Journal of Experimental Botany, Vol. 51, No. 353, pp. 2053-2066,
December 2000
© 2000 Oxford University Press
Original Papers |
Transport and metabolic degradation of hydrogen peroxide in Chara corallina: model calculations and measurements with the pressure probe suggest transport of H2O2 across water channels
Lehrstuhl Pflanzenökologie, Universität Bayreuth, D-95440 Bayreuth, Germany
Received 19 January 2000; Accepted 24 July 2000
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Abstract |
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A mathematical model is presented that describes permeation of hydrogen peroxide across a cell membrane and the implications of solute decomposition by catalase inside the cell. The model was checked and analysed by means of a numerical calculation that raised predictions for measured osmotic pressure relaxation curves. Predictions were tested with isolated internodal cells of Chara corallina, a model system for investigating interactions between water and solute transport in plant cells. Series of biphasic osmotic pressure relaxation curves with different concentrations of H2O2 of up to 350 mol m-3 are presented. A detailed description of determination of permeability (Ps) and reflection coefficients (
s) for H2O2 is given in the presence of the chemical reaction in the cell. Mean values were Ps=(3.6±1.0) 10-6 m s-1 and s=(0.33±0.12) (±SD, N=6 cells). Besides transport properties, coefficients for the catalase reaction following a Michaelis-Menten type of kinetics were determined. Mean values of the Michaelis constant (kM) and the maximum rate of decompositon (vmax) were kM=(85±55) mol m-3 and vmax=(49±40) nmol (s cell)-1, respectively. The absolute values of Ps and s of H2O2 indicated that hydrogen peroxide, a molecule with chemical properties close to that of water, uses water channels (aquaporins) to cross the cell membrane rapidly. When water channels were inhibited with the blocker mercuric chloride (HgCl2), the permeabilities of both water and H2O2 were substantially reduced. In fact, for the latter, it was not measurable. It is suggested that some of the water channels in Chara (and, perhaps, in other species) serve as ‘peroxoporins’ rather than as ‘aquaporins’. Key words: Catalase, Chara corallina, hydrogen peroxide, permeability coefficient, reflection coefficient, water channel.
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Introduction |
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Hydrogen peroxide occurs in important metabolic reactions such as during the action of oxygenases in glyoxysomes and peroxisomes, during photosynthesis in choloplasts, and during the synthesis of lignin in the apoplast (Asada, 1992
; Ishikawa et al., 1993; Schopfer, 1996). The metabolite is usually considered to be toxic either by itself or by the fact that it is a precursor of the even more toxic hydroxyl radical. Plants use H2O2 as a signal and a chemical for defending against attacks by pathogens (‘oxidative burst’; Wojtaszek, 1997; Apostol et al., 1989; Peng and Kuc, 1992). In peroxisomes, H2O2 is produced at high rates. The organelles contain catalase at high concentrations which splits H2O2 into water and oxygen. It has been found that chloroplasts lack catalase, and H2O2 produced during photorespiration is eliminated by reduction to water via the ascorbate/glutathione cycle (Asada, 1992). Although there are, as far as is known, no quantitative data, hydrogen peroxide is usually thought to move rapidly across the membranes of cells and organelles. If the solute was that mobile, the diffusion out of compartments such as peroxisomes could be a problem. The escape of H2O2 from peroxisomes (where it is produced) could be favoured above its degradation because of the small size of the organelles (high surface area to volume ratio). Although this could be compensated for by a high concentration of catalase (as found in peroxisomes), there could be a serious problem depending on the absolute value of the permeability of the membrane for H2O2. Despite the general assumption that H2O2 rapidly crosses membranes, it is nevertheless thought that it may be concentrated in certain tissues during oxidative burst at a level which is sufficient to cause an oxidative stress to pathogens (Apostol et al., 1989; Peng and Kuc, 1992). Obviously, different situations require some regulation of the permeability of membranes to H2O2. The lack of data of the membrane permeability of H2O2 is due to the fact that it is difficult to measure H2O2 fluxes in the presence of substantial activities of catalase and peroxidase, i.e. to separate membrane permeation from reaction flows experimentally. The problem is that of measuring the diffusional permeability of a substrate which takes part in a chemical reaction. Provided that concentrations of H2O2 and its reflection coefficient are rather large, the permeation/decomposition of H2O2 should also affect water flows and cell turgor pressure and should be