Current World Environment

Introduction

Two aspects of global climate change that directly influence plant physiology, growth and productivity; increasing in concentrations of ambient ozone (O₃) and carbon dioxide (CO₂).^1,2 Atmospheric CO₂ is projected to continue rising to at least 550 ppb by 2050.³ The current annual average (O₃) 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. CO2 is the most important greenhouse and carbon source for plant photosynthesis. O3 in troposphere is essentially a pollutant⁶ and it restricts the growth of plant severely. The concentrations of CO2 and O3 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 CO2 and O3 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 CO2 with temperature, moisture^6,7 and effects of O3 with SO2 or NO2^8,9 on plant have been reported. Interactive effects of CO2 and O3 on winter wheat,¹⁰ potato,^11,12 aspen and birch.¹³ However, the research on interactive effects of CO2, O3 and drought, are seldom.¹⁵ Although many studies addressed the effects of CO₂, O₃ 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 CO2 and O3 concentration on wheat yield and biomass production, Photosynthesis, stomatal conductance, anti-oxidative 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 O₃, CO₂ 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 O₃, elevated CO₂ 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.

Materials and Methods

Plant Materials, Growth Conditions and Experimental Design

Grains of wheat (Triticum aestivum L.) plants were sown in pots with 20x20cm² 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) O₃ without CO₂ (O₃), (c) FA with CO₂ (CO₂), (d) O₃ and CO₂ (O₃ +CO₂).

The experiment was split plot Latin square, one chamber was equipped with charcoal filter (FA) and the other was ventilated with FA + Target O₃ (78 ppb/h) and the third was ventilated with FA +Target CO₂ (450 ppm), while the fourth OTC was supplied with O₃ (78 ppb) and CO₂ (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 80℃for 72h. Number of grains per ear was counted. Yield per ear, yield per plant, and 1000-grain weight were determined Stomatal conductance and Net photosyntheti measurements Net CO₂assimilation rate (A) and stomatal conductance (g_s) 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 UV-spectrophotometer (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 80⁰C until the analyses were performed at University of Newcastle, UK.

Samples were weighed and ground at about ⁰C in25 m Tris–HCl buffer containing 3 mM MgCl₂, then the homogenates were centrifuged at 20 000 for 15 min (Centrifuge 17 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 L-methionine, 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.

Catalase (EC, 1.11.1.6) activity was assayed in enzyme extract reaction mixture containing 50 mM phosphate buffer (pH 7.4). The reaction was started by adding 10 mM H₂O₂, and the reduction in absorbance was determined at 240 nm.^14,36

GPX (EC, 1.11.1.7) activity was determined by adding 50 mM phosphate buffer (pH 6.1), 1% H2O2 and 1% guaiacol to the extract, and the absorbance was determined at 470 nm.

APX (EC, 1.11.1.11) activity was determined according to Maehly & Chance (1954). The reaction mixture contained 50 mM potassium phosphate, 0.5 mM ascorbate, 0.1 mM ethylenedimethyl tartaric acid (EDTA) and 0.1 mM H₂O₂, and the absorbance was determined at 290 nm. Protein concentrations of leaf extracts were determined as described earlier.^26,37

Data Analysis

Data were subjected to three-way analysis of variance (ANOVA), using O₃, CO₂ 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.

Results

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 CO₂ and O₃ increased it by 31% (Table 1). There was no significant effect (p > 0.05) of either CO₂ 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 O₃ and 4-fold due to exposure to both O₃and CO₂. Drought stress and CO₂ protected plants against toxic effects of O_3. Plants exposed to D had 39% less leaf injury while exposure to bothCO₂ and D had 50% lower leaf injury than plants exposed to O₃ alone (table 1).  

Table 1: Effect of CO2 and O3, 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) (n=40).  Click here to View table

Effect on Growth and Yield

O₃ 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, CO₂ caused increased by 26% and 18% in these parameters respectively. Moreover, the percentage reduction in 1000 grain weight due to O₃ (40%)was greater than that due to drought(29%),CO₂ alone increased it by 23%,interaction between stresses was less than additive. O₃ 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).  

Table 2: Effects of different treatments on yield parameters of Wheat (Triticum aestivium L.) plants grown under field conditions in open top chambers(OTCS). FA (346 ppm CO2+ CFA); O3(ambient CO2+75 ppb O3 7hd-1(10.00-17.00 h); elevated CO2 (702 ppm1CO2 +CFA) elevated CO2 +O3 (elevated CO2+78 ppb O3 7hd-1).  Plants were harvested 70 d after transfer to open top chamber (OTCs). (*mean P ≤ 0.05;** P < 0.01;*** P < 0.001). Click here to View table

Effect on Stomatal Conductance and Photosynthetic Rates

It was clear that both O₃ 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 O₃ and drought was great reduction more than additive (58% reduction).  On the other hand CO₂ had a synergistic effect it caused increases to 12%, while it ameliorates toxic effects of O₃ and drought when applied with other stresses. The interactive effects of CO₂ and O₃ are contradictory caused greater reduction in g_s than CO₂ and drought by 17 and 11%, respectively. Interactions between different treatments were less than additive (21% reduction). After 10 weeks O₃ and drought exposure negative impacts were evident for Wheat plants revealed a 58% decline in stomatal conductance (g_s). 

