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

Abstract

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...

Action and Inhibition of Endogenous Phospholipases during Isolation of Plant Membranes

“…Phospholipid-containgin spots were removed by scraping and transferred to tubes for determination of lipid phosphorous as described (36). RESULTS Soybean membranes isolated by methods used routinely in our laboratory (40) vary considerably in their phospholipid composition (Table I). Especially conspicuous is the variable content of PA.…”

“…Membranes from about 80-g bean hypocotyls were isolated as described (40). Four membrane fractions were collected finally from a discontinuous coconut milk-sucrose gradient and designated A, B, C, and D. (40). For protection against lipid degradation 4% choline (w/v), 4% ethanolamine (v/v), and 10 mm glycerol-1-P were added to all solutions.…”

“…Even though phospholipase D has been described to be Phospholipid degradation ofmembrane fractions from soybean hypocotyls. Membranes were isolated in unmodified coconut milk-sucrose medium (13,40 Membranes for the phosphatase experiment were isolated in the presence of 4% choline, 4% ethanolamine, and 10 mM glycerol 1-phosphate. The specific activity of phospholipase D was calculated from the experiment in Figure 1, using degradation of the PC and the PE/PG fraction during the first 10 minutes as activity.…”

“…Hypocotyls were homogenized for 45 sec with a Polytron 20 ST (Kinematica, Lucerne, Switzerland) operating at about 5,000 rpm. Membranes from about 80-g bean hypocotyls were isolated as described (40). Four membrane fractions were collected finally from a discontinuous coconut milk-sucrose gradient and designated A, B, C, and D. (40).…”

Rhizoid Differentiation in Spirogyra

“…Four methods are applicable to determine the localization of phytochrome: (a) a method using microspectrophotometer; (b) a physiological method based on the effect of local irradiation of R' or FR in vivo (3); (c) a biochemical method using cell fractionation by density (1,4,6,(9)(10)(11)(12)(13); (d) an immunocytochemical method (5). The first method cannot be used for a cell containing Chl.…”