measurable using the pressure probe technique (Steudle and Tyerman, 1983; Rüdinger et al., 1992; Steudle, 1993; Henzler and Steudle, 1995). Since there is no direct coupling between the decomposition of H2O2 and the transfer of water across the membranes, interactions between the chemical reaction and water flow should be indirect, i.e. mediated by changes of the internal concentration of H2O2. Preliminary observations showed that isolated internodes of Chara corallina could tolerate concentrations of more than 100 mM H2O2. From the osmotic responses measured with the cell pressure probe it was obvious that reflection coefficients were substantially larger than zero. Measurements of steady-state turgor in the presence of hydrogen peroxide indicated that there was a considerable degradation of H2O2 in the cells which was attributed to the action of catalase and peroxidases. In this paper, the permeability and decomposition of H2O2 of these cells is analysed in more detail. By means of the cell pressure probe, osmotic responses of the permeating solute H2O2 have been measured. In order to separate kinetics into components due to reaction flow (degradation of H2O2 in the cell) and due to solute flow across the membranes, a physical/mathematical model was established which predicted the osmotic reactions of internodes (water and solute flows in the presence of catalase action), when H2O2 was added to the external medium. Predictions from the model were tested experimentally. Permeability and reflection coefficients of H2O2 have been worked out as well as kM and vmax of the catalase activity of internodes. The facts that (i) the diffusional permeability of H2O2 (Ps) was smaller by only a factor of two than that of water (Pd) and that (ii) treatment with the channel blocker HgCl2 reduced both Ps and Pd, indicated that H2O2 used water channels on its passage across the plasma membrane. It is suggested that at least some of the water channels present in the cell membrane are H2O2 channels or ‘peroxoporins’ rather than aquaporins.
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Theory |
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The system under investigation is a single cell sitting in a big reservoir of a medium with a constant external concentration of a permeating solute (Co=constant). A cell membrane separates the medium inside the cell (superscript ‘i’) with concentration Ci(t) from the surrounding medium outside (superscript ‘o’). Osmotic pressures of all non-permeating solutes can be comprised into the steady-state turgor pressure to a good approximation (P0=P(t=0)). The membrane has a permeability for water (Lp, hydraulic conductivity) and for the solute (permeability, Ps, and a reflection,
s, coefficient). Any active transport across the membrane is neglected since the time scales of such processes are in ranges which are usually much larger than the rapid processes considered here. At time t=0, step-changes of external concentrations are performed. In the following, an experiment is called ‘exosmotic’ when the external concentration is increased (positive step-change). This induces a water flow out of the cell. An experiment is called ‘endosmotic’ when the external concentration is decreased (negative step-change, and water uptake by the cell). In the system described, water (JV, m3 m-2 s-1) and solute flow (Js, mol m-2 s-1) are given by equations 1 and 2 (Rüdinger et al., 1992):
 | (1) |
 | (2) |
The symbol P denotes cell turgor pressure and R and T are the gas constant and absolute temperature, respectively. 
is the amount of moles of solute crossing the cell membrane during a time interval dt. A and V are the cell surface area and the cell volume, respectively. By convention, a flow out of the cell has a positive sign. Since the cell wall is assumed to be rigid, volume changes (dV) are less than 1% (dV<<V
constant). Therefore, the assumption holds that 
(equation 2
), i.e. the solute flow across the membrane is proportional to the change of internal concentration. The first term on the right side of equation 1 describes the ‘hydraulic’ and the second term the ‘osmotic’ water flow. The first term on the right side of equation 2 represents the diffusion of the solute along the concentration gradient. The second term expresses the coupling between water and solute flow (solvent drag). This latter term is usually negligibly small even when s is substantially smaller than unity (s<0.5; Rüdinger et al., 1992). One can calculate the ratio of diffusion to solvent-drag at the beginning of an osmotic relaxation (t=0) when JV and Js are at maximum. For a Chara internode, typical parameters of transport of water and H2O2 are: Lp=1.7x10-6 m (s MPa)-1, Ps=3.6x10-6 m s-1, and s=0.33 (Table 1). Assuming an initial concentration gradient of Co-Ci(t=0)=Co=100 mol m-3, the solvent drag component comprises always less than 2% of the total solute flow. Therefore, the solvent drag will be neglected in further calculations.
