Modulation of a Mitochondrial Function by Oat Phytochrome in Vitro

Abstract

ABSTRACTresponses of these organelles to actinic R2 and FR were the earliest extensively studied correlations between these two phenomena. In the present paper, we are reporting our tests of whether Manabe and Furuya's results with pea mitochondria can be obtained with oat mitochondria. We also describe tests of whether exogenously added phytochrome, previously shown to bind to mitochondria with a reproducible stoichiometry (12), could influence the activity of the external NADH-dehydrogenases of mitochondria (22) that we supposed would be accessible to exogenous phytochrome. For these experiments, we have used extensively characterized mitochondrial preparations, and discuss why such characterizations are important for interpretation of the results. MATERIALS AND METHODSMitochondrial Isolation. Mitochondria were isolated from 4-day old etiolated oat seedlings (A vena sativa L., var. Garry) by the procedure of Douce et a. (8). This procedure involves two series of low and high speed centrifugations followed by discontinuous sucrose density gradient centrifugation. Approximately I kg of 4-to 5-cm long oat shoots was harvested after the plants had been chilled to approximately 4 C. The shoots were then exposed to 15 min of white fluorescent light (irradiance of 0.1 mw cm-2) while on ice after harvest and prior to homogenization. All subsequent manipulations were carried out in green safelight at 3-5 C. Mitochondria isolated from these plants were termed "light" mitochondria to contrast them from those isolated from etiolated plants that were not irradiated ("dark" mitochondria). The purified mitochondria were determined to be intact and functioning normally by electron microscopy and biochemical tests described by Douce et al. (8). Their purity was estimated by the quantitative morphometry method described by Cedel and Roux (4). Several random, low-magnification fields taken from various portions of a pellet of purified mitochondria were photographed to obtain the electron micrographs used for the morphometric measurements.Phytochrome Isolation. Phytochrome was isolated by the procedure of Roux et al. (26). All phytochrome used was fully photoreversible, exhibited a peak A for Pr at 667 nm, was at least 50%1o pure and showed 120,000 dalton mol wt subunits on SDS gels. Phytochrome spectral measurements were accomplished using a R-2 ratiospectrophotometer or a Cary model 15 scanning spectrophotometer.Determination of NADPH Levels. NADPH levels were determined by a technique that couples NADPH oxidation to the reduction of DCPIP using a diaphorase enzyme (EC 1.6.99) (16

Phytochrome induces photoreversible calcium fluxes in a purified mitochondrial fraction from oats

“…The control of Ca2+ fluxes in mitochondria by phytochrome may be related to its control of a mitochondrial enzyme, NADH dehydrogenase, reported by Cedel and Roux (6). This enzyme is located on the outer surface of the inner membrane of mitochondria, and its activity is strongly stimulated by added Ca2' (21).…”

“…The experiments in ref. 6 an electrochemical gradient (i.e., from inside the mitochondrial matrix to the outside). This movement, just as the Pfr-promoted Ca2+ movement across the plasma membrane, would promote an increased Ca2+ concentration in the cytosol.…”

“…4). We have found evidence for the high-affinity binding of phytochrome to mitochondrial membranes and for the photoreversible modulation ofan inner-membrane enzyme function by the pigment (5,6). Because the regulation of Ca2+ fluxes is also an inner mitochondrial membrane function, it was ofinterest to learn whether phytochrome could influence these fluxes there as it apparently does on the plasma membrane.…”

Phytochrome‐mediated regulation of plant respiration and photorespiration

“…The phytochrome system may regulate mitochondrial respiration through both kinetic mechanisms and modification of gene expression. Cedel & Roux (,) showed that phytochrome could alter the rate of reduction of exogenously added NADP + by plant mitochondrial preparations. Such interaction between phytochrome and mitochondria occurred at the level of the inner mitochondrial membrane and could be seen only if the mitochondria were isolated from plants that were illuminated with white or red light before extraction.…”

Further Characterization of the in Vitro Binding of Phytochrome To a Membrane Fraction Enriched for Mitochondria

“…Given the relatively low number of high affinity sites for phytochrome in the mitochondrial preparation, any amount of contaminant membrane would compromise the confident identification of the binding site, if this identification were based solely on the purity of the preparation. A more important basis for identifying mitochondria as a probable binding site for 125I-P in the data reported here is the fact that exogenously added phytochrome photoreversibly alters a mitochondrial function, NADHdehydrogenase, under the same experimental conditions as used for these binding studies (3). These physiological data in combination with the data in Table II argue strongly for the working hypothesis of this report: That the specific binding of '25I-P to sites in the mitochondrial membrane preparation was to sites on the mitochondria in the preparation.…”