Ion and Glycerol Concentrations in 12 Isolates of<i>Dunaliella</i>

Abstract

Ginzburg, M., and Ginzburg, B. Z., 1985. Ion and glycerol concentrations in 12 isolates of Dunaliella.—J. exp. Bot. 36: 1064-1074. Twelve isolates of Dunaliella with average cell volumes ranging from 50 to 1400 x 1018 m3 were grown in batch culture at 0-5 M or 2 0 M NaCl. Glycerol and ions (Na+, K+, Mg2+, CI, phosphate) were measured in log-phase cultures. The contents of Mg2+, K + and phosphate per cell were found to be a function of cell-volume. Cell glycerol, Na+ and were functions of cell-volume and of the NaCl concentration in the medium. Solute concentrations were calculated from the measured cell-volumes and from the 3H20 content of pellets corrected for intercellular space using Blue Dextran. Cell glycerol was found to account for about one-half of the expected osmolarity, the remainder being largely accounted for by Na+ and Cl~. Key words—Dunaliella, isolates, glycerol, ion concentrations. Correspondence to: Botany Department, Institute of Life Sciences, Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel. INTRODUCTION Dunaliella is a green alga assigned to the order Volvocales, family Polyblepharidaceae. Members of this family are unicellular, oval or lobed organisms which have 1-8 flagella. It is an unusual family in that the cells have no wall but only a firm membrane which is the outer boundary of the cytoplasm. The chloroplast is a parietal cup (Prescott, 1969). Dunaliella forms one of the four genera of this family; Dunaliella cells are oval, biflagellate and lack an eyespot. They are frequently red and store starch. They grow in brackish and salt water. It is their adaptation to growth in salt water which has made them objects of considerable interest to biologists in recent years. The cells have proved to be difficult to manipulate experimentally. Some of these difficulties arise from the absence of cell-wall and the properties of the cell-membrane which is unusually permeable and adsorptive. It is assumed (from the ability of cells to live in highly saline water) that they must regulate their solute content very effectively. Progress is slow, however. There is not even general agreement on the cell solute composition. It is clear that glycerol is a major solute and some biochemists assume that the cell glycerol concentration is enough to balance the salt outside (Borowitzka and Brown, 1974; Ben-Amotz, Sussman, and Avron, 1982). Nevertheless, measurements purporting to support this assumption have depended on indirect methods for the determination of cell-water, e.g. the pellet water This content downloaded from 157.55.39.162 on Thu, 11 Aug 2016 04:53:09 UTC All use subject to http://about.jstor.org/terms Ginzburg and Ginzburg—Ion and Glycerol Concentrations in Dunaliella 1065 measured by 3H20 and intercellular space by some small molecule. Not only has 3H20 been shown to be an unreliable measure of cell-water in a certain isolate (Ginzburg and Ginzburg, 1985) but it is also unproven that the marker molecules remain unabsorbed by the cells. In order to circumvent the difficulties caused by the cell-membrane, it seemed advisable to return to the more direct method for the determination of cell-volume, namely from measurements using the microscope. The cell-volumes so determined might then serve as a check on cell-water measurements made by more conventional means since the numerical values for cell-water and cell-volume may be expected to be close to each other. MATERIALS AND METHODS Materials The isolates, together with their provenance and average cell-volume, are listed in Table 1. Isolates are maintained in axenic condition in the Botany Department of the Hebrew University of Jerusalem by Mr Israel Baldinger. Those used in this study were chosen so as to embrace as wide a range of cell-volume as possible. Table 1. Dunaliella isolates used in the present work Cell axes measured under the microscope; cell volume = d/?>-Ka b2 (a and b represent long and short cell axes respectively). Number of cells measured is indicated in brackets. Isolate Provenance NaCl in medium (M) Cell-volume (x 10~18 m3) Iran 6 Shallow lake near Shiraz, Iran. 0-5 49 (70) 2 45 (54) 14 Rock pools off Mediterranean. 0-5 155 (62) 2 100 E 4 Lagoons of Nabek, S. Sinai. 0-5 117(55) 2 95 (55) A 12 Lagoons near Port Said, 0-5 193 N. Sinai. 2 120 K 1 Rock pools, Eilat. 0-5 193 (99) 2 (a) 178 (62) (b) 100(85) D. parva Dead Sea. 0-5 190 2 210 D. tertiolecta Scripps Institute, La Jolla, Calif. 0-5 273 (53) 2 (a) 237 (56) (b) 157 (82) 19/3 {D. salina) Cambridge Culture Collection. 0-5 685 (43) Teodoresco 2 569 (41) L 10 Salt marshes near Dead Sea. 0-5 807 (18) 2 717 (17) 1644 Texas University Collection. 0-5 1013 2 994 E 1 Lagoons of Nabek, S. Sinai. 0-5 1455 (59) 2 1141 (40) C9AA Pool near Dead Sea. 0-5 — 1 1467 (96) 2 1231 (69) 3 (a) 1147 (75) (b) 1407 This content downloaded from 157.55.39.162 on Thu, 11 Aug 2016 04:53:09 UTC All use subject to http://about.jstor.org/terms 1066 Ginzburg and Ginzburg—Ion and Glycerol Concentrations in Dunaliella Methods of culture The culture medium is described by Ginzburg and Ginzburg (1985) with NaCl added at 0-5 M or 2 0 M. Cells from a sterile agar slant were transferred to a 250 ml conical flask containing 50 ml medium with NaCl at either 0-5 M or 2 0 M. Flasks were put to grow in a growth room at 26 °C with a light intensity of 155 W m~2. When growth had started a little of the suspension was used as inoculum for 500 ml medium at the same NaCl concentration in a 3000 ml conical flask. Growth in this flask was allowed to proceed for 3 d until an optical density of 0 02-0-04 had been reached (mid-log phase). The suspension was then centrifuged and the cells resuspended in 10 ml of the same medium. Samples for analysis were then taken immediately from this concentrated suspension. Cell counts The number of cells per ml suspension was determined using a Levy-Hausser Corpuscule Counting Chamber (Clay-Adams, Parsippany, N.J.). Cell-water and measurement of solutes The methods used are described in Ginzburg and Ginzburg (1985). Calculation of results (a) For substances present at low concentration outside (glycerol, K + , Mg2+, phosphate): the amount per ml suspension was divided by the number of cells per ml so as to obtain the amount per cell. (b) ForNa+ and CI the total Na+ and CI present in cell pellets was corrected for those ions in the intercellular space according to the methods described in Ginzburg and Ginzburg (1985) to give the Na+ and in packed cells. These quantities divided by the number of cells gave Na+ and per cell. In the figures, lines are drawn by the eye. 20Iran 6 14 E4 A12 K1 I ilkk. 0 100 0 100 0 100 0 100 200 0 100 200 20-. 19/3 1644 C9AA El Z 15 V O U o s io H x> s 3 z 5