Journal of Experimental Botany, Vol. 52, No. 355, pp. 223-229,
February 2001
© 2001 Oxford University Press
Original Papers |
Ca2+ and phosphate releases from calcified Chara cell walls in concentrated KCl solution
Division of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan
Received 24 April 2000; Accepted 11 September 2000
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Abstract |
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Ca2+ and Pi (inorganic phosphate) releases from isolated calcified and uncalcified Chara cell walls were measured with a Ca2+-selective electrode and colorimetry, and their ionic relations were analysed on the basis of the electroneutrality rule. The results showed that (1) not only Ca2+ but also Pi can be released from isolated calcified Chara cell walls into pure deionized water and 100 mM KCl solution, and (2) the positive charge due to the Ca2+ released cannot be neutralized only by the negative charge from the simultaneously released Pi. These findings suggest that calcium bands of calcified Chara cell walls are composed of mainly CaCO3 and CaHPO4 and some anions other than Pi should be released simultaneously with the Ca2+ and Pi. More Ca2+ and Pi can be solubilized from isolated Chara cell walls in 100 mM KCl solution than in pure deionized water. The pH value of 100 mM KCl solution in which isolated uncalcified young Chara cell walls have been immersed is a little lower than that of pure deionized water in which the same isolated uncalcified young Chara cell walls have been immersed, suggesting that some acidic substances are solubilized by 100 mM KCl. To explain this from the viewpoint of solution chemistry, the solubilities of pure CaCO3 and pure CaHPO4 in water and 100 mM KCl solution were measured with a Ca2+-selective electrode and their pH values with a glass pH electrode. The conclusion reached was that the Ca2+ release from isolated Chara cell walls is accompanied by the release of Pi, CO2-3 and acidic substances. This suggests that the so-called calcium bands and/or ionic relations, including ion exchange, in Chara cell walls are chemically or physicochemically more complex than they are currently considered to be.
Key words: Calcium band, calcium carbonate (CaCO3), calcium hydrogenphosphate (CaHPO4), cell wall, Chara corallina, electroneutrality rule.
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Introduction |
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Chara internodal cells die in aqueous electrolyte solutions such as 100 mM KCl, 10 mM MgCl2, 1.0 mM BaCl2 (Kiyosawa and Adachi, 1990
), 100 mM NaCl (Katsuhara and Tazawa, 1986; Tufariello et al., 1988), and 0.1 mM HCl (Kiyosawa, 1990). However, adding an appropriate amount of Ca2+ (0.5–10 mM) to such aqueous electrolyte solutions enables the internodal cells to survive for more than a week (Katsuhara and Tazawa, 1986; Tufariello et al., 1988; Kiyosawa and Adachi, 1990; Kiyosawa, 1993). Whittington and Smith have shown that Chara internodal cells can survive in 100 mM NaCl, LiCl, KCl, and RbCl solutions containing 0.1 mM Ca2+ for at least 3 d (Whittington and Smith, 1992). Using the atomic absorption method, combined with the extraction of Ca2+ from Chara cell walls by 100 mM HCl, Kiyosawa and Adachi showed that Ca2+ is released from Chara cell walls in aqueous KCl, NaCl, MgCl2, and BaCl2 solutions, but that the addition of Ca2+ reduces the Ca2+ released (Kiyosawa and Adachi, 1990).
In the present experiments, Ca2+ release from isolated Chara cell walls in pure deionized water and in 100 mM KCl solution was measured with a Ca2+-selective electrode. Along the surface of the Chara cell wall grown under strong light, calcium bands, which are believed to consist of CaCO3 crystals, are formed (Borowitzka, 1982, 1987; Borowitzka et al., 1974; Okazaki and Tokita, 1988).
The present study examined whether or not the Ca2+ released comes mainly from the CaCO3 forming the calcium bands. To do this, the amounts of Ca2+ released from isolated Chara cell walls into pure deionized water and aqueous 100 mM KCl solution were compared, and their pH values were examined in relation to the solubilities of CaCO3 in these fluids. The Ca2+ measurements were done with a Ca2+-selective electrode and the pH values were obtained using a glass pH-electrode.
The present paper also examines whether or not CaHPO4, which is practically insoluble in water, but can neutralize pure deionized water, is involved in the calcium bands of Chara cell walls. The release of 
(Pi) from isolated Chara cell walls in pure deionized water and in aqueous 100 mM KCl solution was measured qualitatively and quantitatively using colorimetry. Also, the solubilities of pure CaHPO4 in pure deionized water and in 100 mM KCl solution were measured using a Ca2+-selective electrode. The pH values of aqueous CaHPO4 solutions and aqueous mixtures of CaHPO4 with 100 mM KCl were also measured with a glass pH-electrode.
