Effects of O

Effects of O3, Doubled CO2 Concentration and drought stress on wheat (Triticum aestivum L.) plants were studied in open-top chambers (OTC). Under doubled CO 2 concentration, grain yield and biomass increased, the SOD activity, and carotenoid (Car) content also increased while relative conductivity yield parameters significantly decreased. But under Elevated O3 concentration, the SOD activity, Carotenoids decreased. The final result was decreased grain yield and plant biomass. Interactive effects of doubled CO2 and O3 concentrations on soybean were mostly counteractive. However, the beneficial effects of concentration-doubled CO 2 is more than compensate the negative effects imposed by doubled O 3 and the latter in its turn partly counteracted the positive effects of the former.


INTRODUCTION
Two aspects of global climate change that directly influence plant physiology, growth and productivity; increasing in concentrations of ambient ozone (O 3 ) and carbon dioxide (CO 2 ) 1,2 .Atmospheric CO 2 is projected to continue rising to at least 550 ppb by 2050 3 .The current annual average (O 3 ) is predicted to continue increasing by 0.5-2% per year over the next century, mainly due to increases in precursor emissions from anthropogenic sources 4,5 .
Greenhouse effect is one of the important concern in present global change and the increase of concentrations of greenhouse gases is the main reason which resulted in the enhancement in the greenhouse effect.CO 2 is the most important greenhouse and carbon source for plant photosynthesis.O 3 in troposphere is essentially a pollutant6 and it restricts the growth of plant severely.The concentrations of CO 2 and O 3 have been increased continually and the responses of plant to them are regarded increasingly.
Ozone diffuses into the leaf apoplast via the stomata where it is rapidly converted into other reactive oxygen species (ROS) that signal a diverse metabolic response (Long and Naidu, 2002;  Kangasjarvi et al., 2005; Hassan, 2006).Stress may promote the formation of harmful reactive oxygen species (ROS) which have the capacity to initiate chlorophyll bleaching, lipid peroxidation, protein oxidation, and injury to nucleic acids (Kangasjarvi  et al., 2005).The effect of CO 2 and O 3 on plant growth and productivity has been determined separately for a large number of plant species, but very little work has focused on their interactive effect 2,6,7,8 .
The studies on combination effects of CO 2 with temperature, moisture 6,7 and effects of O 3 with SO 2 or NO 2 8,9 on plant have been reported.Interactive effects of CO 2 and O 3 on winter wheat 10 , potato 11,12 , aspen and birch 13 .However, the research on interactive effects of CO 2 , O 3 and drought, are seldom 15 .Although many studies addressed the effects of CO 2 , O 3 and /or drought stress on plants in the developed world, no such study, to the best of knowledge, was conducted in developing world.
In this paper, respective and interactive effects of doubled CO 2 and O 3 concentration on wheat yield and biomass production, Photosynthesis, stomatal conductance, antioxidative ability and cell membrane lipid peroxidation of leaves in the context of free radical biology were studied in open-top chambers (OTC).
Though several previous studies report evocation of oxidative stress by water deficit stress in case of wheat 4,9,26 at individual level, information on their comparative response to same degree of stress in terms of their stress sensitivity and functional variation is lacking.In order to fill this gap of knowledge, this study was undertaken to evaluate oxidative response to O 3 , CO 2 and/or drought singly and in combination of wheat plants.The hypothesis behind the work was the sensitivity of plants to water stress might be associated with different abilities for their carbon fixation, and also these stresses may affect to the net assimilation through their impact on stomatal conductance as well as antioxidant enzymes.
Therefore the aims of the present study were to describe interaction of enhanced O 3 , elevated CO 2 and water stress on growth, seed yield, photosynthetic activities, photosynthetic pigments, antioxidant enzymes such as glutathione (GR ), Peroxidase (POD), superoxide dismutase (SOD) and ascorbate Peroxidase (APX) as well as molecular biomarkers in durum wheat (Triticum aestivum L.) plants.Moreover, to study the importance of the accumulation of photosynthetic pigments which could change during exposure to multiple stresses, and this is the novelty of this experiment was a study of triple interaction of the mentioned factors.

