Evidence for Bound Phytochrome in Oat Seedlings

Rubinstein, Benjamin I. P.; Drury, K S; Park, R B

doi:10.1104/pp.44.1.105

Cited by 63 publications

(21 citation statements)

References 14 publications

(6 reference statements)

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“…There is some evidence that phytochrome is a membrane protein (3), and that it exerts its effects by regulating the ionic permeability, and, consequently, the electrical potential of some cell membranes (4,5). We postulated that this regulation could be made possible by differences in membrane conductance induced by the two forms of phytochrome, and thus designed experiments to test this hypothesis (6).…”

Section: Far-red Lightmentioning

confidence: 99%

Photoreversible Conductance Changes Induced by Phytochrome in Model Lipid Membranes

Roux

Yguerabide²

1973

Proc. Natl. Acad. Sci. U.S.A.

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The plant protein phytochrome induces photoreversible conductance changes when added to a black lipid membrane made from oxidized cholesterol. The conductance of the phytochrome-modified membrane is increased by red-light illumination but is decreased by illumination with far-red light. Denatured phytochrome does not affect the conductance of the model membrane.Phytochrome is a widely distributed plant chromoprotein that mediates many developmental changes triggered by light (1, 2). It has two spectrally different forms that can be reversibly interconverted by irradiation with light of the appropriate wavelength: The red-absorbing form, Pr (Xmax 667 nm), and the far-red-absorbing form, Pfr (Xmax 725 nm). The interconversion of the two forms can be described by the scheme: red light PrPfr. far-red lightThere is some evidence that phytochrome is a membrane protein (3), and that it exerts its effects by regulating the ionic permeability, and, consequently, the electrical potential of some cell membranes (4, 5). We postulated that this regulation could be made possible by differences in membrane conductance induced by the two forms of phytochrome, and thus designed experiments to test this hypothesis (6). Here we present the initial results of our studies in which we have attempted to incorporate phytochrome into a model membrane to determine whether Pr and Pfr can differently affect the ionic permeability of a bimolecular lipid membrane.The model membrane that we used was developed by Mueller, Rudin, and coworkers (7). It can be made from various lipids, including phospholipids, and has a conformation and other properties similar to the bilayer regions of biological membranes. The membrane is formed between two aqueous compartments, thus allowing its electrical properties to be readily measured by standard techniques of electrophysiology. The effects of proteins on ionic permeability can be tested by incorporating them into the membrane. The very high resistance of the pure lipid membrane (109-10°1 _-cm2) provides an ideal system for detection of small changes in conductance, such as have been reported after the incorporation in the bilayer of low amounts of a protein called excitability-inducing material (EIM) (8). MATERIALS AND METHODSThe experimental arrangement for forming and studying the membrane is shown in Fig. 1. The membrane was made across a 0.7-mm hole on the side of a 6-ml Teflon cup that was partially immersed in and filled with a solution of 0.1 M KCl, buffered at pH 7 with 5 mM histidine chloride. The temperature of the system was maintained between 250 and 270.

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Section: Far-red Lightmentioning

confidence: 99%

Photoreversible Conductance Changes Induced by Phytochrome in Model Lipid Membranes

Roux

Yguerabide²

1973

Proc. Natl. Acad. Sci. U.S.A.

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“…More interest in plant cell membranes would be generated if it could be demonstrated that phytochrome exerts its control on growth and differentiation through the membrane. Phytochrome has been reported in cell membranes (5). The elicitation of electrical charges on cell surfaces by phytochrome action might eventually prove to be a trivial matter.…”

Section: Resultsmentioning

confidence: 99%

Indoleacetic Acid and Abscisic Acid Antagonism

Tanada

1973

Plant Physiol.