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Materials and methods |
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Isolated cell walls of Chara corallina were used throughout the experiments. Chara corallina was grown in buckets with several centimetres of soil at the bottom. Illumination was provided by one or two 20 W fluorescent lamps several centimetres above the surface of the water in the laboratory. City tap water, which was of neutral pH, was used for the culture. Internodal cells having clear calcium bands, i.e. calcified Chara internodal cells or having no visible calcium bands, i.e. uncalcified Chara internodal cells, were isolated from adjacent cells with scissors on the morning of the day when the experiments were conducted.
Both ends of the internodal cells were cut with scissors and the protoplasm was squeezed out with fingers in deionized water. To remove soluble substances which might interfere with the measurement with a Ca2+-selective electrode and colorimetry, the isolated cell walls from about 40 internodal cells were immersed in pure deionized water for about 20 min. Next, they were transferred into a Teflon beaker in which either pure deionized water or the test electrolyte solution of 25.0 ml had been kept at 25 °C and incubated for 2 h at 25±0.5 °C with stirring with a magnetic bar at a moderate rate. After incubation, the cell walls were removed from the beaker with forceps and the absorbed water was squeezed out onto sheets of filter paper. The cell walls which had first been immersed in pure deionized water were immersed in test electrolyte solution for 2 h at 25±0.5 °C with stirring with a magnetic bar at a moderate rate. In some experiments, the isolated Chara cell walls were directly immersed in test electrolyte solutions after 20 min immersion in pure deionized water to remove interfering substances.
Cell walls isolated from Chara corallina grown outdoors in the sun were also used.
The calcium concentration of pure deionized water and test electrolyte solution in which isolated cell walls had been incubated was measured with a Ca2+-selective combination electrode (6583-10C, Horiba, Ltd., Kyoto, Japan) connected to a pH/mV-meter (Type F-16, Horiba, Ltd., Kyoto, Japan). The concentrations of the aqueous electrolyte solutions were 100 mM KCl, 100 mM NaCl, 10 mM MgCl2, and 10 mM BaCl2. Calibration curves for the relationship between the calcium concentration [Ca2+] and the electrode potential 
E of a Ca2+-selective electrode of a combination type were obtained, respectively, for simple aqueous CaCl2 solutions and aqueous mixtures of CaCl2 with the electrolyte at the same concentration as the test one used for extraction of Ca2+ from isolated Chara cell walls. The Ca2+ concentration of pure deionized water and test electrolyte solutions in which isolated Chara cell walls had been immersed, respectively, were determined by measuring E when a calibrated Ca2+-electrode was dipped into them.
The pH value of pure deionized water and test electrolyte solution in which isolated Chara cell walls had been incubated was measured with a glass pH-combination electrode connected to a pH meter (Type F-16, Horiba, Ltd., Kyoto, Japan) at 25±0.5 °C. Further studies on the Ca2+release from calcified or uncalcified Chara cell walls and on solubilization of CaCO3 and CaHPO4 were performed using pure deionized water and 100 mM KCl solution.
Before measurements of the Ca2+ concentration and pH value of simple aqueous CaCO3, and CaHPO4 solutions or the supernatant of their aqueous suspensions, the mixtures of CaCO3 or CaHPO4 with pure deionized water or 100 mM KCl solution of 1.0 dm3 had been stirred with a stirring bar at a moderate rate for more than 2 d at 25.0±0.5 °C, and then had been left standing for more than 2 d at the same temperature. During the period, almost all of the insoluble CaCO3 and CaHPO4 had precipitated except for some CaCO3 or CaHPO4 which remained floating on the surface of the transparent aqueous supernatants.
The measurements of the calcium concentration by a Ca2+-selective electrode and the pH value by a pH electrode of all of these aqueous solutions were done whilst stirring with a magnetic bar at a moderate rate and the measured values were printed out over 20 min as a function of time. Visual observation was conducted to decide whether or not the CaCO3 or CaHPO4 added had completely dissolved. Their solubilities were also determined by measuring the solubilized Ca2+ concentration as a function of the molar concentration of CaCO3 or CaHPO4 added.
