Effects of Magnesium on Intact Chloroplasts

Abstract

MATERIALS AND METHODS Plant Growth and Chloroplast Isolation. Spinach (Spinacia oleracea L.) plants were grown in the greenhouse or growth chamber-grown (12-h photoperiod, 22°C day/170C night). Leaves were ground briefly in a Waring Blendor2 using an iced slurry containing 0.33 M sorbitol, 10 mm Na4P207 (pH 6.5), 5 mM MgC12, and 2 mm isoascorbate (12). The chloroplasts were pelleted by centrifugation at 2,000g for 5 s. Chloroplasts from greenhousegrown spinach were washed in a buffer containing 0.4 M sorbitol, 50 mM Hepes-NaOH (pH 7.6), 10 mm NaHCO3, and 5 mm EDTA. Chloroplasts were resuspended carefully in a medium containing 0.33 M sorbitol, 50 mm Hepes-NaOH (pH 7.6), 1 mm MnCl2, 1 mM MgCl2, and 2 mm EDTA. The final suspension of chloroplasts (70-80% intact) contained about 0.5 mg/ml of Chl.02 Evolution. Rates of 02 evolution were determined polarographically using a Clark-type platinum O2 electrode placed in a 2-ml. H20-jacketed cuvette at 250C. The reaction mixture contained 0.33 M sorbitol, 50 mM Hepes-NaOH (pH 7.6), 1 mm MnCl2, 1 mm MgCl2, 2 mm EDTA, 250 units catalase, 5 mM NaHCO3, 30 to 60 ,ig Chl, and 0.25 to 0.5 mm Pi. A 75 w flood lamp that emitted 60 nE/cm2.s provided illumination for photosynthesis.Stromal pH and KV. Stromal pH was determined by the equilibration of radioactive bicarbonate ions across the envelope following the method developed by Heldt and Sauer (7). Polyethylene tubes were set up containing 30 ll 2.5 N NaOH in the bottom of the tube with a 70-,ul layer of silicone oil (Wacker AR 200) 2Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.

Development and Use of Chlorotetracycline Fluorescence as a Measurement Assay of Chloroplast Envelope-Bound Mg2+

“…However, the relationship shown in Figure 10 (10) The experiments relating altered chloroplast envelopebound Mg24 with photosynthetic capacity (Figs. 9 and 10) extend some hypotheses developed by Huber (12,17) and Gimmler (8,11). These experiments support the contention that Mg24 bound to the chloroplast envelope, and not necessarily high external Mg2", may affect stromal pH and, hence, photosynthetic capacity.…”

“…Previous research has indicated that incubation of chloroplasts in medium with several millimolar Mg2+ can result in H+ influx into the chloroplast resulting in stromal acidification and inhibition of photosynthesis (8,17). Mg2+ inhibition of photosynthesis is characterized by a class of effects typical of other treatments which result in stromal acidification.…”

“…These include an altered external P043-optima ( 12), relative insensitivity of photochemical reactions and 3-phosphoglycerate reduction (8), stimulation of photosynthesis by addition of K+ and other monovalent cations (12,17), and K+ efflux from the stroma in the absence of high external K+ (17 (Fig. 9A) and photosynthesis in the presence or absence of 40 mm K+ in the reaction medium (Fig.…”

“…However, external Mg2" has been shown to have profound effects on the photosynthetic capacity of isolated chloroplasts (8,12,17 Research was undertaken to address these possibilities. Experiments were performed to characterize the usefulness of using CTC fluorescence to measure chloroplast envelopeassociated Mg2+.…”

Involvement of Na+ in Active Uptake of Pyruvate in Mesophyll Chloroplasts of Some C4 Plants

“…Maury et al (15) postulated a Mg2+-activated reversible K+/H+ exchange. In these studies, it was assumed without any direct measurement of Na+ flux that Na+ also behaved like K+.…”