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cycle is expected to inhibit the synthesis of proteins involved in the repair cycle after photodamage. This is shown in my study by the induction of antioxidative enzymes indicating high ROS formation which in turn inhibit the Calvin cycle as shown by the decline in activity and concentration of RuBisCO (as well as GAPDH). As a consequence, this will inhibit synthesis of D1 protein, and hence, a decline in D1 content and overall photosynthetic activity of the algae. An alternative supply of 3-PGA is from the oxygenation reaction of RuBisCO via the photorespiratory pathway. Thus, photorespiration can help to mitigate inhibition of the synthesis of D1 protein. This is also the case for all the algae in my study where it has been observed that catalase (CAT), an antioxidative enzyme that detoxify H2O2 in the peroxisome is higher than the other two enzymes analysed indicating that photorespiration is activated in the post-irradiated algae (Figs. 11A and 23A, vide infra).
The results obtained from my study also indicate that RuBisCO is more sensitive to high PAR and high UVR (i.e. H1) exposure than GAPDH (p<0.001). Similar results are reported by Bischof and his colleagues (2002b) and they attributed this to the complex holoenzyme structure of RuBisCO and the complex regulation of its activity in comparison to GAPDH (Portis, 1992; Andersson and Backlund, 2008). The reduction of enzymes involved in primary carbon metabolism may also indicates redirection of carbon resources into other pathways, such as those involved in repair or protection processes (Xu et al., 2008), for instance, photorespiratory pathway as explained above or inducing the production of stress proteins (vide infra).
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antioxidant systems the photosynthetic apparatus is further damaged due to destruction of proteins (as in D1 protein, RuBisCO and GAPDH), lipids and nucleic acids (Choo et al., 2004; Häder and Sinha, 2005), which can lead to cell death (Collén and Pedersén, 1996; Franco et al., 2009). Thus, the ecological success of an organism depends on how well and efficient the organism employ mechanisms that can detoxify ROS (Aguilera et al., 2002b).
Data obtained in my study show that presence of UVR greatly induced the antioxidative enzymes such as catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) in all the algae examined indicating that the algae are experiencing oxidative stress. High irradiation with PAR or UVR alone also causes a significant induction in the enzymes but to a lower extent than those with PAR plus UVR.
Furthermore, low irradiance of PAR alone affects GR activity in the red alga S. chordalis indicating that the low PAR has caused a small degree of ROS accumulation in this alga (Fig. 11). A significant reduction in GR and SOD activities are observed in the green alga Acrosiphonia penicilliformis when solar UVR was cut off in the field, indicating low oxidative stress in the absence of UVR (Aguilera et al., 2002b) while APX, CAT and peroxidase were increased in correlation with H2O2 generated from low UVB-flux (Shiu and Lee, 2005).
Build-up of ROS in cells initiates signalling response to induce gene expression of antioxidative enzymes (Strid et al., 1994; Hernández et al., 2006). For instance, UVB disrupts the balance between the production and removal of H2O2 in Ulva fasciata and the accumulation of H2O2 initiates the signalling responses leading to the induction of enzymatic antioxidant defence systems in the alga (Shiu and Lee, 2005). Additionally, as pointed out by Hideg and co-workers (2000), the production of 1O2 is a unique characteristic of acceptor side photoinhibition by excess PAR alone while Shiu and Lee (2005) reported that O2
is the first ROS generated by UVB. Exposure to UVB in combination with PAR may also induce formation of 1O2 but according to Hideg and co-workers (2000), this is only observed in severely damaged leaves after long irradiation and is accompanied by membrane lipid peroxidation. Thus, excess PAR and UV alone or PAR plus UV may initiate different signalling responses to detoxify the ROS. In
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-, H2O2 and hydroxyl radicals can also be produced within the cells. Different from H2O2 metabolism, 1O2 is efficiently quenched by β-carotene, tocopherol or plastoquinone. If not quenched, it can trigger the up-regulation of genes, which are involved in the molecular defence response of photosynthetic organisms against photo-oxidative stress (Krieger-Liszkay et al., 2008) such as genes that expressed the antioxidative enzymes.
Higher induction of the antioxidative enzymes is observed at H1 than at L1 with the red algae exhibiting the highest overall inductions of the enzymes (p<0.001) suggesting that these algae are experiencing higher oxidative stress than the other algal classes. Activities of several antioxidative enzymes including APX, GR and CAT are observed to be much lower in Triticum aestivum L. seedlings grown under low-light conditions than in those grown under high-low-light conditions. Activities of all these enzymes significantly increased within 24 h of transfer of the low-light-grown seedlings to the high-light regime.
The results suggest that the increase in enzyme activities was an adaptive response of the plants to higher amounts of active oxygen species generated at higher light intensities (Mishra et al., 1995). Thus, the increases in activity of CAT, APX and GR observed in the algae are the results of an adaptive response of the algae to high or low PAR and high UV.
Individually, the red alga S. chordalis which inhabits the eulittoral region, exhibits higher activity in the antioxidative enzymes than the middle sublittoral brown alga L. digitata after exposure to H1 (p=0.037).
