Nonenzymatic Antioxidants 331 Ascorbic Acid

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Among the small molecular antioxidants in plants, ascorbate is most abundant and is most concentrated in leaves and meristems (reviewed by Ahmad et al. 2010b) . It is about five to ten times more concentrated than GSH in leaves (Ishikawa et al. 2006) . AsA is present in high concentration in fruits, especially citrus fruits, but the concentration in fruits is not always higher than in leaves (Davey et al. 2000). Some fruits such as blackcurrants and rose hips are famous for their exceptionally high ascorbate content (Ishikawa et al. 2006). AsA occurs in all subcellular compartments, and the concentration varies from 20 mM in the cytosol to 300 mM in chloroplasts (Noctor and Foyer 1998). The synthesis of AsA takes place in mitochondria and is transported to other cell compartments through a proton electrochemical gradient or through facilitated diffusion (Horemans et al. 2000). Franceschi and Tarlyn (2002) reported the presence of ascorbate in the phloem sap of A. thaliana. Other species of plants have also been reported to contain ascorbate in the phloem sap, e.g. cucurbita (Hancock et al. 2008). This led to the conclusion that ascorbate is transported from source (leaves) to sink (meristem) (Ishikawa et al. 2006).

Ascorbate plays an important role in plants as an antioxidant and as a cofactor of many enzymes (Ishikawa et al. 2006) . As an antioxi-dant, ascorbate protects plants from oxidative stress. Ascorbate peroxidase utilizes ascorbic acid and reduces H2O2 to water, thereby generating monodehydroascorbate (MDA) in the ascorbate-glutathione cycle (Pan et al. 2003) . MDA can also be reduced directly to AsA in the presence of the catalytic enzyme MDAR and the electron donor NADPH (Asada 1999). Maddison et al. (2002) have reported that ascorbate plays a role in the defense against ozone. AsA has the capability of donating electrons in various enzymatic and nonenzymatic reactions and is thus a powerful radical scavenger. It can directly scavenge 1 O2, O2% and -OH radicals produced in the cell and can protect membranes against oxidative stress. In plant cells, the most important reducing substrate for H2O2 detoxification is ascorbic acid (Turkan et al. 2005). An increase in oxidized ascorbate during Cd stress has been reported by Demirevska-Kepova et al. (2006) in Hordeum vulgare. Yang et al. (2008) also reported that drought stress increases the ascorbate content in Picea asperata. Water stress results in significant increases in antioxidant AsA concentration in turfgrass (Zhang and Schmidt 2000; Vranova et al. 2002; Jaleel et al. 2007). Ascorbic acid shows a reduction under drought stress in maize and wheat, suggesting its vital involvement in oxidative response (Vertovec et al. 2001; Nayyar and Gupta 2006).

3.3.2 a-Tocopherol

Plants have the capacity to synthesize a lipophylic antioxidant known as a-tocopherol or vitamin E. a-tocopherol scavenges free radicals in combination with other antioxidants (Munne-Bosch and Algere 2003 i Massacci et al. 2008). It has also been reported that a-tocopherol protects the structure and function of PSII as it chemically reacts with O2 in chloroplasts (Lopez-Huertas et al. 2000; Nordberg and Arner 2001). Munne-Bosch and Algere (2003) reported that a-tocopherol helps in membrane stabilization and alleviates the tolerance of plants during oxidative stress. Environmental stresses are responsible for the generation of low molecular mass antioxidants such as a-tocopherol (Lowlor and Cornic 2002; Munne-Bosch and Algere 2003; Mahajan and Tuteja 2005; Martinez et al. 2007).

Falk et al. (2003) reported the upregulation of genes of a- tocopherol synthesis during oxida-tive stress. Water stress resulted in elevated levels of a-tocopherol in Vigna plants (Manivannan et al. 2007) and turfgrass (Zhang and Schmidt 2000).

