subjected to elevated CO2 over extended periods [33]. Prolonged exposure of plants to elevated CO2 reduces the initial stimulation of photosynthesis [35] and as a result suppresses photosynthesis, which reduces growth responses [36]. It has been suggested that crop plants subjected to reduced water will respond positively to elevated CO2 in comparison to crop plants under sufficient water supply, as CO2 causes an increase in stomatal resistance [5]. We found that plants fumigated with elevated CO2 resulted in a decline of the photosynthetic performance (as revealed by the PItotal) under well-watered conditions in both crop species. The decline of the PItotal can be ascribed to exposing the plants to elevated CO2 for more than four weeks. The decrease in the PItotal under well-watered conditions was mainly influenced by the density of reaction centers and efficiency with which an electron from the intersystem electron carriers is transferred to reduce end electron acceptors at the PSI acceptor side (δRo). A low I-P amplitude may indicate a low capacity of electron transport through PSI. It was shown that the activity of PSI can limit photosynthetic electron transport through PSI, and that PSI can be limiting for CO2 assimilation [37,38], which can be especially important under elevated CO2 [39]. Liu et al. [40] reported that elevated CO2 enhanced the photosynthetic performance of cucumber plants under moderate drought stress. We found that none of the water-stressed plants were affected by elevated CO2, but on average the PItotal values were higher than those of the control. The PItotal values were well-maintained by an increase of the parameter δRo, indicating an increased capacity to reduce end acceptors beyond PSI. The increase of the I-P amplitude was shown to be associated also with an

