Metabolism under Microaerobic Conditions of Mitochondria from Cowpea Nodules

Abstract

A method is described for isolating mitochondria from nodules of cowpea ( Vigna unguiculata [L.] Walp.) under completely anaerobic con-ditions. The mitochondria were immediately active when incubated aerobically with substrates, and their respiration rates were higher than mitochondria prepared in air. The mitochondria lacked fumarate reductase and were not inhibited by 5% CO2. When incubated under microaerobic conditions, their respiration could be measured by leghemoglobin spectroscopy. Microaerobic respiration was inhibited approximately 50% by 1 millimolar malonate, and was completely inhibited by cyanide. 02 uptake and the ATP/O ratio declined under microaerobic conditions, and therefore ATP production may be low in the environment of infected nodule cells. The mitochondria of Ascaris can produce ATP in microaerobic conditions. They contain Cyt b and c, but Cyt c,, a, and a3 are absent, as is Cyt oxidase. There is only one coupled phosphorylation during electron transport (1 1).The vigorous respiration ofthe nodule produces a high internal CO2 concentration. Mahon, for example, found that pea nodules respired CO2 even when the rhizosphere CO2 concentration was 3% (12). This indicates that the CO2 concentration within the nodule is even higher. As a result, the bicarbonate concentration must also be high in the nodule. Bicarbonate reduces the activity of mitochondria from potato tubers and spinach leaves (6, 16). We therefore determined if mitochondria from the nodule could tolerate high concentrations of CO2.We described a method for isolating active mitochondria from cowpea nodules (22 A controlled atmosphere glove box (Labconco, model 50004) was modified by incorporating a 2.5 L cold water bath. The bath was kept at 1°C by heat exchange with a refrigerant circulated from an external Lauda K-21RD cooler. The box and its entry port were flushed with prepurified N2 gas, (-15

Expression and kinetics of the mitochondrial alternative oxidase in nitrogen‐fixing nodules of soybean roots

“…The single protein detected by this antibody in roots (Kearns et al 1992) is the A0X3 gene product (Finnegan et al 1997). In each case, the relative amount of the subunit protein largely reflects the relative abundance of the cognate transcript (Finnegan et al 1997), On the other hand, no AOX immunoreactive proteins were observed among mitochon-drial proteins isolated from nitrogen-fixing root nodules of legumes (Kearns et al 1992), and this cori-elates with reports of little or no cyanide-resistant respiration in these tissues (Rawsthorne & LaRue 1986;Day & Mannix 1988;Bryce & Day 1990), leading to the conclusion that AOX expression is suppressed in nodules.…”

“…Nodule mitochondria have lower malic enzytne activity than do root and cotyledon mitochondria (Rawsthorne & LaRue 1986;Day & Mannix 1988) pyruvate within the mauix. This probably explains why eailier studies failed to measure AOX activity in nodule mitochondria.…”

Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role

“…Indeed, the apparent affinity of AOX for O 2 is high (apparent oxygen K m around 1-2 µm) (71-73) and it is lower than the cytochrome oxidase affinity (apparent oxygen K m around 0.1-0.15 µM) (72,74,75). At variance, much higher values of apparent K m of AOX for O 2 have been reported (76), ranging from 10 to 20 µM depending on species, tissues and age of the plants investigated.…”

Products of Dark CO2 Fixation in Pea Root Nodules Support Bacteroid Metabolism

“…The dicarboxylic acids are most likely derived from the host cytosol, as the low 02 tension in the infected zone of the nodule may limit mitochondrial respiration. Thus, it is unlikely that mitochondria can produce dicarboxylic acids at rates suffient to support nitrogenase activity in the bacteroids (12,22).…”