Plant materials, growth conditions and Experimental design
Grains of wheat (Triticum aestivum L.) plants were sown in pots with 20x20cm 2 filled with soil collected from top soil in the field.There were five plants/pot.They were transferred to four Open-Top Chambers (OTCS) 19 when second foliage leaf appeared.The treatments were: (a) control (FA), (b) O 3 without CO 2 (O 3 ), (c) FA with CO 2 (CO 2 ), (d) O 3 and CO 2 (O 3 +CO 2 ).
The experiment was split plot Latin square, one chamber was equipped with charcoal filter (FA) and the other was ventilated with FA + Target O 3 (78 ppb/h) and the third was ventilated with FA +Target CO 2 (450 ppm), while the fourth OTC was supplied with O 3 (78 ppb) and CO 2 (450 ppm).There were 12 pots/chamber.Pots were distributed in a completely randomized block design (CRBD).Half of pots were irrigated to the field capacity while the other half was water-stressed to 0.5 MP 10,12 .They were rotated within each chamber every week.

Biomass and grain yield
5 plants per pot were harvested for determination of grain and seed yield and yield attributes.The grains harvested were air-dried and the shoots, leaves and roots were dried at 80Cfor 72h.Number of grains per ear was counted.Yield per ear, yield per plant, and 1000-grain weight were determined.

Stomatal conductance and Net photosynthetic measurements
Net CO 2 assimilation rate (A) and stomatal conductance (gs) were measured using Infrared Gas Analyzer (IRGA,ciras-1PP System, Hitchin UK).Measurements were carried out on ten attached leaves per treatment on weekly basis.

Measurement of Photosynthetic Pigments
Photosynthetic pigments, chlorophyll a & b and carotenoids were extracted and from flag leaves and were determined by UVspectrophotometer (LKB, UK.) 35,36 .

Antioxidant enzymes assays
Extractions of antioxidant enzymes from the leaves of the four treatments were performed 21 .Leaves were cut from each treatment and immersed in liquid nitrogen and kept in a deep freezer at 80°C until the analyses were performed at University of Newcastle, UK.
Samples were weighed and ground at about °C in 25 m Tris-HCl buffer containing 3 mM MgCl 2 , then the homogenates were centrifuged at 20 000 for 15 min (Centrifuge17 S/RS, Heraeus Sepatech).The supernatants were used for the enzyme assays and the results were expressed on protein basis 35 .
All assays were performed using a final volume of 1 mL, with at least duplicate assays undertaken on each sample.Moreover, the assays were end-point determinations.SOD (EC 1.15.1.1)activity was monitored 21 .The extraction mixture contained 50 mM phosphate buffer solution (pH 7.8), 13 mM Lmethionine, 63 lM nitro blue tetrazolium and 2 lM riboflavin.The ability of the extract to inhibit the photochemical reduction of nitro blue tetrazolium was determined at 560 nm (Schimadzu UV-1201 spectrophotometer).The amount of the extract resulting in 50% inhibition of nitro blue tetrazolium reaction is defined as one unit of SOD activity.

Data analysis
Data were subjected to three-way analysis of variance (ANOVA), using O 3 , CO 2 and drought treatments as factors, followed by a least significant difference test, and P values ≤ ≤ ≤ ≤ ≤ 0.05 were considered significant (using the STATGRAPHICS statistical package, Package 3, UK) based on plot means.

Effects on visible injury symptoms
Visible injury symptoms appeared on the upper surface of flag leaves as point brown spots and by the end of experiment.
Number of injured leaves were increased by 2-fold due to exposure to O 3 , while exposure to both CO 2 and O 3 increased it by 31% (Table 1).There was no significant effect (p > 0.05) of either CO 2 or drought stress and their interaction on number of injured leaves and on degree of injury.Degree of injury increased by 6-fold when exposed to O 3 and 4-fold due to exposure to both O 3 and CO 2 .Drought stress and CO 2 protected plants against toxic effects of O 3 .Plants exposed to D had 39% less leaf injury while exposure to both CO 2 and D had 50% lower leaf injury than plants exposed to O 3 alone (Table 1).