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Equations 1
and 2 are valid only if the permeating solute remains unchanged inside the cell, i.e. when it is not taking part in metabolic processes. When the permeating solute is decomposed inside the cell, this should be taken into account, i.e. equation 2 has to be extended by a term which incorporates the chemical reaction. The most simple case of such a chemical reaction is the decomposition of hydrogen peroxide in the presence of the enzyme catalase inside the cell:
 | (3) |
Of course, there could be also other enzymes such as different peroxidases which would degrade H2O2, but their specific activities are usually smaller than that of catalase (see Discussion). The products of the degradation of H2O2 in the presence of catalase (H2O; O2) are not osmotically active. The enzymatic degradation reduces the internal concentration of H2O2 which is osmotically active. In other words, an additional ‘virtual’ flow of solute is produced (‘reaction flow’ of H2O2). The rate of decomposition (dnicat/dt) should follow a Michaelis-Menten type of kinetics (vmax: maximum rate, in mol (s cell)-1; kM: Michaelis constant, mol m-3). Equation 4 describes the reduction of the inner concentration due to enzymatic reaction according to a Michaelis-Menten equation:
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Equation 2 can be modified by adding the Michaelis-Menten term from equation 4 expressed per unit area:
 | (5) |
Equation 5 implies that the simple two-compartment model is still valid despite the fact that catalase is compartmented in peroxisomes where the splitting of H2O2 takes place. However, the assumption is reasonable, because of the small size of peroxisomes which provides a rapid equilibration of H2O2 in peroxisomes with its surroundings even if the permeabilities of the membranes of organelles were low. There is evidence from earlier data that the permeability of the tonoplast for uncharged small molecules is high which justifies the assumption of a two-compartment model (for a detailed consideration of compartmentation, see Discussion). Equation 5 describes the solute transport across a membrane when a chemical reaction following a Michaelis-Menten type of kinetics decomposes the solute inside the cell. By contrast with the situation in the absence of a chemical reaction, a simple analytical solution for the time-course of the inner concentration Ci(t) after changing the concentration of solute outside at t=0 is not available. It may be possible to calculate a complex formula for Ci(t) involving several substitutions, integrations and solving of a third order polynomial equation (not shown). However, such a complex formula would hardly be of practical use for analysing measured pressure relaxation curves. Nevertheless, three special cases should be discussed which are important in the context of this paper. For these cases, analytical solutions are given in the following paragraphs. Solutions are analysed for an exosmotic experiment, but the procedure for the endosmotic case is similar.
Case I (t
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After changing the osmotic gradient between inside and outside at t=0, a new steady-state will be reached when water and solute flow have vanished again (JV, Js=0 at t). For the new steady-state, it follows from equation 5:
 | (6) |
is the final steady-state concentration inside. According to equation 6, the substrate moves into the cell at a rate which is just balanced by enzymatic degradation. Equation 6 shows that, in the presence of a chemical reaction, the internal concentration is always lower than the external. From equation 1 it follows that the final steady-state pressure (P=P(t)) would also then be lower than the initial (P0), i.e.:
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is difficult to measure. However, equation 6 can be used to express as a function of Co. Substituting 
into equation 7 leads to a relation between 
P and Co that can be measured:
 | (8) |
shows plots of P as a function of either (equation 7) or Co (equation 8) which have been generated by a computer. The figure illustrates that P is a measure of the rate of decomposition by the enzyme, and therefore, follows a Michaelis-Menten type of kinetics. By definition, a Lineweaver-Burk plot of 1/P as a function of 
yields a straight line (see inset of Fig. 1). However, the inset also shows that this type of plot is of no use for evaluating vmax and kM when 1/P is plotted as a function of 1/Co. At saturating concentrations of substrate, the pressure difference approaches a maximum value proportional to vmax:
 | (9) |
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Half of the maximum pressure difference is reached when the concentration inside the cell equals kM. Since the concentration outside is always higher than inside, a plot of
P versus Co is shifted to the right as compared with that of P versus . The difference between the two plots depends on the ratio of the rates of decomposition to permeation of substrate (vmax/(kMPs)). When treating a cell with a series of solutions with different concentrations, the corresponding P can be obtained (see Results). From these data, it is then possible to evaluate vmax and kM using equation 8.
