Plant Cell Physiot. 32(8): 1137-1144 (1991) JSPP © 1991 The Density of the Cell Sap and Endoplasm of Nitellopsis and Chora Randy Wayne and Mark P. Staves Section of Plant Biology Cornell University, Ithaca, NY 14853, U.S.A. We measured the densities of the cell sap, endoplasm and cell wall of Nitellopsis obtusa and Chara corallina using interference microscopy, refractometry, immersion refractometry, equilibrium sedimentation and chemical microanalysis techniques. These values are important for the determination of many rheological properties of the cytoplasm as well as for understand- ing buoyancy regulation, dispersal mechanisms and how cells respond to gravity. The average densities of the cell sap, endoplasm and cell wall are 1,006.9, 1,016.7 and 1,371 kgm"3 for Downloaded from Nitellopsis and 1,005.0, 1,013.9 and 1,355.3 kgm"3 for Chara. Key words: Cell sap — Chara corallina — Density — Endoplasm — Interference microscopy — Nitellopsis obtusa. http://pcp.oxfordjournals.org/ We have proposed a novel model for the way plant needed to understand how cells respond to gravitational cells sense gravity in which the plant cell protoplast brings a and centrifugal accelerations. The densities of the endo- pressure to bear against the cell wall, providing a direction- plasm and cell sap determined by Kamiya and Kuroda al signal for gravitational responses. By this model, all the (1957b) have been used previously to calculate the motive cell contents, including the amyloplasts, act as ballast force of streaming (Kamiya and Kuroda 1958b, Wayne et by guest on November 12, 2015 (Wayne et al. 1990, Staves et al. 1991). The falling of the al. 1990), the viscosity of the cytoplasm (Kamitsubo et al. protoplast consequently causes a compression of the plas- 1988), the tension at the surface of a protoplasmic droplet ma membrane of the lower part of the cell and a tension on (Kamiya and Kuroda 1958a, 1965) and other rheological the plasma membrane of the upper part of the cell. The properties of cytoplasm (Kamitsubo et al. 1989, Kamiya difference between these forces is sufficient to activate the and Kuroda 1957a). A knowledge of the densities of the ion channels necessary for plant cells to respond to gravity endoplasm and cell sap is also important in the understand- (Wayne et al. 1990). The magnitude of the graviresponse ing of buoyancy regulation (Raven 1984, Walsby 1975) and depends on the volume of the protoplast as well as the dispersal mechanisms (Gregory 1961). density of the protoplast relative to the density of the solu- tion in the wall. Since the densities of the endoplasm and Materials and Methods cell sap now take on added importance in gravitational work, we have measured the densities of the endoplasm Culture conditions—Nitellopsis obtusa (Devs. in and cell sap of Nitellopsis obtusa and Chara corallina. Lois.) J. Gr. and Chara corallina Klein ex Willd., em. These data extend the early measurements of the densities R.D.W. (= Chara australis R. Brown) were grown in a of the endoplasm and the cell sap done by Kamiya and soil/water medium under continuous white fluorescent Kuroda (1957b) on Nitella flexilis and Collander (1930) on light at 26°C. Under these conditions, CaCO3 does not Chara ceratophylla, which heretofore provided the basic deposit on the cell wall and there is not any visible banding knowledge on protoplasmic components which may be pattern. The soil water medium contained 3.75 mM Ca, 1.71 mM Mg, 1.37 mM S, 0.98 mM Cl~, 637 /mK, 508 (M Abbreviations: a, specific refractive increment; ACS, ar- Na, lll^MSi, 277^MMn, 12.9/mP, 6.51 /atB, 2.47/m Al, 1.96 (MV, 1.13/mNi, 756nMFe, 465 nM Ag, 356 riM tificial cell sap; Da, density of cell sap; Dra, density of endoplasm; Do, concentration of organic dry matter; D,, concentration of the Co, 223 nM Cr, 152 nM Mo, 112nM As, 76.5 nM Zn, and 3 salt solution, OPD, optical path length; S, total solids; t, diameter 69.7 nM Y. The density of the medium is 1,000.5 kg m~ of endoplasmic drop; W, percentage of water; W,, percentage of as determined by refractometry and gravimetricalry. Ex- salt solution. periments were carried out at 25°C. 1137 1138 R. Wayne and M. P. Staves Measurement of the density of the cell sap recorded on a strip chart recorder (Model 2125M, Allen Refractometry method—The density of the cell sap Datagraph, Inc. Salem, NH, U.S.A.). was determined by measuring the total solids (S in g (100 In order to calculate the density of the cell sap, we ml)"1) of the cell sap with a hand held refractometer assumed that the average specific volume of the cell sap