Chloroplast, illuminated, ascorbic

These reactions may proceed nonenzymatically or through the action of glutathione dehydrogenase (ascorbate) (E.C. 1.8.5.1) and ascorbate-specific peroxidase (Halliwell, 1978). Despite these plausible arguments for the reduction of H2O2 by GSH, Jablonski and Anderson (1978) were unable to demonstrate a sustained reduction of either H2O2 or dehydroascorbate by chloroplasts illuminated in the presence of catalytic amounts of GSSG and NADPH. 

Finally superoxide radicals can also be generated photochemically in chloroplasts in the presence of ascorbate or of paraquat. The formation was demonstrated by spin trapping on illumination of spinach chloroplasts in the presence of oxygen and paraquat Superoxide radicals are formed, moreover, in the near-ultraviolet photooxidation of tryptophan, as indicated by the increase of the HjO production in the presence of SOD and on irradiation in aerated solutions of protoporphyrin at 400 nm and of melanin with light of 320—600nmas shown by spintrapping. 

The digitonin preparation of PSI-RC has also been demonstrated to be able to catalyze a light-induced proton uptake when incorporated in phospholipid liposomes and illuminated in the presence of ascorbate and phenazine methosulphate [47] incorporation of chloroplast ATPase in the same system yielded the reconstitution of photophosphorylation in a model system. The PSI-RC preparation therefore seems to possess all the functional features of PSI for the vectorial transmembrane electron transfer [48] (see Fig. 4.7). 

Therefore, illuminated chloroplasts must be protected against hydrogen peroxide and singlet oxygen. Ascorbic acid plays a key role in such protection, as summarized in the next section. 

Ascorbic Acid for Removing Hydrogen Peroxide in Chloroplasts. In 1976, it was suggested (70) that hydrogen peroxide could be removed in illuminated chloroplasts in vivo by a nonenzymic reaction with ascorbic acid ... 

This nonenzymatic reaction occurs at a significant rate in crude chloro-plast or plant cell extracts (71, 72). At pH 8, the pH of the stroma in the illuminated chloroplast, dehydroascorbate is reduced to ascorbate by glutathione (GSH), which is present in the stroma of spinach chloroplasts at millimolar concentrations (70). Although nonenzymatic, the reaction is extremely swift at this pH (73). 

Figure 3. An ascorbate-glutathione cycle for removal of H2O2 in illuminated chloroplasts.

Enzymes involved I, ascorbate peroxidase and 2, glutathione reductase. Reactions 3 and 4 are nonenzymatic. Reaction 3 proceeds rapidly at pH 8, the pH of the stroma in illuminated chloroplasts. 

While ascorbate is transported into isolated intact chloroplasts (Anderson et al., 1983b Beck et al., 1983 Foyer and Lelandais, 1996), the thylakoid membranes appear to have no carrier system to transport ascorbate into the thylakoid lumen (Foyer and Lelandais, 1996). This is surprising since the enzyme VDE, located inside the thylakoid, requires ascorbate to convert violaxanthin to zeaxanthin (Fig. 2). Upon illumination the pH ofthe lumen falls and VDE binds to the lumenal side of the thylakoid membrane and becomes active (Hager and Holocher, 1994 Rockholmand Yamamoto, 1996). The affinity of VDE for ascorbate is strongly dependent on pH, because ascorbic acid is the true substrate of the enzyme (Bratt et al., 1995). The addition ofHjOj to illuminated thylakoid membrane preparations induces a transient inhibition of zeaxanthin formation (Neubauer and Yamamoto, 1992), suggesting competition for ascorbate between the Mehler-peroxidase cycle and the VDE reaction. Since the ascorbate pool in the chloroplast stroma is substantial (10-50 rnM), sufficient amounts of ascorbic acid may be able to aoss the membrane by diffusion (Eoyer et al., 1983 Foyer, 1993 Foyer and Lelandais,... 

Foyer CH and Lelandais M (1996) A comparison ofthe relative rates of transport of ascorbate and glucose across the thylakoid, chloroplast and plasmalemma membranes of pea leaf mesophyll cells. J Plant Physiol 148 391-398 Foyer CH, Rowell J and Walker D (1983) Measurement ofthe ascorbate content of spinach leaf protoplasts and chloroplasts during illumination. Planta 157 239-244 Foyer CH, Furbank R, Harbinson J and Horton P (1990) The mechanisms contributing to photosynthetic control of electron transport by carbon assimilation in leaves. Photosynth Res 25 83-100... 

Some further aspects of the toxic effects of unscavenged H2O2 will be discussed below (Section 1.6.1). Experiments with isolated chloroplasts have shown that during illumination in the presence of paraquat, there is a rapid oxidation of both ascorbate and glutathione. ... 

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