Case II (kM>>Co, Ci)
When the external (and therefore also the internal) concentration is much lower than the Michaelis constant kM, the rate of decomposition is proportional to the inner concentration to a good approximation (linear range of Michaelis-Menten kinetics). Equation 5 reduces to:
 | (10) |
can be integrated using standard procedures. This yields the following time-course for Ci(t):
 | (11) |
describes an exponential increase of Ci(t) up to a steady-state concentration lower than Co. The rate constant of the process (ks*) is higher than that in the absence of a chemical reaction (i.e. vmax=0, kcat=0). In this latter case, equation 11 reduces to:
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reflects the sum of the two single processes (see Discussion). In the presence of a chemical reaction, the final pressure difference is:
 | (13) |
that the absolute value of P depends on the relative contribution of the rate of degradation (kcat) to the overall rate (ks*). For example, when, transport dominates, i.e. when vmax>>(PsAkM), P0 is obtained. This has been often verified in the absence of a chemical reaction. On the other hand, if vmax>>(PsAkM) holds, the solute entering the cell is rapidly degraded. At an extreme, this should result in a P=sCoRT. In this case, the second solute phase would be completely missing. This result has been obtained in this paper when the transport of H2O2 was blocked in the presence of HgCl2.
Case III (kM<<Ci, Co)
Under these conditions, maximum rates of decomposition of substrate should be attained. The second term on the right side of equation 5 reduces to the constant factor vmax/A. The integration of equation 5 then yields:
 | (14) |
It can be seen that the kinetics is independent of kM. The rate constant of the exponential process 
is that of the solute permeation only. This is so because, at high concentrations, membrane permeation is the limiting step (see Discussion). The final pressure difference is then P=Pmax (equation 9) which can be directly approximated from equation 8.
Cases II and III show that, in both extremes, time-courses of inner concentrations Ci(t) can be approximated by single exponential functions. Exponential curves just differ in their rate constants (ks+kcat and ks, respectively). This leads to the idea that, in general, the main part of a solution for equation 5 is a single exponential function, and its rate constant decreases with increasing concentrations outside (Co). To test this idea, a numerical solution of equation 5 must be found and analysed.
Basis of numerical simulation
In order to simplify expressions, it is useful to rewrite equation 5 by substituting time and concentration in terms of dimensionless variables (
=kst and x()=1+Ci()/kM), i.e.:
 | (15) |
is therefore normalized. In an exosmotic experiment, the external concentration changes from xo=1 to a constant xo>1 at =0. Boundary conditions for a numerical solution are:
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 | (16) |
In the case of an endosmotic experiment, step changes of concentration are in the opposite direction, i.e. at =0, the external concentration is changed from a constant xo>1 to xo=1. The differential equation is modified since the actual value of xo is xo=1:
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 | (18) |
s*=ks*/ks). When the process is dominated by permeation it follows that ks*ks and s*1. |
Materials and methods |
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Plant material
Chara corallina was grown in artificial pond water (APW; composition in mol m-3: 1 NaCl, 0.1 KCl, 0.1 CaCl2, 0.1 MgCl2) in tanks which contained a layer of natural pond mud (Henzler and Steudle, 1995
). Tanks were placed in a greenhouse without additional illumination. Internodes used in pressure probe experiments were 50–100 mm in length and 0.8–1.0 mm in diameter.
Determination of transport parameters
Transport parameters were measured and calculated from ‘hydrostatic’ (Lp: hydraulic conductivity in m s-1 MPa-1) and ‘osmotic’ (Ps: permeability coefficient in m s-1; s: reflection coefficient) pressure relaxations. Relaxation curves have been measured using a conventional cell pressure probe as previously described (Henzler and Steudle, 1995). Numerical analysis of the time-course of solute concentration inside the cell indicated that kinetics can be described by single exponential functions to a good approximation (see Results). Therefore, overall rate constants (ks*) were evaluated from the second part of exosmotic pressure relaxations, i.e. from the ‘solute phase’. Rate constants ks used to calculate the solute permeability (Ps=ksV/A) were extrapolated from a series of exosmotic relaxations with different external concentrations of hydrogen peroxide to Co. In some cases, only a small if any correlation between ks* and Co could be detected. Hence, ks was calculated as a mean of ks*. In other cases, ks* decreased with increasing Co, and ks was determined from the extrapolated value of a single exponential fit of ks* as a function of Co.
