Effect of Glycidate on Glycolate Formation and Photosynthesis in Isolated Spinach Chloroplasts

Abstract

Preparation of Chloroplasts. Chloroplasts were isolated from leaves of spinach (Spinacia oleracea L., var. Virginia Blight Resistant) grown in a growth chamber at 21 C with a 10-hr day (2100 ft-c) and 16 C night. The light-dark cycle in the chamber was adjusted so that the plants received about 30 min of illumination before chloroplast isolation. Chloroplasts were prepared essentially as described by Jensen (12) with some modifications. The rinsed leaves, about 10 g after deribbing, were chopped gently at 4 C with razor blades in 45 ml of a grinding medium containing 330 mm sorbitol, 50 mm MES, 2 mM Na2EDTA, 1 mM MgCI2, I mm MnCI2, 0.5 mm K2HPO, (entire solution adjusted with NaOH to pH 6.5 at 4 C) plus 5 mm freshly prepared isoascorbate. The slurry was squeezed through two layers of Miracloth and centrifuged for 50 sec at 2000g. The chloroplast pellet was resuspended and stored in the dark at 4 C in the sorbitol-salt solution described above except that 50 mm HEPES (pH 6.7) replaced MES and the isoascorbate concentration was reduced to 2.5 mm. Chl was determined by the method of Arnon (1), and the chloroplast preparations contained 110 to 650 ,ug Chl/ml.Photosynthetic "C02 Assimilation. Photosynthetic CO2 fixation by the isolated chloroplasts was determined by H'4CO3-incorporation at 25 C and 3700 ft-c (74 nanoeinsteins/cm2 sec, 400-700 nm) under various He/02 atmospheres. Unless noted otherwise, the reaction vessels contained chloroplasts (5-32 tig of Chl), resuspension medium adjusted to pH 7.8, 5 mm freshly prepared Na4P207, +3 mm glycidate, and 1 mm NaH 4CO3 (2)(3)(4) Ci/mole) in a final volume of I ml. Vessels containing all the reaction medium components except chloroplasts and "4C-bicarbonate were sealed and repeatedly evacuated and refilled with the appropriate He/02 mixture and chilled, after which the chloroplasts were introduced with a 20-gauge syringe needle.Following a 4-min preillumination without shaking in the photosynthetic Warburg bath, the reactions were initiated by injecting NaH14CO3 and terminated at varying times by injecting 0.1 ml of 6 N acetic acid. Contents of the flasks were thoroughly mixed, aliquots dried at 25 C under a stream of N2, and dpm 237 www.plantphysiol.org on May 11, 2018 -Published by Downloaded from

Effects of Glycidate on Carbon Dioxide Fixation with Isolated Spinach Chloroplasts

“…Several other communications reported the observation that glycidate did not affect photorespiration by directly inhibiting RuBP oxygenase (2,23,24). Results of this paper also support earlier observations by Chollet (7), that GL, the photorespiratory end product of the chloroplasts, was produced in even higher amounts in the presence of glycidate, but that its ratio to other photoassimilates was not affected.…”

“…A controversy arose between the results of Zelitch (23) and Chollet (7) with respect to the effect of glycidate on the formation of GL. As already mentioned above, glycidate was introduced as an inhibitor of GL formation (23 (Fig.…”

“…The enhancement of CO2 fixation due to the presence of glycidate (7,23) might be explained by an increased level of ATP and NADPH in the illuminated chloroplasts. It was of interest to study the effect of this compound on electron transport and photophosphorylation.…”

“…Glycidate stimulates net CO2 fixation in leaf segments (23) and isolated chloroplasts (7). The action of glycidate on isolated chloroplasts could be linked to a decrease of photorespiration, ie.…”

Comparative Growth Analyses of Panicum Species with Differing Rates of Photorespiration

“…The C02/02 mixtures were continuously flushed through the open flow system (18) CO2 Compensation Concentration Determinations. The r of excised leaves was determined at 30 C and 2150 ft-c as described previously (6). In order to vary PO2, the bags of Mylar polyester film were flushed and inflated with the appropriate commercially prepared 02/N2 mixture.…”