The concentration of phosphate was measured by colorimetry (Taussky and Shorr, 1953) with some modification (Kiyosawa, 1979). This method showed no interference from KCl, KNO3, MgSO4, CaCl2, (NH4)2SO4 or TRIS pH buffer at concentrations used in usual biochemical and physiological experiments.
The pure deionized water was above 17 M
cm-1. CaCO3 of reagent grade (99.9%) and CaHPO4.2H2O of reagent grade (min 98.0%) were purchased from Wako Pure Chemicals Industries, Ltd., Japan and Nakarai Tesque, Inc., Japan, respectively.
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Results |
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The electrode potential
E read by a pH/mV meter equipped with a Ca2+-selective electrode decreases or increases monotonically as a function of time without reaching constant values. One of the possible origins of drift in electrode potential E would be that in the boundary potentials of a calcium selective combination electrode. However, plotting the E at around 15 min after the start of measurement against the logarithm of the calcium concentration, log[Ca2+], gives a good linear curve expressed by the equation
 | (1) |
). For aqueous mixtures of CaCl2 with an electrolyte, the constants a and b change depending on the species and the concentration of the electrolyte used. The constant b also varies greatly from electrode to electrode.
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After obtaining the calibration curve expressed by Equation (1) for simple aqueous CaCl2 solutions or aqueous mixtures of CaCl2 with the same electrolyte as the test solution such as 100 mM KCl, it is possible to determine the calcium concentrations [Ca2+] of samples by measuring
E after dipping a Ca2+-selective electrode into them. Since E changes slightly from day to day, calibration curves for the calcium concentration [Ca2+] and the electrode potential E expressed by Equation (1) were obtained before every experiment.
Table 1 shows the calcium concentrations of 25.0 cm3 pure deionized water and aqueous test electrolyte solutions of 100 mM KCl, 100 mM NaCl, 10 mM MgCl2, and 10 mM BaCl2 after the isolated calcified Chara cell walls had been immersed for 2 h under stirring. The Ca2+ released from the cell walls during the 2 h changed the pure deionized water of 25 cm3 to about 0.17–0.29 mM Ca2+. The Ca2+ released from the cell walls during 2 h of subsequent immersion in aqueous electrolyte solutions of 25 cm3 made them 0.68–0.91 mM Ca2+. Direct immersion of the cell walls in aqueous electrolyte solutions also had brought their calcium concentrations to 0.66–0.70 mM (Table 1).
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Table 2
shows the Pi release from isolated mature calcified, mature uncalcified and young uncalcified Chara cell walls in pure deionized water and 100 mM KCl solution together with the simultaneous Ca2+ release, and the pH values of the water and the solution. Clearly, the table shows that (1) Pi is released from isolated calcified Chara cell walls both in pure deionized water and in 100 mM KCl solution, but little Pi is released from uncalcified cell walls in either solution, (2) the amounts of Pi and Ca2+ released from cell walls in pure deionized water during 2 h of immersion are less than that in 100 mM KCl solution, (3) the Pi concentrations are always lower than those of the released Ca2+, and (4) the pH values of the water and the solution in which isolated mature Chara cell walls have been inmersed are neutral, not weakly acidic as is the case for pure deionized water and aqueous 100 mM KCl solution saturated with atmospheric CO2, but (5) the pH value of 100 mM KCl solution in which young uncalcified Chara cell walls have been immersed is weakly acidic.
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These observations indicate that calcium compounds, which can dissolve very sparingly into pure deionized water and more into 100 mM KCl solution, are rather abundantly bound to Chara cell walls.
To examine whether or not these calcium and phosphate ions come from CaCO3 and CaHPO4, the solubilities of pure CaCO3 and CaHPO4 were measured in both pure deionized water and 100 mM KCl, and the pH values were also determined. These pH values were compared with those of pure deionized water and of the test electrolyte solution in which Chara cell walls had been immersed for 2 h.
Figure 2 shows the Ca2+ concentrations of aqueous CaCO3 solutions and supernatants of their suspensions measured with a Ca2+-selective electrode. The Ca2+ concentrations of aqueous CaCO3 solutions and suspensions are expressed as a function of the molar concentration when all of the CaCO3 added to 1.0 dm3 pure deionized water or 100 mM KCl solution is assumed to have dissolved completely.
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Mixtures of CaCO3 up to about 0.28 mM in pure deionized water become transparent when they are stirred with a magnetic bar at a moderate speed for about 2 d. However, at above 0.28 mM, white floating material, probably CaCO3, is first observed on the surface and if the CaCO3 concentration continues to increase, a white precipitate, probably CaCO3, can be observed at the bottom of a beaker which has been stirred for 2 d and left standing for a further 2 d. The Ca2+ concentrations of the supernatant of aqueous CaCO3 suspensions are around 0.28 mM and almost constant above this concentration.