The definiton for calcluation of reflection coefficients is derived from equation 1. Since JV vanishes at the minimum of the curve (JV(t=tmin)=0) it holds that:
 | (19) |
(equation 7), the time-course of Ci(t) can be espressed as:
 | (20) |
and 20 at t=tmin, yields an expression which was used to calculate reflection coefficients from exosmotic relaxation curves:
 | (21) |
corrects for the change of the initial osmotic pressure gradient (RTCo) caused by permeation and degradation of solute. Different from earlier formulae used to estimate s, the overall rate constant ks*, and not ks, had to be used for the correction, because ks* describes the change of the concentration gradient (equations 11 and 14). It can be seen from equation 21 that, in the absence of a chemical reaction (ks*=ks and P0=P), equation 21 is identical with the equation used earlier to evaluate reflection coefficients from osmotic pressure relaxations (Steudle and Tyerman, 1983).
Analysis of enzyme kinetics
To determine parameters for enzyme kinetics, a concentration series of osmotic experiments was performed with each of six different internodes. Starting with low concentrations of hydrogen peroxide, several subsequent exosmotic and endosmotic pressure relaxations with four to five different concentrations were performed (Fig. 3). Using equation 8, final steady-state pressure differences of the exosmotic experiments (P) were fitted to the corresponding concentrations of H2O2 in the medium (Co). Michaelis constants (kM) and maximum rates of decomposition (vmax
Pmax) were obtained from the fitted parameters either directly (kM) or using equation 9 (vmax). Since the external concentrations of substrate (Co) which could be measured here, were higher than internal concentrations at steady-state (Ci), a double reciprocal plot (Lineweaver-Burk type) was of no use (see inset of Fig. 1and equations 7 and 8).
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Numerical simulation
A numerical calculation of the solute concentration inside a cell during an exosmotic or endosmotic pressure relaxation was provided by solving the normalized equations 15
and 17 by means of a computer (PC, Pentium-II, 300 MHz). Since start values were known (equations 16 and 18), time-courses were calculated by stepwise iteration. During exosmotic water flow, values of x(+) were calculated from previous values x() using equation 15: x(+)=x()+[x()-xo+ß-ß/x()]. Commercial data processing software was employed in the simulations (Transform-tool of SigmaPlot 2.01, Jandel Scientific, Erkrath, Germany). Time-steps of =5x10-5 were used that corresponded to 1–5 ms in ‘real’ time. Maximum changes of x(+)-x() occurred at the beginning of calculations (0) for the high values of xo. These changes corresponded to changes in ‘real’ concentration of smaller than 0.05 mol m-3 (50 µM) at external concentrations of 7–1000 mol m-3 (7 mM to 1 M). Therefore, a sufficient resolution of time for numerical calculations and the linearity of step-changes of x() was guaranteed. Numerical calculations of time-courses of x() for 30 different values of xo took about 3 h.
Fitting of curves
In the context of this paper, fitting of curves just means to determine numerically the optimal parameters of a pre-selected function for the measured data points by means of a least-square algorithm (Marquardt-Levenberg). No searches for appropriate mathematical functions were performed. Commercial software for data processing was employed for the fits (Curve-fit-tool of SigmaPlot 2.01) which also provided asymptotic standard errors for the parameters determined.
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Results |
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Numerical simulation
A stepwise numerical solution of equations 15 and 17 for 30 different values of the normalized external concentration (xo) is shown in Fig. 2
. Exosmotic as well as endosmotic experiments were simulated (Fig. 2A). For the exosmotic case, a semi-logarithmic plot showed that, for the whole range of external concentrations (xo=1–12; xoCo), x() can be described by a single exponential function to a good approximation (Fig. 2B, left). This has already been proposed in the theoretical section. Deviations from a single exponential could be seen only in endosmotic experiments (Fig. 2B, right). In this case, deviations increased with increasing external concentrations. At the beginning of the endosmotic curves (=6–7), initial slopes were smaller than mean slopes. Towards the end of relaxations (>10), slopes became bigger. The asymmetry between exosmotic and endosmotic experiments originated from either increasing (exosmotic) or decreasing (endosmotic) internal concentrations. Therefore, the two processes (permeation and degradation) influenced the curves differently during different time intervals. This resulted in a more complex behaviour in the case of an endosmotic experiment. Hence, for the sake of simplicity, only the exosmotic case was investigated further.