Similarly, the antioxidative enzymes are higher in the upper sublittoral red alga P. palmata than L.
digitata as well (p=0.011). These results are in accordance to that obtained by Aguilera and co-workers (2002b) with several Arctic marine macroalgae collected on field and concluded that eulittoral and upper sublittoral Arctic marine macroalgae showed higher antioxidant enzyme activities than species from lower sublittoral. However, two red algae from subtropical region, namely Gelidium amansii and Ptercladiella capillacea showed a reverse reaction on exposure to UVB. G. amansii which inhabits lower subtidal region exhibits higher antioxidative enzymes, thus, experiencing greater oxidative stress than P.
capillacea which inhabits upper subtidal region (Lee and Shiu, 2009). In comparison, P. palmata in my
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study has a higher concentrations of antioxidative enzymes than S. chordalis after the post-irradiation treatment with additional UVB at H1 but with no significant differences (p=0.581). Furthermore, lower antioxidant enzyme activities are measured in the green alga U. lactuca compares to the others after exposure to H1 (p<0.001) indicating that the alga can tolerate with the changing environment conditions.
This can be attributed to the high antioxidative enzymes in the pre-irradiated algae (Table 16). Aguilera and co-workers (2002b) also observed that Arctic marine green algae generally show higher antioxidant enzyme activities than the brown or red algae.
The increase in the activity of GR is to regenerate GSH needed for the reduction of dehydroascorbate (DHA) to ascorbic acid (AsA). AsA is then used as a substrate in APX activity. APX has been considered as the main H2O2 scavenging enzymes in the cytosol and chloroplast (Asada, 1992). According to Shiu and Lee (2005), the first ROS generated by UVB is the superoxide anions (O2
-). O2
- can be converted to H2O2 by a dismutation process by the enzyme superoxide dismutase (SOD). CAT, on the other hand, is also one of the enzymes that can detoxify H2O2 but is important in peroxisomes for scavenging H2O2
created by photorespiration (Asada, 1992). Thus, in the case of my study, an increase in all the three antioxidative enzymes can be correlated to an increase in the H2O2 and since CAT activity is the highest induced by all the algal classes particularly under the high PAR and high UVR conditions (H1, p=0.008 for red algae, p<0.001 for brown and green algae), perhaps that the oxidative stress during the experiments was mainly caused by photorespiration.
Photorespiration is one of the alternative electron sinks to reduce the over-reduction of electron carriers which can excite PSII to photoinhibition (Melis, 1999; Niyogi, 2000). It is believedthat consumption of photochemical energy, such as ATP and NADPH,through the photorespiratory pathway helps avoid the photo-oxidativedamage to PSII via the acceptor-side photoinhibition by 1O2 (Osmond and Grace, 1995;
Osmond et al., 1997). For instance, the photorespiratory pathway helpsavoid inhibition of the synthesis of D1 protein, which isimportant for the repair of photodamaged PSII upon interruptionof the Calvin cycle (Takahashi et al., 2007, vide supra). Thus, it appears that all the algal classes examined in my study
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employ the photorespiratory pathway in order to mitigate photodamage to PSII from the inhibitory conditions of H1 and L1. Another mechanism that can act as electron sinks is the water-water cycle (Asada, 1999; Niyogi, 2000). In the water-water cycle, electrons generated by oxidation of water at PSII are used to reduce O2 to water at PSI via O2
- and H2O2, thus helping to scavenge ROS in the chloroplast (Asada, 1999; Asada, 2006). In addition to this function, the water-water cycle can also dissipates excess excitation energy by the generation of proton gradient across the thylakoid membrane (Asada, 1999;
Asada, 2006). GR and APX are the two main enzymes involved in this pathway. Therefore, the algae examined may also employ this mechanism to detoxify H2O2 that is accumulated in the chloroplast. As Collén and Davidson (1999) as well as Dummermuth and co-workers (2003) pointed out, the key element in reactive oxygen metabolism might be the balance between production and protection in individual compartments, such as peroxisomes and chloroplasts rather than protection integrated over the entire cell.
Similar results were also demonstrated by a 2-fold increased in CAT activity compared to that of APX in Cladophora glomerata incubated under 600 µmol m-2 s-1 PAR in the laboratory (Choo et al., 2004).
Nevertheless, in the field samples, APX activity was observed to be higher than CAT activity in C.
glomerata (Choo et al., 2004) and in the brown alga Fucus spp. (Collén and Davison, 1999) and the green alga Ulva rigida (Collén and Pedersén, 1996) as well, suggesting APX as the main scavenging enzyme for H2O2.
When UVR or high PAR is not a burden anymore, H2O2 ceased to accumulate and excess H2O2 has been quenched, thereby slowing down the activity of the protective antioxidative system. Thus, a recovery in the antioxidative enzymes is observed under the dim light (Figs. 11 and 23). Concomitant to the reductions in the antioxidative enzymes activity, there are recovery of Fv/Fm, D1 protein, RuBisCO and GAPDH activity and TSPs. For instance, higher ability of U. lactuca to completely recover from the light stress may be due to the higher ability of this alga to induce antioxidative enzymes to detoxify the ROS which can induce more damage to PSII if left unchecked.
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