Effect on growth and yield
O 3 had greater effect on the numbers of ears per plant and the number of grains per ear than drought as these parameters was reduced by 20 and 26% respectively (Table 2).However, CO 2 caused increased by 26% and 18% in these parameters respectively.Moreover, the percentage reduction in 1000 grain weight due to O 3 (40%) was greater than that due to drought(29%),CO 2 alone increased it by 23%,interaction between stresses was less than additive.O 3 and Drought caused significant reductions in all yield parameters measured in this experiment (Reductions reached maximum of 63% in dry mass and 54% in 1000 grains weight).

Effect on stomatal conductance and photosynthetic rates
It was clear that both O 3 and drought had the greatest negative effects on stomatal conductance (g s ) caused reduction by 41 and 50% respectively (over the entire period the experiment) (Fig. 1).Moreover, interaction between O 3 and drought was great reduction more than additive (58% reduction).On the other hand CO 2 had a synergistic effect it caused increases to 12%, while it ameliorates toxic effects of O 3 and drought when applied with other stresses.The interactive effects of CO 2 and O 3 are contradictory caused greater reduction in g s than CO 2 and drought by 17 and 11%, respectively.Interactions between different treatments were less than additive (21% reduction).After 10 weeks O 3 and drought exposure negative impacts were evident for Wheat plants revealed a 58% decline in stomatal conductance (g s ).
It was clear that O 3 and drought had the greatest effect on net photosynthetic rates (A) (Fig. 2).O 3 and drought caused reduction in net photosynthetic rates (A) by 70% and 80%, respectively.On the other hand, CO 2 increased net photosynthetic rates A by 6%.Interaction between O 3 and CO 2 decreased it by 8%, while O 3 and drought decreased it to 12%.Exposure of Wheat plants to O 3 and drought simultaneously had the greatest decline effect on the net photosynthetic rates A by 88%.Drought had more pronounced negative effects than those other parameters.CO 2 mitigates toxic effects of drought and O 3 , it mitigated O 3 and reduced its toxicity by to 23 % and drought to 19 %.Interactions between different treatments were less than additive.

Effects on antioxidant enzymes
A significant increase was observed in POD by 1.5-fold in Wheat plant treated with elevated CO 2 ; in contrast, elevated CO 2 had a reducing effect on GR and SOD by 11and 9 %, respectively.There was no significant (Pd≤0.05)effect of all treatments on APX.O 3 caused increases in activities of GR, POD by 18 and 36 % respectively, while SOD was decreased by 11%.Drought had more pronounced effect on these enzymes as it caused increases by    injurious as indicated by the fast decrease in SOD activity, pigments content and increase in MDA accumulation and ion leakage 3,5,31 .The decrease in the anti-oxidative ability and increased production induced by high O 3 exposure is an indication of senescence.This is consistent to the fact that the plant exposed to doubled O 3 shed their leaves 7-8 days earlier than others.
It was found in the present study that elevated CO 2 confer some protection against O 3 , and it was clear that stress caused by CO 2 predisposed leaves to injury caused by O 3 and not vice versa and this explain how they interact to alleviate foliar injury 39 .
In the present study, increased GR concentration and POD activity in ambient air can be interpreted as response to oxidative stress imposed by O 3 (Chernkova et al. 2000).However, the lack of significant changes in activities of SOD and AA in ambient air, which also observed in other O 3 studies 40 , differed from studies where activities of these enzymes increased in response to O 3 41 .The variability in the response of antioxidants to elevated O 3 and CO 2 among studies reflects differences in the magnitude of the perceived oxidative stress, the species-specific mechanisms involved in responses to changes in redox status, the plant capacity to cope with additional stress(s), experimental protocols and environmental conditions.Therefore, further studies are needed to further the understanding of the response of antioxidant metabolism to elevated O 3 , CO 2 and drought singly and in combination 17,18,24,25,27,30 .