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): endosmotic; (•): exosmotic) and external concentrations is analysed. Data were taken from fits of the numerical calculations in (A). The value of Figure 2C
shows a plot of the normalized overall rate constant (s*=ks*/ks) as a function of xo. Data were determined from the fits in Fig. 2A. It can be seen that s* decreased with increasing xo. For the exosmotic experiment, values varied from 1+ß (low xo1) to unity (high xo) as was expected before (Cases II and III in the Theory section). Due to the bigger slopes at the end of the curves (see above), values for s* were, in general, larger in endosmotic than in exosomotic relaxations. The functional relation between s* and the external concentration was exponential in nature (exponential fits in Fig. 2C). The numerical analysis verified that the rate constant of the sum of the processes (permeation and degradation of H2O2) was bigger than that of permeation only. Absolute values depended on the actual concentration of hydrogen peroxide in the medium. Only at high concentrations of H2O2, measured rate constants could be regarded solely as those of the process of permeation only.
Pressure probe experiments
In Fig. 3, a typical series of time-courses of osmotic pressure relaxations is shown for five different concentrations of hydrogen peroxide (40–265 mM). After quickly replacing the medium with a solution containing a certain amount of H2O2, cell turgor pressure decreased due to a flow of water out of the cell. After reaching a horizontal tangent at the minimum of the curve (P(tmin)=Pmin and JV=0), pressure increased again due to permeation of hydrogen peroxide into the cell. However, turgor pressure did not come back to its initial value. Since H2O2 was decomposed inside the cell, a final steady-state pressure difference (P) was maintained. Changing the medium back to a solution containing no H2O2 resulted in a response curve which was symmetrical to the exosmotic one, but was in the opposite direction.
Although high concentrations of hydrogen peroxide of up to 350 mol m-3 were used with some cells, there was a decrease of cell turgor of only 3–9% of the initial pressure during 1–2 h of experiment with a given cell. After one ex- and endosmotic experiment with a given concentration, a decrease of steady-state pressure of about 0.3–1% was found resulting in a slight ‘undershoot’ of the endosmotic curve (as compared with the initial pressure before the exosmotic experiment). Although this pointed to a stress of the cell caused by the treatment, its effect was fairly small. Therefore, it was concluded that the intregrity and stability of the cell membrane was maintained.
In Fig. 4, the evaluation of the rate constant of solute permeation is shown. Figure 4A summarizes biphasic exosmotic pressure relaxation curves for six different external concentrations of H2O2 as subsequently measured with a cell pressure probe. Fits for the second phase are plotted which represent the increase of concentration inside the cell. The fit procedure was used to determine the optimal values for Pfit(t=0), Pfit=Pfit(t), and ks* of a single exponential curve. In Fig. 4B, a semi-logarithmic plot of the data of Fig. 4A is given which shows that curves were single exponentials to a good approximation (correlation coefficient r2>0.97). At the end of relaxations, differences between P and Pfit were small which resulted in a marked scatter of the semi-logarithmic plot. This systematic error at the end of the curves was cut off for evaluation.
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0.974). In (C), the extrapolated hydrostatic pressure difference at t=0 (from fit in A) is plotted versus the osmotic pressure of the medium (RTCo) that caused the response in cell pressure. The very good correlation of the linear regression shows the consistency of results determined with a fit procedure for different concentrations (r2=0.999; solid line; dotted lines: prediction interval at 95% level). The slope of the regression line is a measure of the reflection coefficient (In Fig. 4C
, a plot of the extrapolated initial pressure differences caused by the different osmotic pressure gradients of H2O2 is shown. The extrapolated value of Pfit(t=0) is a theoretical value that would be attained in an experiment if the half-time of water exchange was close to zero (Tw1/20) and therefore tmin0. Then from equation 21, it follows that (Pfit(t=0)=P(t=tmin=0)=Pmin):
 | (22) |
The high correlation (r2=0.999) indicated that fitting the curves yielded consistent results. The slope of the regression line is a measure for the reflection coefficient (s; equation 22). Alternatively, this parameter was determined from the pressure minimum of the curve (equation 21). A similar value was obtained (cell no. 5 in Table 1). Figure 4D presents values for the rate constant (ks*) of the change of the internal concentration of H2O2 determined by two slightly different procedures. Both fitting the curve and regressions of a semi-logarithmic plot yielded nearly the same values of the overall rate constant (ks*).
In Fig. 5, a summary of the dependence of ks* on the concentration of H2O2 in the medium is given for all measured cells. It can be seen form the figure that with four cells (1, 2, 4, 5), ks* decreased with increasing concentration. This can be attributed to the fact that, at high concentrations, substrate permeation became limiting (see Discussion). Values for ks used to calculate Ps were obtained by extrapolating ks* to high concentrations of hydrogen peroxide in the medium, either from mean values or from exponential fits (see Materials and methods). The mean value for six internodal cells was Ps=(3.6±1.0)x10-6 m s-1 which is high compared with other permeating solutes, including the diffusional permeability of water (Pd) as measured with isotopic water (HDO; Tables 1, 2).