Discussion
Elevated CO 2 increases the photosynthetic performance of plants, which in turn results in higher biomass production [33,34], but often declines with time when plants are subjected to elevated CO 2 over extended periods [33]. Prolonged exposure of plants to elevated CO 2 reduces the initial stimulation of photosynthesis [35] and as a result suppresses photosynthesis, which reduces growth responses [36]. It has been suggested that crop plants subjected to reduced water will respond positively to elevated CO 2 in comparison to crop plants under sufficient water supply, as CO 2 causes an increase in stomatal resistance [5]. We found that plants fumigated with elevated CO 2 resulted in a decline of the photosynthetic performance (as revealed by the PI total ) under well-watered conditions in both crop species. The decline of the PI total can be ascribed to exposing the plants to elevated CO 2 for more than four weeks. The decrease in the PI total under well-watered conditions was mainly influenced by the density of reaction centers and efficiency with which an electron from the intersystem electron carriers is transferred to reduce end electron acceptors at the PSI acceptor side (δ Ro ). A low I-P amplitude may indicate a low capacity of electron transport through PSI. It was shown that the activity of PSI can limit photosynthetic electron transport through PSI, and that PSI can be limiting for CO 2 assimilation [37,38], which can be especially important under elevated CO 2 [39]. Liu et al. [40] reported that elevated CO 2 enhanced the photosynthetic performance of cucumber plants under moderate drought stress. We found that none of the water-stressed plants were affected by elevated CO 2 , but on average the PI total values were higher than those of the control. The PI total values were well-maintained by an increase of the parameter δ Ro , indicating an increased capacity to reduce end acceptors beyond PSI. The increase of the I-P amplitude was shown to be associated also with an increase of capacity in alternative electron transport pathways [41]. In addition, above ground biomass in both crops was not significantly affected by elevated CO 2 when subjected to well-watered and water-stressed conditions. However, previous studies reported that elevated CO 2 significantly increased biomass in canola and wheat plants [42][43][44]. The difference can be attributed to the decline in the photosynthetic capacity of the plants, as indicated by the PI total , and that the response of crops to elevated CO 2 varies between genotypes [5]. Even though there was no significant difference in the water-stressed plants, the CO 2 treatment enhanced the biomass of both crops. The biomass stimulation caused by elevated CO 2 can also result from reduced water loss and water stress, and/or from decreased respiration [45]. The greater biomass in dry conditions in response to elevated CO 2 has been discussed by Fitzgerald et al. [46]. It should be noted that the beneficial effect of elevated CO 2 in plants subjected to water-stress not only increases biomass production, but also translates into higher crop yield [14,47]. Elevated CO 2 stimulated yield of water-stressed wheat plants (data not shown). Our results suggest that elevated CO 2 concentrations may counteract the negative effect of droughts on canola and wheat. Therefore, it appears that canola and wheat crops grown in limited water availability will benefit more from elevated CO 2.
The appearance of the positive ∆Land ∆K-bands indicates that both crops are more sensitive to elevated O 3 with reference to lower energetic connectivity and inactivation of the OEC, respectively [26,31,48]. Similar findings were reported by Desotgiu et al. [49] in poplar plants subjected to O 3 and water stress. The amplitude of the ∆K-band was higher in the well-watered plants when compared with the water-stressed plants. This suggests that limited water availability can reduce inactivation of the OEC. The OEC represents one of the sensitive components of the PSII (Photosystem II) [50,51]. Furthermore, the appearance of the K-band revealed that elevated O 3 upset the functioning of OEC in the PSII [52]. The transients were further analyzed with the JIP-test equations, which led to the calculation of several photosynthetic parameters and the PI total . In the present experiment, both crop species-canola and wheat-showed a tendency to reduce the PI total in the O 3 treatment (80 ppb) particularly under well-watered conditions. Under drought conditions, the decrease was significant only in wheat plants. This indicates that the effect of O 3 was minor under drought conditions in canola plants [53]. The reduction of the PI total was caused by the efficiency with which an electron from the intersystem electron carriers was transferred to reduce end electron acceptors at the PSI acceptor side, as well as the decline of the density of reaction centers, which, taking into account that ϕ Po remained nearly unchanged, represented an increase of the functional PSII antenna size [26]. The efficiency that an electron moves further than Q A¯w as relatively less affected by elevated O 3 in both plants. Although a temporarily enhanced ψ Eo was detected in canola plants, it did not influence the PI total . The increased efficiency was linked to the activating of repair processes, but when it was linked to a reduced end acceptor capacity in combination with a reduced Calvin cycle, energy demand led to over-excitation of the photosynthetic apparatus [27]. These results support the findings obtained from other studies that the I-P region (as revealed by the relevant parameters) is sensitive to stress caused by O 3 [27,54]. The biomass was significantly affected by O 3 fumigation in both crop plants under well-watered conditions. There was no significant difference under water-stressed treatment, but based on average values, the biomass was reduced in O 3 treatment. In the experiment, where fumigation with 60 ppb of O 3 was applied in canola, the biomass was barely affected [43], and similar findings were reported in a study using four canola cultivars [44]. On the contrary, Feng et al. [55] found that elevated O 3 decreased above ground biomass by 18%. The response to O 3 achieved in the present study can be interpreted by the higher than 60 ppb concentration applied, and to cultivar differences [56]. Similarly, only two out of five wheat cultivars showed a decrease in above ground biomass when subjected to O 3 [57] . Based on the radar, it was suggested that the drop in biomass production was associated with the decline of the PI total [58,59].
The ∆L-band exhibited differences in energetic connectivity among the PSII units [26]. The energetic connectivity among PSII units improved in the CO 2 + O 3 treatment under water-stressed conditions. This was demonstrated by the appearance of the negative ∆L-band, which indicated higher energetic connectivity. A higher energetic connectivity resulted in an improved use of the excitation energy and a higher stability of the photosynthetic system [31,60]. This suggests that elevated CO 2 and reduced water supply could improve the utilization of excitation energy and the stability of photosynthetic systems. Furthermore, the appearance of the negative ∆K-band under water stress showed that plants fumigated with elevated CO 2 + O 3 have either a more active oxygen evolving system or a smaller PSII antenna size [60]. These effects should be regarded as favorable for the photosynthetic apparatus [60]. As shown by the PI total , fumigation with elevated CO 2 + O 3 increased the photosynthetic efficiency of canola plants under both well-watered and water-stressed conditions. In wheat plants, the increase was significant only under water-stressed conditions. The increase in the PI total was mostly caused by the parameter δ Ro . The combinations of CO 2 and O 3 did not reveal any significant reductions in biomass. This suggests that elevated CO 2 can ameliorate the detrimental effects of elevated O 3 and droughts upon canola and wheat. In addition, the photosynthetic process was not compromised as a result of the combined effects of elevated CO 2 and O 3 . The increase in photosynthetic performance (as revealed by the JIP-test parameters) was associated with the increase in biomass production. This indicates that biomass enhancement was most probably caused by the increase in photosynthetic efficiency of the plants.