con- (Reichert-Jung, Model 10441, Cambridge Instruments, stituents was 0.5040. The total density of the cell sap (Da Buffalo, NY, U.S.A.) and assuming that KC1, with a in g (100 ml)"') is then obtained using equation 1 and the specific volume of 0.5040 (which is derived from the percent of water (W in g (100 ml)"') is obtained by using reciprocal of the specific gravity), made up the majority of equation 2. the solute. The total density of the cell sap (D^ in g (100 ml)"1) is then obtained with the following equation: Measurement of the density of the endoplasm Quantitative interference microscopy method—We DCS=W+S (1) used an AO-Baker interference microscope with a 40 x shearing type objective lens (NA=0.7) and a Zeiss Jamin- where the percentage of water (W in g 100 ml ') is given by Lebedeff interference microscope with a 40 x objective lens the following equation: (NA=0.65) to determine the density of the endoplasm Downloaded from (Beneke 1966, Davies et al. 1954, Hale 1958). We express- W=100-0.5040(S) (2) ed the endoplasm from internodal cells of Chara into a solu- tion of artificial cell sap (ACSJ containing 80 mM KC1, 10 Equilibrium sedimentation method—In order to mini- mM CaCl2, 30 mM NaCl and 10 mM MgCl2. Using the mize the importance of the assumption that the specific vol- specific volumes of these salts (0.5040, 0.4651, 0.4619 and ume of the solute of the cell sap is 0.5040, we also measured 0.4304, respectively), we calculated that the density of the http://pcp.oxfordjournals.org/ the density of the cell sap using an equilibrium method artificial cell sap was 1,004.23 kg m"3. Likewise, we ex- where the cell sap was expressed, collected with a plastic pressed the endoplasm from internodal cells of Nitellopsis pipette tip (ca. 3 //I) and released in solutions of various su- into a solution of artificial cell sap (ACS,,) containing 90 crose concentrations that were all made isotonic (0.617 mM KC1, 10 mM CaCl2, 30 mM NaCl and 10 mM MgCl2. MPa for Chara; 0.702 MPa for Nitellopsis) with ethylene Again, we calculated that the density of the artificial cell glycol. We then determined whether the drop floated, sap was 1,006.22 kg m"3. In both cases, the endoplasmic sank or did not move. The density of the solution in drops were spherical and the chloroplasts retained their which the drops neither sank nor floated was considered to natural shapes, indicating that the osmotic strengths of the by guest on November 12, 2015 be equal to the density of the cell sap. This method is artificial cell saps were appropriate for the endoplasmic similar to that used by Kamiya and Kuroda (1957b). drops of each species (Wayne and Tazawa 1988). Chemical analysis method—Lastly, in order to apply Quantitative phase-contrast method—Endoplasmic an independent check on the density of the cell sap, we ana- drops were effused into drops of ACS, appropriate for lyzed chemically the individual constituents in order to find each species, to which various amounts of Bovine Serum the concentration of the individual solutes (S in g (100 Albumin (BSA, Fraction V, Sigma, St. Louis, MO, ml)~'). The elements (except Cl~), were measured with an U.S.A.) had been added. We then determined the percent- inductively coupled argon plasma atomic emission spec- age of droplets that were darker (using negative phase con- trometer (Model ICAP 61, Thermo Jarrel Ash Corp., trast) or lighter (using positive phase contrast) than the me- Franklin, MA. U.S.A.) and the total and free amino acids dium. This gave us the percentage of drops that had a were measured with an amino acid analyzer (Pico-tag lower refractive index than the medium. We plotted this System, Waters Chromatography, Milford, MA, U.S.A.). percentage against the concentration of BSA in the medi- Cl~ was measured with a Ag/AgCl electrode using a um. The density of the drops was calculated by assuming glass microcapillary electrode filled with 100 mol m~3 that the concentration (in percent) of organic dry matter in KNO3 as a reference electrode. The electrodes were the drop was equivalent to the percentage of BSA when pulled with a microcapillary pipette puller (Model PP-83, 50% of the droplets had a higher refractive index than the Narashige Scientific Instrument Lab., Tokyo, Japan). medium and 50% of the droplets had a lower refractive in- The reference electrode was either connected directly to a dex than the medium. The concentration of BSA (in g Ag/AgCl pellet or first passed through a 3 M KC1 bridge. (100 ml)"1) is the concentration (in kg m~3) divided by 10. Both methods worked equally well.