Figure 1: Effects 0f different treatments on stomatal conductance (gs) mmol m-2s-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. Click here to View figure

It was clear that O₃ and drought had the greatest negative effect on net photosynthetic rates (A) (Fig.2). O₃ and drought caused reduction in net photosynthetic rates (A) by 70% and 80%, respectively. On the other hand, CO₂ increased net photosynthetic rates A by 6%. Interaction between O₃ and CO₂ decreased it by 8%, while O₃ and drought decreased it to 12%. Exposure of Wheat plants to O₃ 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. CO₂ mitigates toxic effects of drought and O₃, it mitigated O₃ and reduced its toxicity by to 23 % and drought to 19 %. Interactions between different treatments were less than additive.

Figure 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). Legends as Figure 1. Click here to View figure

Effects on antioxidant enzymes

A significant increase was observed in POD by 1.5-fold in Wheat plant treated with elevated CO₂; in contrast, elevated CO₂ had a reducing effect on GR and SOD by 11and 9 %, respectively. There was no significant (P≤0.05) effect of all treatments on APX.

O₃ 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 21% in POD while GR and SOD were decreased by 11 and 15%, respectively. The antioxidant enzymes; GR and SOD were significantly decreased in  Wheat plants exposed to  elevated CO₂ + elevated O₃ by 15 and 50%, respectively than in plants grown under normal conditions. CO₂ followed the same pattern of O₃. Interaction between O_3, CO₂ and drought and their multiple interactions were less than additive.  

Table 3: Activities of glutathione reductase(GR) guaicol peroxidase (POD),superoxide dismutase (SOD) and Ascorbic peroxidase (APX) in extract Wheat leaves (70 DAP).  Click here to View table

Discussion

The results support the previous reports^22,23,24,25 that biomass and yield of crop plant were significantly increased under high CO2 level but decreased under high O3 level. In our case, the increased yield and biomass under doubled CO2 were more than sufficient to eliminate the doubled O3-indced yield decrease as shown in the treatment of the combination of doubled CO2 and O3 concentration.

It is known that enriched CO2 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 concemyraions. This would enhance the resistance of the plant to environment stresses, such as exposure to high O3. The enhancement of the antioxidative ability is especially important since O3 itself is a strong oxidant. Enriched CO2 also decrease stomata conductance of the leaves. This would reduce the flux of O3 into leaves though stomata. Take these two factors together, the effect of enriched CO2 in amelioration of the harmful effect of O3 is reasonable.

O₃ 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 O₃ 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 O₃ 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 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 O₃ exposure is an indication of senescence. This is consistent to the fact that the plant exposed to doubled O₃ shed their leaves 7-8 days earlier than others.

It was found in the present study that elevated CO₂ confer some protection against O₃, and it was clear that stress caused by CO₂ predisposed leaves to injury caused by O₃ 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 O₃ (Chernkova et al. 2000). However, the lack of significant changes in activities of SOD and AA in ambient air, which also observed in other O₃ 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 O₃ and CO₂ 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 O₃, CO₂ and drought singly and in combination.^{17,18,24,25,27,30}

Conclusions

Ozone exposure reduced corn grain yield in response to 0₃ damage during the flowering process. Both O₃ and CO₂ 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 CO₂ enrichment and significantly reduced in response to O₃-induced stress. While water use efficiency (WUE) was reduced in O₃- stressed plants grown under water stress and elevated CO₂. Similar results were observed for the control treatment and the high-O₃/enriched-CO₂ treatment, indicating that the damaging effect of 0₃ air pollution was counteracted by the beneficial effect of CO₂ enrichment without any interactive effects between the two gases for the measured variables except for grain yield, during the first wheat experiment, where the CO₂-enriched environment more than overcame the O₃-induced stress.

Stomata closed under water stress treatment and decreased influx of O₃ and CO₃, which would affect growth, yield and physiology of both crops. Closure of stomata is beneficial as less O₃ influx but reduction in CO₂ influx would reduce photosynthetic rates of both crops and hence lowering yield

Acknowledgements

Part of this work was funded by a fellowship grant from UNESCO to RHB. Thanks to Late Prof Adel Aal (Alexandria University) for his suggestions and help at early stages of the work.

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