3.3.3 Reduced Glutathione

Glutathione (L-glutamyl-L-cysteinylglycine, GSH) is a thiol compound composed of L-glutamic acid,

L-cysteine, and glycine. GSH is distributed universally in animals, plants, and microorganisms and has an established role as an essential compound of a free radical scavenger (Monneveux et al. 2006). GSH participates in numerous cellular processes and protects cells from the toxic effects of many ROS (Petropoulos et al. 2008). Additionally, GSH is involved in other biological functions, such as regulation of protein and DNA synthesis, protein activities, and maintaining membrane integrity (Cabuslay et al. 2002). Meyer et al. (2005) reported that levels of H2O2 are controlled by the action of glutathione. Reduction of glutathione (GSH) and oxidation of glutathione (GSSG) are necessary for controlling H2O2 levels in cells and have an important role in redox signaling (Pastori and Foyer 2002. Reduced glutathione is directly involved in the reduction of ROS in plants. Transgenic tobacco expressing glutathione gene withstands oxidative stress (Singh and Verma 2001).

Glutathione is a tripeptide (a-glutamyl cystei-nylglycine) and is found in the cytosol, chloro-plasts, ER, vacuoles, and mitochondria (Sankar et al. 2007a, b). The nonprotein thiols are nucleo-philic in nature and thus are important for the formation of mercaptide bonds with metals and for reacting with selective electrophiles (Rodriguez et al. 2005). In most plants, the major source of these nonprotein thiols is glutathione. Glutathione is considered the most important nonenzymatic antioxidant due to its relative stability and high water solubility (Samarah 2005). It can protect plant cells from environmental stress-induced oxidative stress (Samarah 2005).

4 The Effect of Elevated Atmospheric CO2 Concentration on Antioxidants and Osmolytes Under Environmental Stress

Elevated atmospheric CO2 concentration leads to a higher CO2 concentration gradient between the outside air and the intercellular spaces of the leaves, so that the diffusion of CO2 into the leaves and the pCO2/pO2 ratio at the sites of photoreduc-tion is increased (Robredo et al. 2007). Therefore, usually photorespiration and the rates of oxygen activation and ROS formation are reduced due to an increased NADPH utilization, whereas the net photosynthetic rate and thus the carbon supply is enhanced, especially in C3 plants (Polle 1996; Urban 2003; Kirschbaum 2004; Long et al. 2004; Hikosaka et al. 2005; Ignatova et al. 2005). Furthermore, we often find a lower stomatal resistance (Hsiao and Jackson 1999; Li et al. 2003 ; Marchi et al. 2004; Rogers et al. 2004), which together with the higher net assimilation also leads to a better water use efficiency of photosynthesis (Amthor 1999; Morgan et al. 2001; Urban 2003). As a consequence of these effects, on the one hand there might be less need for anti-oxidants as elevated CO2 ameliorates oxidative stress (Schwanz et al. 1996) ; On the other hand more energy can be provided for energy-dependent stress tolerance mechanisms such as the synthesis of osmolytes and antioxidants. Due to both effects mentioned earlier, elevated CO2 can increase plant survival under abiotic stress conditions (Ball and Munns 1992; Rozema 1993; Drake et al. 1997; Fangmeier and Jäger 2001; Wullschleger et al. 2002; Urban 2003; Geissler et al. 2010).

Regarding oxidative stress, the antioxidant system can respond differently to elevated CO2 depending on species or even genotype as well as on treatment duration and growth conditions such as mineral nutrition (Schwanz et al. 1996; Polle et al. 1997; Sanita di Toppi et al. 2002; Perez-Lopez et al. 2009). Varying responses can be related to a species-specific differential regulation in order to maintain an adequate balance between ROS formation and antioxidant ability under the actual conditions (Perez-Lopez et al. 2009). However, many studies have reported an increased tolerance to various abiotic stresses under elevated CO2 due to an alleviation of oxi-dative stress:

