The effect of ozone (03) on ribulose bisphosphate carboxylase/ oxygenase (Rubisco) activity and quantity and net photosynthesis in greenhouse-grown Solanum tuberosum L. cv 'Norland' foliage was studied in relation to oxidant-induced premature senescence. Plants, 26 days old, were exposed to 0.06 to 0.08 microliters per liter 03 from 1000 to 1600 hours for 4 days in a controlled environment chamber. On day 5, plants were exposed to a 6-hour simulated inversion in which 03 peaked at 0.12 microliters per liter. Net photosynthesis declined in response to 03 but recovered to near control levels 3 days after the exposure ended. Rubisco activity and quantity in control potato foliage increased and then decreased during the 12-day interval of the study. In some experiments foliage studied was physiologically mature and Rubisco activity had peaked when 03 exposure commenced. In those cases, 03 accelerated the decline in Rubisco activity. When less mature foliage was treated with 03, the leaves never achieved the maximal level of Rubisco activity observed in control foliage and also exhibited more rapid decline in initial and total activity. Percent activation of Rubisco (initial/total activity) was not affected significantly by treatment. Quantity of Rubisco decreased in concert with activity. The decrease in activities is most likely due to a decrease in available protein rather than a decrease in the percentage of Rubisco activated in vivo. The reduction in the quantity of Rubisco, an important foliar storage protein, could contribute to premature senescence associated with toxicity of this air pollutant.Ozone (03)
The gene encoding the E subunit (atpf?) of the chloroplast ATP synthase of Spinacia oleracea has been overexpressed in Escherichia COK The recombinant protein can be solubilized in 8 M urea and directly diluted into buffer containing ethanol and glycerol t o obtain E that is as biologically active as E purified from chloroplastcoupling factor 1 (CF,). Recombinant E folded i n this manner inhibits the ATPase activity of soluble and membrane-bound CF, deficient in e and restores proton impermeability to thylakoid membranes reconstituted with CF, deficient i n E . Site-directed mutagenesis was used to generate truncations and single amino acid substitutions i n the primary structure of E. I n the five mutants tested, alterations that weaken ATPase inhibition by recombinant E affect its ability t o restore proton impermeability t o a similar extent, with one exception. Substitution of histidine-37 with arginine appears to uncouple ATPase inhibition and the restoration of proton impermeabilty. As in the case of E. coli, it appears that N-terminal truncations of the 4 subunit have more profound effects than C-terminal deletions on the function of E. Recombinant E with six amino acids deleted from the C terminus, which is the only region of significant mismatch between the E of spinach and the E of Pisum sativum, inhibits ATPase activity with a reduced potency similar t o that of purified pea E . Four of the six amino acids are serine or threonine.These hydroxylated amino acids may be important in E-CF, interactions.The chloroplast ATP synthase (CF,CFo) catalyzes the phosphorylation of ADP to ATP at the expense of the proton gradient. The ATP synthase is biochemically separable into two parts: CF,, a membrane-integrated protein complex that translocates protons, and CF,, the catalytic portion of the CF,CFo complex. CF, consists of five different types of subunits, denoted a, p, y, 6, and E, in order of decreasing molecular weight, with a stoichiometry of a,P,y8~. On illuminated thylakoid membranes, CF, catalyzes ATP synthesis at high rates. In the dark, the enzyme usually does not catalyze ATP hydrolysis, even though hydrolysis is thermodynamically favorable. Thus, when thylakoid membranes are illuminated, the ATP synthase activity is rapidly switched on (McCarty et al., 1988).The transition of CF,CFo from an active state to an inactive state is tightly regulated. The suppression of wasteful ATPase activity in the dark requires the presence Supported by National Science Foundation grants MCB 94
The activation of spinach (Spinacia oleracea) chloroplast coupling factor 1 (CF(1)) by thioredoxin (ThR) was characterized using membrane-bound and soluble CF(1). Light generates an electrochemical proton gradient across the thylakoid membrane, which increases the accessibility of the disulfide bond on the gamma-subunit of CF(1) to reduced ThR. The proton gradient substantially accelerates the activation of CF(1) compared with thylakoids incubated in the dark with similar concentrations of dithiothreitol and ThR. The interaction of soluble CF(1) with ThR was studied using fluorescent probes. CF(1) in solution, with and without its associated epsilon-subunit, was labeled at Cys-322 of the gamma-subunit with fluoresceinyl maleimide. ThR from Escherichia coli was labeled with eosin isothiocyanate. Labeled ThR and CF(1) showed normal activities. Fluorescence energy transfer between donor fluoresceinyl maleimide and acceptor eosin isothiocyanate, manifested by a quenching of the donor fluorescence, was detected, suggesting that ThR and CF(1) form an intermolecular complex. When the epsilon-subunit was absent, quenching of donor fluorescence was approximately doubled, indicating that labeled ThR could approach more closely to the gamma-subunit of CF(1). The distance between the fluorescent probes on CF(1) and ThR was calculated to be approximately 65 A when epsilon-subunit was present and 52 A when epsilon was absent. These values are consistent with other distance measurements and energy transfer values reported previously for fluorescent probes on CF(1). Whereas the extent of quenching increased by removal of the epsilon-subunit, the apparent dissociation constant was unchanged. The quenching effect was reversed when the epsilon-subunit was added back to the titration mixture. Similarly, the addition of unlabeled ThR decreased donor quenching.
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