The Ca2+ concentrations of aqueous mixtures of CaCO3 with 100 mM KCl are plotted in the same figure as the function of the apparent CaCO3 concentration. On addition of 100 mM KCl, the saturated Ca2+ concentration measured with a Ca2+-selective electrode seems to increase up to around 0.38 mM. However, if measurement errors are taken into consideration, a more reasonable explanation would seem to be that the saturated Ca2+ concentration of CaCO3 does not change significantly with the addition of 100 mM KCl. Little salt-out or salt-in occurs in aqueous CaCO3 suspensions on addition of 100 mM KCl.
Figure 3 shows the pH values of aqueous CaCO3 solutions and supernatants of its aqueous suspensions as functions of the apparent CaCO3 concentration and time. The pH value of pure deionized water saturated with atmospheric CO2 is also shown as a function of time. The pH values read by a pH meter change with time. The pH value of an aqueous solution having only weak buffering ability measured with a pH meter was found to change with time, not maintaining a constant equilibrium value, but displaying scattering around a value. Thus, it is difficult to determine pH values precisely (Ozeki et al., 1998). However, the pH values of simple aqueous CaCO3 solutions read with a pH meter equipped with a glass pH electrode change greatly as a function of time, for reasons not yet known.
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What can be said is that the pH value of pure deionized water saturated with atmospheric CO2 is around 5.4, i.e. weakly acidic, but that of aqueous CaCO3 solutions and supernatants of its aqueous suspensions is weakly alkaline at all concentrations examined. At below and above 0.28 mM, the greater the amount of CaCO3 added, the higher is the pH of the supernatant of the aqueous mixtures (complete data not shown). This is curious because the Ca2+ concentration of the supernatant of aqueous CaCO3 suspensions is almost constant above 0.28 mM (cf. Fig. 2
).
Figure 4 shows the Ca2+ concentrations of simple aqueous CaHPO4 solutions and supernatants of its suspensions, and those of mixtures with 100 mM KCl as a function of the apparent CaHPO4 concentration. Here, ‘apparent’ CaHPO4 concentration is that when all of the CaHPO4 added is assumed to dissolve in 1.00 dm3 pure deionized water or 100 mM KCl solution.
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The saturation concentration of CaHPO4 is about 0.37 mM in water and 0.70 mM in 100 mM KCl solution (Fig. 4
). Clearly, a greater molar concentration of CaHPO4 can be dissolved in pure deionized water than CaCO3 and the solubility of CaHPO4 can be increased by the addition of 100 mM KCl. In other words, salt-in occurs in aqueous CaHPO4 suspensions by the addition of 100 mM KCl.
The pH values of aqueous CaHPO4 solutions are shown in Fig. 5 as a function of time. Clearly, they change less over time than those of CaCO3. The pH values of aqueous CaHPO4 solutions and supernatants of its suspensions are almost neutral. The pH values of aqueous CaHPO4 solutions and suspensions seem to be lower than those of aqueous CaCO3 solutions and its suspensions, but are nearly equal to those of pure deionized water and 100 mM KCl solution in which isolated Chara cell walls have been immersed.
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Discussion |
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Phosphate is present in Chara cell walls. Since potassium and sodium salts of phosphate are very soluble in water and little K+ and Na+ remain bound to Chara cell walls during short-term immersion in pure deionized water preceding 2 h immersion in pure deionized water and 100 mM KCl solution (cf. Kiyosawa and Adachi, 1990
), the phosphate can be regarded to exist in Chara cell walls as calcium salts which are practically insoluble in water. One of the calcium salts of phosphate is CaHPO4.
The solubility of CaHPO4 can be increased in water by the addition of 100 mM KCl. However, the solubility of CaCO3 does not change significantly in water on addition of 100 mM KCl. Ca2+ release from isolated Chara cell walls can be increased in 100 mM KCl solution (Table 1). Pi release from isolated Chara cell walls can also be increased in 100 mM KCl solution (Table 2). However, when the balance between the positive charge due to cations and the negative charge due to anions based on the electroneutrality rule was examined, the amount of Pi released from isolated calcified Chara cell walls together with the Ca2+ release is not large enough to neutralize the positive charge due to the Ca2+.
