Formate Oxidation and Oxygen Reduction by Leaf Mitochondria

Abstract

Mitochondria isolated from the leaves of several plant species were investigated for the presence of NAD-linked formate dehydrogenase. The NADH produced was oxidized by the electron transport sequence and was coupled to ATP synthesis. The amounts of formate dehydrogenase, and, thereby, the capacity for formate-dependent 02 uptake, varied greatly among species. While no activity was detectable in mitochondria from soybean leaves, the rate of formate oxidation by spinach mitochondria was about one-half the rate of malate oxidation. In spinach, only mitochondria from green tissues oxidized formate. These last two observations raise questions as to the role of this reaction and the possible sources of the formate metabolized.The metabolism of formate in higher plants appears to proceed by three independent mechanisms. Formate is reduced to hydroxymethyl tetrahydrofolate, in which state it can participate in serine synthesis by the serine hydroxymethyl transferase reaction (16). Alternatively, formate can be oxidized to CO2 by two separate reactions. These are the peroxidatic action of catalase (7,8) and the activity of NAD+-dependent formate dehydrogenase (7,9). Both the serine hydroxymethyl transferase and the NAD-dependent formate dehydrogenase activities are mitochondrial, whereas the catalase reaction is peroxisomal (7,15 MATERIALS AND METHODS Tobacco (Nicotinia tabacum), lentils (Lens culinaris), and soybeans (Glycine max) were grown in a greenhouse. Spinach (Spinacia oleracea) was either greenhouse-grown or purchased locally.Peas (Pisum sativum), bush beans (Phaseolus vulgaris), chard (Beta vulgaris cicla), beets (Beta vulgaris), and lettuce (Lactuca sativa) were grown in a garden plot.Mitochondria were isolated by a modification of the method of Douce et al. (2). Leaf tissue was homogenized for 4 s in a WaringBlendor in 250 ml partially frozen slurry containing 0.3 M mannitol, 2 mm EDTA, 30 mm Hepes (pH 7.5), 0.2% (w/v) BSA, 0.6% (w/v) polyvinylpolypyrolidone, and 0.05% (v/v) 2-mercaptoethanol. Better coupling ratios were obtained if the BSA level was increased to 1% for bush beans and beets. The homogenate was rapidly filtered through four layers of cheesecloth and two layers of Miracloth (Chicopee Mills, Milltown, NJ) and centrifuged at 1,000g for 10 min. The supernatant was then centrifuged at l0,OOOg for 10 min. The pellet was resuspended in about 30 ml reaction mix, which was comprised of 0.3 M mannitol, 5 mm MgCl2, 10 mm KCI, 10 mm K-phosphate (pH 7.2), and 0.1% BSA (2). Resuspension was greatly facilitated by use of a No. 5 red sable artist brush. The mitochondrial preparation was centrifuged again at l,000g for 10 min, and the mitochondria were collected from the supernatant by centrifuging at 6,000g for 10 min (1). This pellet was either resuspended in the reaction mix and used directly or further purified by phase partitioning (4). Chloroplast protein contamination was corrected as described (2). (11,12). Formate dehydrogenase activity was assayed by solubilizing 0.025 to 0.200 ml of the mi...

A Formate Dehydrogenase Confers Tolerance to Aluminum and Low pH

“…S4). This result is in accord with previous reports concerning the localization of FDH proteins in various higher plant tissues (Halliwell, 1974;Oliver, 1981;Colas des Francs-Small et al, 1993). Question then arises concerning the nature of the pathways to formate formation in the mitochondrial matrix under Al and H + stress.…”

“…FDHs catalyze the oxidation of formate into CO 2 , reducing NAD + to NADH in the process. In higher plants, FDHs are mainly localized in the mitochondrial matrix (Halliwell, 1974;Oliver, 1981;Colas des Francs-Small et al, 1993). These observations raised questions as to whether VuFDH is involved in formate catabolism in rice bean roots, and whether the induction of VuFDH by Al is an Al stress response whose function is to reduce Al toxicity.…”

“…Most FDHs in higher plants are localized to mitochondria (Halliwell, 1974;Oliver, 1981;Colas des Francs-Small et al, 1993). In order to determine the subcellular localization of VuFDH, we used 35S:: VuFDH::GFP fusion constructs to be transiently expressed in N. benthamiana leaves and to examine whether the GFP signal is colocalized with the mitochondria-staining fluorescence dye TMRM (tetramethyl rhodamine methyl ester).…”

Effects of Irradiance on the in Vivo CO2:O2 Specificity Factor in Tobacco Using Simultaneous Gas Exchange and Fluorescence Techniques

“…Formate and CO2 are produced by reaction of hydrogen peroxide with glyoxylate. Formate dehydrogenase is normally present at low levels in leaf tissue (20) so that the probable fate of any formate synthesized is entry into the C, pool following ATP-dependent activation by formyltetrahydrofolate synthetase (27 …”

Effects of Glycolate Pathway Intermediates on Glycine Decarboxylation and Serine Synthesis in Pea (Pisum sativum L.)

“…Although enzymes catalyzing formate oxidation exist in leaf tissue ( 18), the formate carbon is not readily lost as C02, but is metabolized further, possibly via serine synthesis (3,7,9,10,28).…”