In chestnut trees, photoinhibition due to high irradiance stress was ameliorated, and higher GSH levels were found in juvenile leaves (Carvalho and Amancio 2002). Sgherri et al. (2000) reported that CO2 enrichment led to an improved water use efficiency and a decreased photorespiration in Medicago sativa under drought stress. As a consequence, the cells showed a higher reducing status, increased ascor-bate/dehydroascorbate and GSH/GSSG ratios. There was no demand for a higher GR activity (no CO2 effect) and less requirement for Ca2+ ATPase activity to maintain Ca2+ homeostasis under stress conditions. Similarly, in cold stressed maize elevated CO2 had no effect on SOD, CAT, and APX activities, but the formation of superoxide radicals and membrane injury was reduced (Baczek-Kwinta and Ko cielniak 2003). The alleviation of oxidative stress was probably due to a higher CO2 assimilation, overcoming the Rubisco limitation under low temperature. In some cases elevated CO2 even leads to reduced activities of antioxidative enzymes because there is less need for antioxidants: Perez-Lopez et al. (2009) reported that barley plants exposed to NaCl stress under ambient CO2 exhibited enhanced activities of SOD, APX, CAT, GR, and dehydroascorbate reductase (DHAR), which was accompanied by ion leakage and lipid peroxidation. Furthermore, the expression ratio of enzyme isoforms changed, e.g. a relatively higher contribution of GR1 relative to GR2 and of Cu/Zn-SOD (which seems to be especially important for salt tolerance in Hordeum vulgare) was observed. Elevated CO, ameliorated ion leakage and lipid peroxidation, while the plants showed a lower upregulation of the antioxidant enzymes and an even higher relative contribution of GR1 and of Cu/Zn-SOD. The authors explain these results with less ROS generation and a better maintenance of redox homeo-stasis due to an enhanced photosynthesis and a reduced photorespiration. Similar results were found for Solanum lycopersicum by Takagi et al. (2009) . NaCl salinity decreased plant biomass, net assimilation, and the transport of assimilates to the sink (stem), while CAT and APX activities increased. Under elevated CO2 the negative effects of salinity were alleviated, especially when the sink activity was relatively high, and CAT and APX activities decreased compared to ambient CO2. The improvement of oxidative stress (and of water relations) seemed to cause an activation of sink activity under elevated CO2.

Fig. 1.6 Antioxidant enzyme expressions (relative volume percentages of the spots) in controls and salt treatments (75% seawater salinity) of Aster tripolium under ambient and elevated CO2. (a) Superoxide dismutase, (b) ascorbate peroxidase, (c) glutathione-S-transferase. Values represent mean ± SD values of eight gels per treatment. Significant differences (P£0.05) between the salinity treatments (within one CO2 treatment) are indicated by different letters, significant differences between the CO2 treatments (within one salt treatment) are indicated by an asterisk. ctr control, sal salt treatment

Fig. 1.6 Antioxidant enzyme expressions (relative volume percentages of the spots) in controls and salt treatments (75% seawater salinity) of Aster tripolium under ambient and elevated CO2. (a) Superoxide dismutase, (b) ascorbate peroxidase, (c) glutathione-S-transferase. Values represent mean ± SD values of eight gels per treatment. Significant differences (P£0.05) between the salinity treatments (within one CO2 treatment) are indicated by different letters, significant differences between the CO2 treatments (within one salt treatment) are indicated by an asterisk. ctr control, sal salt treatment

In contrast to the studies mentioned earlier, in some cases antioxidant activities are enhanced by elevated CO2. In ozone stressed Betula pendula, elevated CO2 eliminated the chloroplastic accumulation of H2O2, which could be explained by a higher photosynthetic rate, leading to a higher NADPH formation and a more efficient enzymatic detoxification (e.g., via the ascorbate-glu-tathione cycle; Oksanen et al. 2005). Marabottini et al. (2001) found a higher activity of catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD) in drought-stressed Quercus under elevated CO2 . Rao et al. 2 1995) observed a more persistent high activity of glutathione reductase (GR), APX, und SOD in ozone-stressed wheat. Schwanz and Polle (2001) examined the drought tolerant species Quercus robur and the sensitive Pinus pinaster. They found out that Q. robur generally exhibits a higher activity of several antioxidative enzymes; furthermore, elevated CO2 concentration ameliorated damage caused by drought stress in both species due to a higher stability of antioxidative enzymes and an enhanced SOD activity. Similar results were reported for the facultative halophyte A. tripolium2 Under ambient CO2 concentration salt stress led to an overexpression and thus to higher relative activities of the antioxidative enzymes APX, SOD, and glutathione-S-transferase (GST), while under elevated CO2 the expression and activities of these enzymes were further increased (Fig. 1.6; Geissler et al. 2010). Similarly, elevated CO2 concentration led to a significantly higher content of carotenoids -nonenzymatic antioxidants - in the salt treatments (Geissler et al. 2009b). These results implicate that the enhancement of enzyme expression and activity and the carotenoid content were not high enough to sufficiently eliminate ROS under ambient CO2 concentration. Under elevated CO2, however, a higher supply of energy-rich organic substances due to a significantly enhanced net assimilation rate (Geissler et al. 2009a, b) enabled the plants to invest more energy in the energy-dependent synthesis of enzymatic and nonenzy-matic antioxidants. Therefore, ROS could be detoxified more effectively, so that salinity tolerance could be improved, manifesting itself in a higher survival rate of the salt-treated plants (Geissler et al. 2009a).

