Photoreduction of Oxygen in Mesophyll Chloroplasts of C₄ Plants

Abstract

Mesophyll chloroplasts of three C4 sub types, Panicum miliaccum (NAD-malic enzyme), Panicum maximum (PCK), and Zea mays (NADPmalic enzyme), were prepared from protoplast extracts and used to study the photoreduction of 02. The processes of 02 uptake and evolution in these preparations, which lack ribulose 1,5-bisphosphate carboxylase/ oxygenase, were studied simultaneously using stable isotopes of 02 and mass spectrometry. The responses of 02 uptake to 02 tension and addition of various substrates (3-phosphoglycerate, pyruvate, and oxaloacetate) were studied in detail. sumably dependent upon the utilization of NADPH relative to the ATP demand as has been proposed for some time (15). Few measurements have been made of chloroplast pyridine nucleotide levels (23) and manipulation of these levels in an intact system is difficult. Hence, there is little evidence of such a regulation in vivo. Although there is substantial physiological evidence for light and ATP-dependent ion transport associated with photoreduction of 02 ( 13), there is little direct evidence of the capacity of the Mehler reaction to generate ATP for photosynthetic carbon assimilation in vivo.In this study, 02 uptake and evolution in chloroplasts in mesophyll protoplast extracts from Zea mavs (NADP malic enzyme), Panicum maximum (PEP carboxykinase), and Panicum miliaceuim (NAD malic enzyme) are examined using 1802 and a mass spectrometer. The photoreduction of 02 in this system is characterized and its response to the addition ofvarious substrates is examined in order to study the regulation of the process and its ability to produce ATP. MATERIALS AND METHODSLight-dependent 02 uptake in higher plants has previously been studied in a variety of systems: isolated thylakoids (11), intact chloroplasts and cells (5,9,10,16,21), and in the intact leaf (3,6,12,22,24 Approximately 10 g of leaves were harvested for each preparation. The leaves were sliced into approximately 1-mm strips with a razor blade and digested at 30°C for 2 to 2.5 h, with illumination (1 50-w flood lamp), in 50 ml of 0.5 M sorbitol, 0.2 mM CaCI2, I mM MgC92, 0.2 mM KH2PO4, 10 mM Mes, 0.2% BSA, 2% cellulase (Onozuka S-3), and 0.2% pectinase (Macerozyme R-10) at pH 5.5 with HCI. Leafstrips were examined under a microscope each hour to determine the correct digestion time. When gentle agitation of the tissue resulted in mesophyll protoplast release, the incubation medium was carefully decanted and the leaf strips washed in a plastic tea strainer with 50 ml of 0.6 M sorbitol, 5 mM Hepes, 1 mM MgCl2, 0.2 mM CaCl2, 0.2 mM KH2PO4, and 0.1 % BSA, pH 7.8. The filtrate was passed through two nylon nets (250 ,um net and then an 80 ,um) and the filtrate was centrifuged at 350g for 3 min. The pelletted protoplasts were gently resuspended in 20 ml of the solution previously described but with 0.6 M sucrose substituted for sorbitol. The suspension 1038 www.plantphysiol.org on May 12, 2018 -Published by Downloaded from

Oxygen Uptake and Photosynthesis of the Red Macroalga, Chondrus crispus, in Seawater

“…Furthermore, with 02 concentrations exceeding 20%, net photosynthesis was 02 insensitive although it was very CO2 sensitive; also, 02 uptake was 02 insensitive, weakly CO2 sensitive, and never inhibited by high CO2 concentration, contrary to theoretical predictions of such conditions (2). All (16,18). The affinity for 02 of 02 uptake observed in C.…”

“…But also, these discrepancies suggest that: (a) the Rubisco enzyme differs either as it is isolated or placed in its functional cellular environment; or (b) the Rubisco enzyme is not involved in all of the 02 photoconsumption. From several reports on higher plants (2,(16)(17)(18)25) and microalgae (7,11,15,21,26,(28)(29)(30) organisms. Furthermore, some microalgae have presented gas exchange kinetics in contradiction with the expected competition between 02 and CO2 at the Rubisco enzyme level for photochemically generated reducing equivalents (15,29,30), and exhibit no detectable photorespiration as measured by CO2 release in the light (24,30).…”

“…But also, these discrepancies suggest that: (a) the Rubisco enzyme differs either as it is isolated or placed in its functional cellular environment; or (b) the Rubisco enzyme is not involved in all of the 02 photoconsumption. From several reports on higher plants (2,(16)(17)(18)25) and microalgae (7, 11, 15, 21,26,[28][29][30], the Mehler reaction appears to be a reaction common to all photosynthetic 'Abbreviations: RuBP, ribulose 1,5-bisphosphate; FW, fresh weight; Pc, net CO2 photosynthesis; Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase; PCR, photosynthetic carbon reduction; PCO, photorespiratory carbon oxidation.…”

Regeneration of Ribulose 1,5-bisphosphate and Ribulose 1,5-bisphosphate carboxylase/oxygenase Activity Associated with Lack of Oxygen Inhibition of Photosynthesis at Low Temperature

“…McVetty and Canvin (1981) suggested that pseudocyclic photophosphorylation may be decreased at low p(O 2 ). Pseudocyclic phosphorylation due to photoreduction of oxygen has been shown to be necessary for balancing ATP and NADPH requirements in mesophyll chloroplasts of C 4 -plants (Furbank, Badger, and Osmond, 1983). No pseudocyclic electron flow can occur in the absence of oxygen.…”

Our Eclectic Adventures in the Slower Eras of Photosynthesis: From New England Down Under to Biosphere 2 and Beyond

“…John Andrews and Murray Badger predicted and proved that C 4 RuBisCO would show higher K m for CO 2 (9), Paul Hattersley pioneered immunofluorescent localization of both carboxylases (53) (42). Tom Neales and I were determined to put CAM into the picture of the prickly pear (Opuntia inermis/stricta) invasion of eastern Australia (1846-1933), otherwise remembered only for the success of biological control (114,128).…”