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(molar volume of water: 
m3 mol-1). The diffusional permeability of water (Pd) denotes the permeability of HDO (Pd=Ps(H2O)). By definition, In Fig. 6
, the analysis of enzyme kinetics from a typical plot of a series of exosmotic pressure relaxations is shown. Values for kM and vmax were evaluated by fitting the steady-state pressure differences (P=P0-P) to the external concentrations used (Co), according to equation 8. Mean values for six cells were: kM=(85±55) mol m-3 and vmax=49±40 nmol (s cell)-1 (Table 1). Values are in line with data for other species reported in literature (see Discussion).
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The hydraulic conductivity was measured from hydrostatic pressure relaxation curves (not shown). The mean value for six internodes was Lp=1.7±0.7x10-6 m (s MPa)-1. This value can be converted to an osmotic water permeability Pf=LpRT/
m s-1 
m3 mol-1, molar volume of water; Table 2). It should be noted that Pf was larger by a factor of 30 than Pd. The ratio of Pf/Pd has been used as a measure for the number of water molecules aligned in a single file in water channels (Steudle and Henzler, 1995; Hertel and Steudle, 1997).
The absolute value of the permeability of hydrogen peroxide was rather high. It was smaller by only a factor of two than that of heavy water (HDO: Pd=7.7x10-6 m s-1; Henzler and Steudle, 1995). This may suggest that, because of the similarity in the chemical structure, H2O2, uses water channels to cross the plasma membrane. In order to test this possibility, the channel blocker mercuric chloride (HgCl2) was used to inhibit water channels. If the permeability of H2O2 were affected, one should expect a decrease of the rate constant of the solute phase. Figure 7 shows that, upon treatment with mercuric chloride, the second (solute) phase was completely absent. This may indicate, that in the presence of the channel blocker, the rate of chemical degradation could compete with that of membrane permeation, i.e. the amount of H2O2 arriving in the cell was immediately degraded in the presence of the enzyme. Assuming that, under these conditions, the concentration of the substrate in the cell was close to zero, a reflection coefficient could be evaluated (equation 21; ks*0) which was smaller than that measured in the absence of the blocker. In terms of the composite transport model of the membrane (Steudle and Henzler, 1995), this may indicate that the reflection coefficient of the bilayer of H2O2 is smaller than that of the water channel array (see Discussion). The half-time of water exchange (first phase of biphasic pressure relaxations) increased indicating a decrease of Lp(Pf) besides the reduction of the rate of uptake of H2O2. To date, an inhibition of solute transport in the presence of the channel blocker HgCl2 has only been shown for heavy water (Henzler and Steudle, 1995; Steudle, 1993). An effect on the permeability of other small uncharged solutes such as monohydric alcohols, amides and acetone was not detectable, although there was some slippage of these solutes across water channels (Hertel and Steudle, 1997). The finding that transport of H2O2, HDO and water (Lp) were similarly affected by a closure of water channels, strongly suggests that there was a substantial movement of H2O2 across water channels. Figure 7C shows that the scavenger 2-mercaptoethanol reverted the effect of HgCl2. However, the figure also indicates that in the presence of two stresses (mercury and high hydrogen peroxide levels), cells tended to become leaky and turgor slowly but inexorably tended to decline. The combination of two stresses could only be tolerated over periods of time which were much shorter than that used during the application of high concentration of H2O2 (up to 3 h).
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The mathematical model given in this paper describes the combination of the permeation of a solute (H2O2) and of its enzymatic decomposition. Experiments are presented which are in line with the model. According to the results, H2O2 permeates membranes at a rate which is comparable to that of diffusional water flow. This and the fact that the chemical structure of H2O2 resembles that of water suggests that hydrogen peroxide uses water channels to cross membranes. To the best of the authors’ knowledge, these data are the first rigorous theoretical and experimental analyses of a permeation/reaction system, which may be of some importance because, on one hand, H2O2 is a precursor of other toxic oxygen compounds. On the other hand, rapid membrane transport of H2O2 should affect the intracellular concentration of H2O2 and, hence, all metabolic reactions in which this compound is involved. The most simple case of a transport combined with a chemical reaction has been investigated. The permeating solute (H2O2), which is osmotically active, is decomposed inside the cell by the enzyme catalase into products (H2O and O2) which are not osmotically active. However, even in this simple case, a general simple analytical solution is not available. Therefore, a numerical simulation is shown, that yields quantitative predictions which were sucessfully tested in experiments. The experimental results indicate that the model is adequate to describe the system under investigation.
