Isolated, intact spinach (Spinacia okracea L. var. 'Long Standing Bloomsdale") chloroplasts were heated in the dark and the effect of this treatment on photosynthetic activities was determined at 25°C. Dark incubation of the chloroplasts for 10 minutes at 35°C and pH 8.1 resulted in a 50% decline in CO2 photoassimilation. This decline in photosynthetic performance was dependent upon time, temperature, and medium pH with the optimum effect at acidic pH values. Photosynthetic decline was not observed if MgATP, MgADP, or a mixture of fructose 1,6-bisphosphate, aldolase, and oxaloacetate or ribose 5-phosphate and oxaloacetate was added prior to but not after the temperature pretreatment. A chloroplast preparation reconstituted with thylakoids and stroma from pretreated (35°C, 10 minutes, pH 8.1) intact chloroplasts and supplemented with ferredoxin, ADP, and NADP was photosynthetically competent, indicating that ATP-coupled electron flow and the enzymes comprising the Benson-Calvin cycle remained stable during the dark treatment. In contrast, exposure of isolated thylakoids to 35°C for 10 minutes uncoupled photophosphorylation from NADP and ferricyanide reduction. We propose that the decline of intact chloroplast photosynthesis is the result of a decrease in the content of or a change in the ratios of the adenine nucleotides. Maintenance of an adequate supply of adenine nucleotide is the effect of the externally added MgATP or of chloroplastic respiration of a sugar phosphate.Many studies have been reported on the effect of temperature pretreatment on photosynthetic electron transport and phosphorylation in isolated thylakoidal preparations (13,16, 25, 27), but less attention has been paid to relate these findings to CO2 photoassimilation in the parent chloroplast (7,(28)(29)(30). Of the thylakoidal reactions measured, there appears to be a collective agreement that phosphorylation is the most susceptible to thermal inactivation, followed by PSII and with PSI the most stable. The isolated stromal enzymes that comprise the Benson-Calvin cycle when assayed individually were found to be comparatively stable to high temperature (18, 28, 29). This approach makes evaluation of the temperature-sensitive steps of photosynthesis within the intact chloroplast difficult inasmuch as there is evidence that soluble stromal compounds (sugar, proteins) are able to protect cell structures against heat inactivation (18, 26, 27).We show here a quantitative study on the effects of preheating up to 40°C in the dark on the photosynthesis ofthe intact spinach chloroplast at 25C. The pH of the medium during the pretreatment was varied since the stability of the thylakoidal reactions is 'Supported by National Science Foundation PCM 83-04147. 2Present address: Roche Institute of Molecular Biology, Nutley, NJ 07110.reported to be pH sensitive (6). We then examined thylakoidal and stromal performance both individually and combined in a reconstituted system fortified with adenine and pyridine nucleotides, Fd, and Pi in order to determine...

Spinach chloroplasts were used to study the relationship between photosynthetic CO2 fixation and temperature from 30 to -15°C. In saturating light and high concentrations of CO2, the temperature coefficients (Q,.) above 20°C were less than 2 in the intact chloroplast. Below 15°C, the Qlo values were greater than 2 and gradually increased with decreasing (down to 0WC) temperature to approximately 4.4. Photosynthesis responded similarly to temperature in a reconstituted chloroplast preparation fortified with ribose 5-phosphate. In the intact chloroplast, temperature did not alter the Qlo value in low light and high CO2.Elevating the temperature to 25°C after photosynthesizing at -15°C (46 minutes) or 0°C (17 minutes) restored the temperature-depressed photosynthetic rate without a lag in the intact chloroplast to the rate of a chloroplast continually at 25°C. At 0°C, the intact chloroplast photosynthetic rate responded slightly to the inorganic phosphate concentration (0.1-1.0 millimolar) and to pH (7.04.6). Relative to 25°C, the levels of ribulose 1,5-bisphosphate and glycerate 3-phosphate were increased 1300 and 200%, respectively, whereas glycolate decreased 57% during intact chloroplast photosynthesis at 0°C. Chilling temperature impeded the transport of photosynthetic intermediates from the stromal compartment to the external medium. Ethylene glycol was shown to be an appropriate additive to prevent freezing of the reaction mixture down to -15°C for photosynthetic CO2 assimilation.The influence of temperature on photosynthesis is a complex phenomenon. In experiments involving short exposures to low temperature (minutes rather than hours or beyond) where no apparent time-dependent inhibition effects occur, it should be possible to obtain results of the direct effect of temperature on the photosynthetic process. To eliminate the complication of the stomates well known to be influenced by temperature as well as CO2 concentration, light intensity, and the water status of the plant, the algae rather than the higher plant became the principal experimental material. Whenever the illumination was weak and the supply of CO2 high, the rate of photosynthesis was found to be practically equal over a range of temperature from 4 to 30C.At high light intensity, the photosynthetic rate increased with temperature more so in high than in C02-limiting concentrations (see Ref. 24 for a review of the early literature).To