The amount of Pi released from calcified Chara cell walls averages 0.15 mEq dm-3 if the Pi is and 0.30 mEq dm-3 if it is 
in pure deionized water. On the other hand, the amount of Ca2+ simultaneously released averages 0.56 mEq dm-3 (Table 2). Therefore, to neutralize the excess positive charge due to the Ca2+, some other anions amounting to 0.26–0.41 mEq dm-3 should also be solubilized from calcified Chara cell walls in pure deionized water. One of these anions should be 
. However, the pH value of simple aqueous CaCO3 solutions and that of the supernatant of its aqueous suspensions are weakly alkaline (Fig. 3).
The pH value of 100 mM KCl solution in which isolated young uncalcified Chara cell walls had been immersed was weakly acidic (Table 2) while that of pure deionized water in which the same young uncalcified Chara cell walls had been immersed was neutral (Table 2), indicating that some acidic substances can be released by 100 mM KCl solution (Gillet et al., 1989). This result does not deny that such acidic substances are released from young uncalcified Chara cell walls even in pure deionized water and that such acidic substances are released from mature uncalcified and calcified Chara cell walls more or less in pure deionized water and 100 mM KCl solution.
These acidic substances may make pure deionized water and 100 mM KCl solution, in which isolated Chara cell walls have been immersed, less alkaline than that of simple aqueous CaCO3 solutions, even bringing the pH to neutral. Thus, anions other than which can neutralize excess positive charge due to Ca2+ over the negative charge due to Pi are probably organic acids (Gillet et al., 1989, 1998).
The amount of Pi released from the same Chara cell walls in 100 mM KCl is 0.22 mEq dm-3 or 0.44 mEq dm-3 depending on whether the Pi is or . On the other hand, the released Ca2+ amounts to 1.68 mEq dm-3 in 100 mM KCl solution. Thus, some other anions amounting to 1.24–1.46 mEq dm-3 should be released simultaneously. These anions also seem to be organic acids (Gillet et al., 1989, 1998) and . Numerical values of the charges due to Ca2+, Pi and organic acids in ionic relations including ion exchange (Métraux and Taiz, 1977; Van Cutsem and Gillet, 1981; Gillet et al., 1989; Reid and Smith, 1992) based on the electroneutrality rule differ among experiments using Chara cell walls which have different amounts of calcium bands and are of different ages (Table 2). Reid and Smith have already shown that the amount of calcium in Chara cell walls varies depending on the age of the cell and the culture conditions (Reid and Smith, 1992). The content of phosphate in calcium bands on Chara cell walls is also thought to have much to do with its concentration in culture media.
The pH value alone cannot show how much has been released and how much Pi has been released from isolated Chara cell walls during the Ca2+ release in pure deionized water and 100 mM KCl solution, respectively. Also, it is usually not very easy to quantify the concentration of .
The pH value of pure deionized water is weakly acidic under atmospheric conditions because CO2 in air dissolves into it. The pH values of aqueous CaHPO4 solutions and the supernatant of its aqueous suspensions are approximately neutral. CaHPO4 probably dissociates to make OH- in water as follows:
 | (2) |
 | (3) |
; cf. Ogata, 1983):
 | (4) |
 | (5) |
 | (6) |
Judging from the results of the present findings, the so-called calcium bands and/or ionic relations in Chara cell walls are not chemically or physicochemically simple (Katz, 1973; Borowitzka, 1982, 1987). This suggests that chemical and physiological processes around and in the cell membrane from which calcium bands are formed in/on Chara cell walls under strong light should be more or less modified from those proposed thus far. However, this does not necessarily deny the hypothesis that calcium bands would be formed by the simultaneous deposition of calcium and anions such as in/on the cell wall due to localized pH changes through transmembrane transport of H+ and 
(Lucas, 1975; Lucas and Nuccitelli, 1980; Borowitzka, 1987; Fisahn et al., 1989).
It is worth noting that Smith has suggested that the mechanism of the formation of calcium bands on the cell wall is not physiologically so simple (Smith, 1985a, b). The present study seems to suggest one direction for further studies on the mechanism of the formation of calcium bands on Chara cell walls.
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Acknowledgments |
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This work was supported in part by a Grant-in-Aid (No. 10640632) for Scientific Research from the Ministry of Education, Science and Culture of Japan.
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Notes |
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1 Fax: +81 6 6850 6557. E-mail: kiyosawa{at}bpe.es.osaka\|[hyphen]\|u.ac.jp
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