Furthermore, investigations about A. tripolium showed that elevated CO2 concentration does not

Fig. 1.7 Content of osmolytes in controls and salt treatments (75% seawater salinity) of Aster tripolium under ambient and elevated CO2. (a) Proline, (b) total soluble carbohydrates. Values represent mean ± SD values of six measurements per treatment. Significant differences

(P < 0.05) between the salinity treatments (within one CO2 treatment) are indicated by different letters, significant differences between the CO2 treatments (within one salt treatment) are indicated by an asterisk. ctr control, sal salt treatment

Fig. 1.7 Content of osmolytes in controls and salt treatments (75% seawater salinity) of Aster tripolium under ambient and elevated CO2. (a) Proline, (b) total soluble carbohydrates. Values represent mean ± SD values of six measurements per treatment. Significant differences

(P < 0.05) between the salinity treatments (within one CO2 treatment) are indicated by different letters, significant differences between the CO2 treatments (within one salt treatment) are indicated by an asterisk. ctr control, sal salt treatment only have an effect on antioxidants, but on osmolytes as well. This halophyte employed its additional carbon gain under elevated CO2 concentration also for a higher synthesis of compatible solutes (Geissler et al. 2009a): Salinity (under ambient CO2) led to an accumulation of proline in all plant organs and of soluble carbohydrates in the main roots (Fig. 1.7). Under elevated CO2 concentration, the plants accumulated a higher amount of proline, especially in the leaves which are the primary areas of influence of CO2. In the main root, there was no necessity of an additional accumulation of proline because this organ is well protected against salt damage due to a high content of compatible solutes even at ambient CO2 concentration. Furthermore, a higher amount of soluble carbohydrates under elevated CO2 was found in all plant organs due to the increased photosynthesis and a lower conversion of saccharides to starch. These results are in accordance with the study of Abdel-Nasser and Abdel-Aal (2002) investigating Carthamus mareoticus under drought stress: Elevated CO) concentration increased the accumulation of total soluble carbohydrates in well watered as well as in stressed plants due to a higher amount of assimilates. The drought-induced inhibition of the sucrose phosphate synthase activity was annihilated under elevated CO2, and the drought-induced increase in sucrose content was further enhanced. The content of total amino acids and especially of proline behaved similarly to sucrose, as well as the activities of the proline synthesizing enzymes 1-pyrroline-5-carboxylate reductase (P5CR) and the ornithine aminotransferase (OAT). In contrast, the activity of the proline degrading enzyme proline dehydrogenase (PDH) was reduced by drought stress and further decreased under elevated CO2.

In contrast to C. mareoticus, proline (and other amino acids) do not seem to contribute to salt tolerance in barley, but to reflect a reaction to stress damage, as shown by Perez-Lopez et al. (2010): Although a better osmotic adjustment (more negative osmotic potential) of salt-stressed plants was recorded under elevated CO2, the proline content decreased, showing less stress damage. Instead, the accumulation of soluble sugars and other unidentified osmolytes (possibly polyols and/or quaternary nitrogen compounds) was actively enhanced under elevated CO2, and these substances played an important role in osmotic adjustment and as compatible solutes under saline conditions. Elevated CO2 provided a higher carbon and ATP supply for salt tolerance mechanisms, enabling the plants to actively increase their compatible solute concentration, which in turn leads to a better water uptake and turgor maintenance for plant growth.

As a summary, it can be concluded that elevated CO2 concentration can enhance salt and drought tolerance of plants by alleviating oxida-tive stress, increasing the activity of the antioxi-dative system, and/or increasing the accumulation of compatible substances, having a positive effect on their suitability as crops on dry and saline soils in future.

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