Leaf Respiration

supporting

confidence: 91%

Mitochondria of Isolated Plant Cells (Acer pseudoplatanus L.)

Bligny¹,

Douce²

1977

“…Such a strong dependence of respiration on the external 02 concentration is well known for many types of bulky plant organs. This phenomenon has generally been ascribed to diffusion problems, although data obtained with tissue pieces of varying size were found to be inconsistent with such an explanation (10,11,18).…”

Section: Methodsmentioning

confidence: 99%

“…This is essentially what Moyse (22) and Moyse and Jolchine (23) found for intact Bryophylluin leaves, but the situation in their particular case was somewhat more complex. Ducet and Rosenberg (10) An often discussed supposition invokes the involvement of more than one terminal oxidase system as an explanation for the observed effects of increasing oxygen concentrations on the respiration rate (10,11,42). One might expect that these oxidases differ in their sensitivity to cyanide.…”

Section: Methodsmentioning

confidence: 99%

The Relation between Photosynthesis, Respiration, and Crassulacean Acid Metabolism in Leaf Slices of Aloe arborescens Mill

Denius¹,

Homann²

1972

Leaves and leaf slices from Aloe arborescens Mill. were used to study the interrelations between Crassulacean acid metabolism, photosynthesis, and respiration. Oxygen exchange of leaf slices was measured polarographically. It was found that the photosynthetic utilization of stored malic acid resulted in a net evolution of oxygen. This oxygen produetion, and the decrease in acid content of the leaf tissue, were completely inhibited by amytal, although the rate of respiratory oxygen uptake was hardly affected by the presence of this inhibitor of mitochondrial electron transport. Other poisons of respiration (cyanide) and of the tricarboxylic acid cycle (trifluoroacetate, 2-diethyl malonate) also were effective in preventing aciddependent oxygen evolution. It is concluded that the mobilization of stored acids during light-dependent deacidification of the leaves depends on the operation of the tricarboxylic acid cycle and of the electron transport of the mitochondria.A mits a closure of the stomata during the hot daytime by making CO, available for photosynthesis from the endogenous acid pools (24).Many striking resemblances exist between the mechanism of CO. assimilation in CAM and that in the C-dicarboxylic acid pathway of CO, fixation which is characterized by the appearance of labeled dicarboxylic acids as first stable products of the photosynthetic assimilation of 'CO2. Many data and concepts have recently been published on the physiological differences which separate C-plants from plants with the "classical" Calvin-Benson pathway of CO2 fixation (C,-plants). Investigations on CAM, however, have been few. This situation is reflected in Hatch and Slack's (13) review on the mechanisms of CO2 assimilation. The photosynthetic use of CO2 from stored malate in chlorophyllous tissue with CAM should be reflected in a light-dependent net evolution of oxygen even in the absence of external CO,. It was the aim of this work to devise a rapid and reproducible method for the measurement of 02 exchange in such tissue under various conditions. This approach was hoped to permit an analysis of the physiological conditions regulating the storage and photosynthetic utilization of CO2 in plants with CAM.The use of intact leaves was not desirable because their gas exchange is influenced by the activity of the stomata and by the diffusion of gases through the bulky tissue. Sectioning of the leaf would lessen, or eliminate, such problems and facilitate the entry of added inhibitors and other substances. Bruinsma (6) had observed, however, that CAM was adversely affected when Bryophyllum leaves were sliced. This appeared to have been due to actions of leached tannins and other phenolic compounds. Therefore, we had to find a plant which was relatively free of such potentially inhibitory compounds. This criterium eliminated nearly all plants which had been used by other investigators in the past, e.g., from the genera Bryophyllum, Sedum, and Kalanchoe. Leaves from Aloe arborescens Mill. (Liliaceae), however, had a very low conten...

“…In marked contrast, relatively few methods are available in the literature for the isolation of intact mitochondria from the leaves of higher plants (8,12,17,18). Therefore, little information is available about the oxidative and phosphorylation properties of mitochondria from green leaves (10,12,17,18).…”

Section: Introductionmentioning

confidence: 99%

Isolation and Oxidative Properties of Intact Mitochondria Isolated from Spinach Leaves

Douce¹,

Moore²,

Neuburger³

1977

121

A procedure was described for preparing intact mitochondria from spinach (Spinacia oleracca L.) leaves. These mitochondria oxidized succinate, malate, pyruvate, a-ketoglutarate, and NADH with good respiratory control and ADP/O ratios comparable to those observed with mitochondria from other plant tissues. Glycine was oxidized by the prepartions. This oxidation linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport and phosphorylation inhibitors.Cyanide completely inhibited the oxidation of NADH. The oxidation of succinate, malate, and glycine was only partially inhibited.The development of techniques for the isolation of tightly coupled mitochondria from etiolated and storage tissues (12,17,18) has resulted in considerable progress toward understanding the physiological properties of the plant mitochondria (11,12,17,18). In marked contrast, relatively few methods are available in the literature for the isolation of intact mitochondria from the leaves of higher plants (8,12,17,18). Therefore, little information is available about the oxidative and phosphorylation properties of mitochondria from green leaves (10,12,17,18).The object of this paper is to describe the preparation, oxidative capacities, and response to various inhibitors of spinach leaf mitochondria. MATERIALS AND METHODSPreparation of Mitochondria. Fresh spinach (Spinacia oleracea L.) leaves were obtained from a local market and used immediately. Young spinach leaves (0.8 kg after deribbing) were cut into 3 liters of chilled medium containing 0.3 M mannitol, 4 mM cysteine, 1 mM EDTA, 30 mm MOPS3 buffer (pH 7.5), 0.2% defatted BSA, and 0.6% insoluble PVP. The leaves were disrupted at low speed for 2 sec in a 1-gallon Waring Blendor. The homogenate was squeezed through six layers of cheesecloth and mitochondria were isolated as fast as possible by differential centrifugation according to the method of Bonner (4). We found that very short grinding times in a Waring Blendor were the most successful. Longer grinding times had a deleterious effect on the mitochondrial oxidative and phosphorylative capacities.Mitochondrial Assays. Mitochondrial respiration was mea- sured polarographically using a Clark 02 electrode in a closed cell at 25 C. Oxygen consumption measurements were performed in a reaction medium (3.3 ml) containing 0.3 M mannitol, 5 mM MgCl2, 10 mm KCI, 10 mm Na-phosphate buffer (pH 7.2), and 0.1% defatted BSA. The 02 concentration in airsaturated medium was taken as 240 UM (9).Rotenone (Sigma), FCCP (Boehringer), oligomycin (Sigma), and antimycin A (Sigma) were dissolved in absolute ethanol. Salicyl-hydroxamic acid dissolved in formamide was a gift of Dr. Vignais.Mitochondrial Protein Determination. Total protein was determined by the Folin-Ciocalteau phenol reagent (16). Chl was extracted from the mitochondrial pellet in 80% acetone and measured according to Arnon (1). If we assume a protein to Chl ratio of 7, in broken thylakoids (15), the amount of mito...