Blue light-induced Absorbance Changes in Membrane Fractions from Corn and Neurospora

Abstract

Blue light-induced absorbance changes were measured from differentially centrifuged membrane fractions from dark-grown coleoptiles of Zea mays L., and mycelia from an albino mutant of Neurospora crassa Actinic irradiation caused changes in absorbance consistent with a flavinmediated reduction of a b-type cytochrome. Both corn and Neurospora showed similar light-minus-dark difference spectra, dose response curves, and kinetics of dark recovery after irradiation. The photoreducible cytochrome system from Neurospora showed the same distribution as the activity of a sodium-stimulated adenosine triphosphatase, thought to be a plasma membrane marker, in differential centrifugation experiments. The fraction showing the absorbance change did not co-sediment with the mitochondria, nor with the endoplasmic reticulum. Comparison of absorption spectra of fully oxidized, partially reduced, and fully reduced preparations showed that approximately a 30% reduction of the cytochromes involved with the process was needed to obtain the lightinduced absorbance changes.There is currently a substantial body of literature concerning the effects of blue light on biological processes in higher plants and fungi. Only recently has any real progress been made toward the identification of the blue light photoreceptor associated with these effects (see ref. 6). Poff and Butler (16) ation in that report was the separation of soluble from pelletable components. Mufioz and Butler (15) did report light-inducible Cyt reduction in soluble extracts of Neurospora, but they studied supernatant rather than pelletable activity, observed reduction both of Cyt b and c, and had to add FMN or FAD in order to observe the reaction. It seems likely that they were studying the same phenomenon as Schmidt and Butler (20) who found photoreduction of Cyt c in the presence of added flavins in a model system. Widell and Bjorn (24) have reported light-induced A changes in another higher plant system, intact wheat coleoptiles, but the responses measured were not the same as those monitored by Briggs (5) or Munioz and Butler (15).The purposes of the study reported here were: (a) to isolate and characterize partially purified membrane fractions of Neurospora with a photoreduction system similar to that found in intact mycelia by Munoz and Butler; (b) to study further the blue light-induced A changes found in corn membrane fractions and compare them to similar changes found in Neurospora; and (c) to determine qualitatively the redox state necessary for observation of these changes. A preliminary account of these studies has appeared elsewhere (3, 7). To prepare the mycelia, approximately 70-ml liquid suspensions were made of the 7-day-old agar cultures, and added to a 2,000-ml Erlenmeyer flask, filled with 1,000 ml of liquid medium (the same as solid medium except dextrose instead of sucrose, and no agar). The cultures were grown aerobically in the dark at 30 C for 24 hr at 250 rpm in an incubating rotary shaker (New Brunswick Scientific,. MATERIALS AND METHODSA...

Plasma Membranes from Oats Prepared by Partition in an Aqueous Polymer Two-Phase System

“…A blue light-induced absorbance change indicating reduction of a membrane bound b-Cyt (and with a possible connection to blue light physiology) has been proposed to be located in plasma membranes (6). It has also been shown that red light and methylene blue can be used as substitutes for blue light and endogenous photoreceptor, respectively (7).…”

Redox enzymes in the plant plasma membrane and their possible roles

“…Although the presence of b-type cytochromes in higher plant PM was first reported by Brain et al (1977) and Jesaitis et al (1977), conclusive evidence was obtained only by Asard et al (1989) and Askerlund, Larsson & Widell (1989).The amount of b-type cytochromes in plant PM vesicles varies from 60 pmol mg -1 protein (Asard et al 1989) to 800 pmol mg -1 protein (Widell & Sommarin 1991). Spectra of reduced-minus-oxidized b-type cytochromes in plant PM show a typical absorption maximum between 555 and 563 nm (a-band of the spectra), depending on temperature and species .…”

“…When cytochromes were discovered to reside in the mitochondria, the theory of 'anion respiration' lost prominence (Robertson 1960(Robertson , 1991. The plant PM redox studies started again after two observations in the 1970s; it was shown that (i) iron acquisition of plants required an Fe 3+ reduction step (Chaney, Brown & Tiffin 1972), and (ii) a blue-light reducible b-type cytochrome was associated with membrane fractions from corn and Neurospora (Brain et al 1977). Marker enzyme studies demonstrated that the so-called light-inducible absorbance change (LIAC) activity cosedimented with the oligomycininsensitive Mg 2+ -ATPase and glucan synthase II activities, two proposed positive marker enzymes for the PM in higher plants Leong, Vierstra & Briggs 1981).…”

Effect of Xenon on the Excited States of Phototropic Receptor Flavin in Corn Seedlings

“…The preferential quenching of the flavin triplet over the singlet excited state Flavins have been strongly implicated as the chromphore responsible for the myriad of blue light responses found in plants (10). Evidence concerning phototropism in corn (1,7,8) and Phycomyces (2, 5) and phototaxis in Euglena (3), in particular, have been interpreted to suggest that the flavin triplet state is the active species (2, 3). This conclusion is based mainly on the observations that potassium iodide, which effectively quenches flavin photoreactions in solution (9), inhibits the photoresponses of both corn and Euglena.…”

“…Evidence concerning phototropism in corn (1,7,8) and Phycomyces (2,5) and phototaxis in Euglena (3), in particular, have been interpreted to suggest that the flavin triplet state is the active species (2,3). This conclusion is based mainly on the observations that potassium iodide, which effectively quenches flavin photoreactions in solution (9), inhibits the photoresponses of both corn and Euglena.…”