CONCLUSIONS
Ozone exposure reduced corn grain yield in response to O 3 damage during the flowering process.Both O 3 and CO 2 had a major impact on physiological processes that are independent on PAR absorption.Therefore, radiation use efficiency (RUE) in response to gases treatments, in wheat, was significantly increased in response to CO 2 enrichment and significantly reduced in response to O 3 -induced stress.While water use efficiency (WUE) was reduced in O 3 -stressed plants grown under water stress and elevated CO 2 .Similar results were observed for the control treatment and the

DISCUSSION
The results support the previous reports 22,23,24,25 that biomass and yield of crop plant were significantly increased under high CO 2 level but decreased under high O 3 level.In our case, the increased yield and biomass under doubled CO 2 were more than sufficient to eliminate the doubled O 3 -induced yield decrease as shown in the treatment of the combination of doubled CO 2 and O 3 concentration.
It is known that enriched CO 2 increases photosynthesis 1,12 by providing more carbon source.This is the basis for the increase in yield and biomass.The increased photosynthesis also provides more reducing power and more biosynthesis of chlorophyll and carotenoids as well as enhancement of antioxidants concentrations.This would enhance the resistance of the plant to environment stresses, such as exposure to high O 3 .The enhancement of the antioxidative ability is especially important since O 3 itself is a strong oxidant.Enriched CO 2 also decrease stomata conductance of the leaves.This would reduce the flux of O 3 into leaves though stomata.Take these two factors together, the effect of enriched CO 2 in amelioration of the harmful effect of O 3 is reasonable.
O 3 as a strong oxidant, is highly injurious to the plant tissues.It inhibits photosynthesis 1,2,12 , decrease yield and biomass production 35,36,38 .It directly attacks the cell membranes inducing increase in lipid peroxidation and ion leakage.By inhibiting photosynthesis, the biosynthesis of the antioxidants or active oxygen scavengers may also be affected.The imbalance between the generation and scavenge of active oxygen would decrease the stress resistance of the plant.
It is interesting to note that when plants exposed to O 3 was less than 20 days, they responded in a way that it induced higher SOD activity, higher chlorophyll and carotenoids content.This indicates that small dosage O 3 or a short duration of exposure might not result in damage but induce acclimation response of the plant.But soon the accumulated dosage had increased to the level that intolerable to the plant and became high-O 3 /enriched-CO 2 treatment, indicating that the damaging effect of 0 3 air pollution was counteracted by the beneficial effect of CO 2 enrichment without any interactive effects between the two gases for the measured variables except for grain yield, during the first wheat experiment, where the CO 2enriched environment more than overcame the O 3induced stress.
Stomata closed under water stress treatment and decreased influx of O 3 and CO 3 , which would affect growth, yield and physiology of both crops.Closure of stomata is beneficial as less O 3 influx but reduction in CO 2 influx would reduce photosynthetic rates of both crops and hence lowering yield

Fig. 2 :
Fig. 2: Effects of different treatments on net photosynthetic rates (A) µmol m -2 s -1 of Wheat plants (Triticum aestivum L.) grown under field conditions in open top chambers(OTCS).

Fig. 1 :
Fig. 1:Effects of different treatments on stomatal conductance (gs) mmol m -2 s -1 of Wheat plants grown under field conditions in open top chambers(OTCS).Values are means of 10 replicates ± 1SE.An arrow indicates data of re irrigation of water -stressed plants.Duration of expose (weeks)

Table 1 : Effect of CO 2 and O 3 , drought, singly and in combination on foliar injury symptoms of Wheat leaves in year 2005. (Means not followed by the same letter in each row are significantly different from each other at P≤
≤ ≤ ≤ ≤0.05) (