Experimental Site and Plant Materials
The experiments were conducted in Open-Top Chambers situated at the North-West University, South Africa. The canola and wheat experiments were performed from June to August in 2014 and 2015, respectively. Heyneke et al. [61] has discussed the design and operation of the OTCs system used in the current study. Canola (Brassica napus L. cv. Rainbow) and wheat (Triticum aestivum L. cv. SST875) seeds were sown in pots with a diameter of about 30 cm. The pots were watered manually prior to the start of fumigation with elevated CO 2 and O 3 to ensure that the seeds germinated successfully. Six-month slow release fertilizer (25 g) was added to each pot comprising 17 nitrogen:11 phosphorus:10 potassium:2 magnesium oxide:TE (Osmocote Pro, The Netherlands). The growth medium was composed of topsoil, river sand and vermiculite (2:1:1). The pots were placed into eight OTCs. Two chambers were used per treatment. The treatments were the control (carbon filtered air, OTC 1 and 2); CO 2 (700 ppm, OTC 3 and 4); O 3 (80 ppb, OTC 5 and 6) and CO 2 + O 3 (700 ppm + 80 ppb, OTC 7 and 8). The carbon filtered air was used only for the control treatment, from which O 3 and other pollutants were removed.

Fumigation and Water Treatment
Plants were exposed to elevated CO 2 (700 ppm) and O 3 (80 ppb) and the combination of these two gases (CO 2 + O 3 ) from 08:00am to 17:00pm for 4 weeks. Elevated CO 2 levels inside the OTCs were monitored with a CO 2 monitor (Model 174687 CO 2 _temp-relative-humidity monitor, Scientific Associates, Inc., China). Ozone levels were continuously monitored using an O 3 monitor (Model 205 Ozone Monitor, 2B Technologies, Inc., USA). The temperatures varied between 23 • C and 17 • C during the fumigation period. The ambient ozone levels were below 35 ppb over the entire fumigation period.
The plants were exposed to two water regimes, namely water-watered and water-stressed (water deficit) conditions. All the plants absorbed water through glass fiber wicks that were projected into water reservoirs. In the well-watered treatment, four wicks were placed at four levels within the pots, while in the water-stressed treatment, one glass fiber wick was positioned at the middle level of each pot [43]. The pots were positioned into reservoirs that were connected to a drip irrigation system that filled up the water.

Chlorophyll a Fluorescence
The fast chlorophyll a fluorescence transients were measured with a Handy PEA (Plant Efficiency Analyser) fluorimeter (Hansatech Instruments Ltd, UK) on the leaves of canola and wheat. Before measurements were taken, the leaves were dark-adapted for an hour. The OJIP transients were induced by red light (peak at 650 nm) of 3000 µmol photons m −2 s −1 provided by an array of three light-emitting diodes and recorded for 1 s with 12 bit resolution. The data acquisition occurred at every 10 µs, from 10 µs to 0.3 ms; every 0.1 ms, from 0.3 to 3 ms; every 1 ms, from 3 to 30 ms; every 10 ms, from 30 to 300 ms and every 100 ms, from 300 ms to 1 s. The OJIP transients were analyzed by the JIP-test [19,50] using the PEA Plus ver. 1.10 Program (Hansatech Instruments Ltd, UK). The following fluorescence data from the original measurements were used by the JIP-test: the minimal intensity at 20 µs (O step); the intensities at 50 and 300 µs (F 300 and F 50 ) used for calculation of the initial slope (M 0 ); the intensity at 2 ms (J step); the intensity at 30 ms (I step) and the maximal measured intensity when all PSII reaction centers (RCs) were closed (F m , P step). The following JIP-test parameters were derived from the OJIP transients, all

Biomass
Plants were harvested after four weeks and the fresh plant material was oven-dried at 60 • C for 72 h and weighed.

Statistical Analysis
Statistical analysis were carried out with STATISTICA 13 (Stat Soft. Inc., Tulsa, OK, USA). The data were analyzed using one-way analysis of variance (ANOVA) and significant differences between treatment means were determined by the Tukey's honest significant difference (HSD) post-hoc test.

Conclusions
In conclusion, elevated O 3 led to a decrease in biomass of canola and wheat plants. This reduction was caused by a decline in the photosynthetic efficiency as revealed by the total performance index (PI total ). The results of the current study indicate that elevated O 3 would pose a threat in the future to agricultural crops. The decline in the PI total was mostly influenced by the efficiency with which an electron from the intersystem electron carriers was transferred to reduce end electron acceptors at the PSI acceptor side. The present study also suggests that the PSII was damaged and the photosynthetic apparatus was compromised due to elevated O 3 . Elevated CO 2 reduces the drought effect both in the absence and presence of O 3 . This was also supported by above ground biomass results, which showed higher values under elevated CO 2 . Our findings suggest that elevated CO 2 can reduce the negative effect of abiotic stress, such as droughts and O 3 in canola and wheat plants. The measurement of Chl a fluorescence can be used to screen the effect of elevated CO 2 and O 3 in canola and wheat plants cultivated locally. Further studies should seek to investigate several local varieties of canola and wheat responses to elevated CO 2 , O 3 and droughts and their interaction.