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“…7). Rubinstein et al (29) concluded that membrane-associated phytochrome, because of its sensitivity to detergents and lability, was distinct from soluble phytochrome. They isolated particulate fractions in a buffer containing EDTA and without Mg2+ which was comparable to our Mg'+-free buffers.…”

Section: Discussionmentioning

confidence: 99%

“…Information concerning its location has been mainly inferential, relating rapid phytochromemediated responses to physiological events at the plasma membrane (10-12) or the nuclear envelope (7). A part of the extracted phytochrome is associated with a particulate fraction (25,26,29); yet, a membrane or membrane fraction which binds phytochrome has not been identified.We examined different fractions obtained from soybean hypocotyls by differential and sucrose gradient centrifugation and observed that phytochrome content in the presence of Mg2+ cor- related with the presence of endoplasmic reticulum with attached ribosomes. In the absence of Mg2+, phytochrome was found in the so-called "heavy" plasma membrane fraction, possibly as a result of contamination of this fraction by fragments of endoplasmic reticulum.…”

mentioning

confidence: 99%

“…Information concerning its location has been mainly inferential, relating rapid phytochromemediated responses to physiological events at the plasma membrane (10)(11)(12) or the nuclear envelope (7). A part of the extracted phytochrome is associated with a particulate fraction (25,26,29); yet, a membrane or membrane fraction which binds phytochrome has not been identified.…”

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confidence: 99%

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Association of Phytochrome with Rough-surfaced Endoplasmic Reticulum Fractions from Soybean Hypocotyls

1975

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Distribution of phytochrome (as Pfr) among membranes from soybean hypocotyls (Glycine max L. cv. Wayne) was determined by the combined techniques of cell fractionation, difference spectrometry, and electron microscopic morphometry. More than 90% of the phytochrome was found in the soluble fraction. With homogenates prepared in the presence or absence of Mg2+, the portion associated with membrane was only 6.5% and 1%, respectively. In the presence of Mg2+, the content of particulate phytochrome correlated with the amount of endoplasmic reticulum with attached ribosomes in the fractions but not with mitochondria or other membranes (including endoplasmic reticulum membranes from which the ribosomes may have been lost during cell fractionation). In the absence of Mg2+, phytochrome was associated with a "heavy" plasma membrane fraction. The phytochrome content was sufficiently low to be accounted for by a contamination of less than 10% by rough-surfaced fragments of endoplasmic reticulum. The findings show association of phytochrome with a particulate fraction enriched in rough-surfaced fragments of endoplasmic reticulum but do not rule out cosedimentation of some unknown or unspecific phytochrome aggregate with this fraction.Since the discovery of phytochrome and the elaboration of its ubiquitous role in the regulation of growth and development of flowering plants, its location within the cell and immediate mode of action have remained unknown. Information concerning its location has been mainly inferential, relating rapid phytochromemediated responses to physiological events at the plasma membrane (10-12) or the nuclear envelope (7). A part of the extracted phytochrome is associated with a particulate fraction (25,26,29); yet, a membrane or membrane fraction which binds phytochrome has not been identified.We examined different fractions obtained from soybean hypocotyls by differential and sucrose gradient centrifugation and observed that phytochrome content in the presence of Mg2+ cor- related with the presence of endoplasmic reticulum with attached ribosomes. In the absence of Mg2+, phytochrome was found in the so-called "heavy" plasma membrane fraction, possibly as a result of contamination of this fraction by fragments of endoplasmic reticulum. MATERIALS AND METHODSPlant Material. Soybean seeds (Glycine max L. cv. Wayne) were soaked in water for 6 hr and grown in moist vermiculite (9). After 4 days in darkness at 29 C, the cotyledons were removed, and the hypocotyl apices (approximately 1.5 cm) were harvested.Membrane Isolation. Isolations of membrane fractions were either in fluorescent room light (Table I) or in dim green light after a 5-min exposure of the tissue to red light (Tables II to IV). By either procedure, the phytochrome was expected to be predominantly in the far red-absorbing form at the time of isolation. Fifty to 100 g of tissue were finely chopped with a razor blade and suspended in cold (0-4 C) homogenizing medium (1 ml/g tissue).For isolations in the absence of Mg2+, the homogenizatio...

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Evidence for Bound Phytochrome in Oat Seedlings

Cited by 63 publications

References 14 publications

Photoreversible Conductance Changes Induced by Phytochrome in Model Lipid Membranes

Photoreversible Conductance Changes Induced by Phytochrome in Model Lipid Membranes

Indoleacetic Acid and Abscisic Acid Antagonism

Association of Phytochrome with Rough-surfaced Endoplasmic Reticulum Fractions from Soybean Hypocotyls

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