Activation of O₂ Uptake and NAD-Specific Isocitrate Dehydrogenase in Mitochondria Isolated from Cotyledons of Castor Bean by cis, trans-Abscisic Acid

Abstract

The rate of 02 uptake and the activity of NAD-specific isocitrate dehydrogenase (NAD-ICDH) of mitochondria isolated from castor bean cotyledons were increased by added cis,trans-abscisic acid (ABA) in an in vitro system, while the NADP-specific isocitrate dehydrogenase (NADP-ICDH) was not affected by cis,trans-ABA. Trans,trans-ABA showed only a slightly inhibitory effect on 02 uptake. The Vmax value for the isotherm of isocitrate by the enzyme was also increased by cis,trans-ABA. The isocitrate Km value for the enzyme with cis,trans-ABA was calculated to be approximately 249.8 micromolar, while the S05 for the enzyme without the ABA was 151.6 micromolar. The n value calculated from the slopes of Hill plots of the reaction velocity of NAD-ICDH against isocitrate concentration was 1.5 in the mitochondrial fraction in the absence of ABA, and cis,trans-ABA treatment decreased the value to 1.0. Cis,trans-ABA also partly overcame the inhibition of NAD-ICDH activity by ATP. It has been reported that the respiratory rate (10) and ABA content (9) in grape, expressed on a per berry basis, increased during the ripening process after veraison (which means the onset of maturation [13]). These phenomena suggest a close relationship between ABA content and respiratory metabolism in mature tissues and organs of plants.Goldthwaite (5) observed that ABA stimulates oxygen consumption during senescence of Rumex leaf discs. Hourmant and Penot (6) reported that the rate of respiration of aerated potato tuber discs increased after a 24-h aging period with ABA in the dark. Tillberg et al. (16) found that respiration in Lemna gibba L. was significantly stimulated 2 to 7 d after ABA treatment. In the unicellular green algae, Chlorellafusca and Ankistrodesmus brauni, dark respiration was increased by ABA (17).In the present work, we studied the stimulating action of ABA on respiration of mitochondria isolated from cotyledons of castor beans and found evidence for the regulation of enzyme activity by ABA in an in vitro system. MATERIALS AND METHODS Plant MaterialsCastor beans (Ricinus communis L.) were grown under the conditions described previously by Kondo and Sugahara (7).The plants were grown for 2 to 3 weeks at 25 ± 0.5°C during the day and 20 ± 0.5°C at night with 70 ± 5% RH in an environmentally controlled glasshouse under natural light conditions. The growth substratum was composed of vermiculite: peat moss:perlite:fine gravel (2:2:1:1, v/v/v/v). A nutrient solution containing 8 g of magnesia lime and 4 g of hyponex per liter was supplied twice a week to each 1.9 L pot. Isolation of MitochondriaFive pairs of castor bean cotyledons were cut into small pieces (about 15 g) and homogenized with 45 mL of 25 mM Hepes buffer (pH 7.8) containing 0.35 M mannitol, 0.25 M sucrose, and 0.1 mg Na-2-mercaptobenzothiazole and 1 mg BSA per mL (14) using a Homo blender (18,000 rpm, 100 W, Sakuma Seisakusho, Ltd., Tokyo) at a quarter of maximum speed for 20 s. The homogenate was squeezed through four layers of gauze and centrifuged for 10...

Changes in the activities of carbon metabolizing enzymes with pod development in lentil (Lens culinaris L.)

“…Sucrose synthase in cleavage direction was measured by incubating 200 mol.m -3 of HEPES buffer (pH 6.5), 4 mol-m -3 of UDP and 100 mol.m -3 of sucrose and enzyme extract at 37 °C for 30 min and fructose released was estimated (Nelson 1944). Activities of isocitrate dehydrogenase, NADP malic enzyme and PEP carboxylase were determined by procedures described by Tezuka et al (1990), Iglesias andAndreo (1990) andChristeller et al (1977) respectively and described in details earlier (Chopra et al 2002).…”

Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii1

“…Matrix and membrane forms of the enzyme have been detected in potato (Tezuka and Laties, 1983) and pea (McIntosh, 1997). Although it is an allosteric enzyme that exhibits sigmoidal kinetics with respect to isocitrate (Cox and Davies, 1967;McIntosh and Oliver, 1992) and is activated in vitro by ABA (Tezuka et al, 1990), the regulatory importance of NAD-IDH in photosynthetic organisms is still under debate.…”

Effect of LAB 173 711, an ABA analogue, on low-temperature resistance of mung bean seedlings

“…The ABA level increased linearly with respiration rate in ripening grapefruit (Niimi and Torikata 1979). ABA has been reported also to have enhanced respiration rate and NADP-specific isocitrate dehydrogenase activity in mitochondria which had been previously isolated from castor bean cotyledons (Tezuka et al 1990). Higher metabolic activity was indicated to be present as a result of enhancement of oxygen consumption.…”