Hydrogen peroxide is produced during different metabolic processes such as during photorespiration in chloroplasts or during the formation of lignin in cell walls (Asada, 1992; Ishikawa et al., 1993; Takeda et al., 1995; Schopfer, 1996). Hydrogen peroxide affects the integrity of cells because it is a precursor of highly reactive oxygen species such as the hydroxyl radical which attacks proteins, lipids and nucleic acids (Foyer et al., 1994; van Rensburg et al., 1992). Oxidative bursts caused by high levels of H2O2 play a role during the defence of plants against pathogens (Wojtaszek, 1997; Apostol et al., 1989; Peng and Kuc, 1992). Hydrogen peroxide and other reactive oxygen compounds have also been discussed in relation to senescence and the development of cancer (van Rensburg et al., 1992; Sinha et al., 1987). By means of the enzymes superoxide dismutase, ascorbate peroxidase and catalase, organisms keep the levels of toxic compounds low. According to the literature, catalase (largely concentrated in peroxysomes) has the highest specific activity (in U mg-1: catalase (EC 1.11.1.6): 7800–282000; peroxidase (EC 1.11.1.7): 262–290; L-ascorbate peroxidase (EC 1.11.1.11): 34–254; Schomburg et al., 1994).
These experiments show that Chara cells can tolerate H2O2 at concentrations as high as 350 mol m-3, when these concentrations are applied at time intervals of 5–10 min that are necessary to attain a steady-state (P). Experiments with an individual cell treated repeatedly with H2O2 lasted for up to 3 h. During this period of time, initial cell turgor (which is a good indicator of membrane integrity) was lost by only a few per cent if any. At first sight, the finding that Chara internodes can tolerate H2O2 at concentrations of a few hundred millimoles may be astonishing. Although there are other examples that plant cells can tolerate H2O2 in the range of some millimoles (Baker and Orlandi, 1995; Schopfer, 1994, 1996), the very high concentrations found for Chara may be exceptional. It was reported that, in contrast to higher plants, the photosynthesis of algae is more resistant to H2O2 due to structural differences of their thiol-mediated enzymes (Takeda et al., 1995). The findings presented here indicate that H2O2 by itself is not very toxic to Chara as long as the concentration of OH radicals is kept low. This is in line with experiments in which much lower concentrations of H2O2 were added to the medium in the presence of Fe2+. This increased the level of OH radicals and caused an immediate loss of cell turgor and big changes in the permeabilty of the internodes for water and solutes (Fenton reaction; E Steudle and T Henzler, unpublished results). Eventually, cells were irreversibly damaged.
In the calculations of transport coeffcients and of kinetic parameters of the splitting of H2O2, it was assumed that the cell interior and the medium can be treated as a two compartment system. The assumption may be questioned, because catalase is compartmented in peroxisomes which would require that the substrate has to cross another membrane. The tonoplast could represent another substantial barrier for H2O2. Since the surface area to volume ratio of cell organelles was bigger by three orders of magnitude than that of the entire cell (diameter of peroxisomes1 µm), concentration differences of H2O2 between cytoplasm and organelles should be rapidly equilibrated, although there may be differences in the permeability between the plasma membrane and that of peroxisomes. In Chara, there is experimental evidence that, compared to the plasma membrane, the permeability of the tonoplast for small uncharged solutes such as H2O2 is high. When the solute permeability was evaluated from the solute phase of biphasic pressure relaxations, the ‘cytoplasmic’ compartment may be overlooked, because it could be ‘buried’ within the water phase. For the experiments reported here, an appropriate compartment analysis is, therefore, not possible. The solute phase and the Ps derived from it would refer to the sum of the permeation resistances of plasma membrane and tonoplast. However, data of solute permeability measured from the solute phase agreed quite well with those obtained by the pressure minimum technique which allowed the evaluation of Ps from the shape of pressure/time-courses around the minimum, i.e. during periods of time of about 20–30 s (Tyerman and Steudle, 1984). Values of Ps determined from the solute phase also agreed with data which were obtained from measuring the initial uptake of radioactive tracers (Dainty and Ginzburg, 1964). In both cases, permeabilities were measured during periods of time, where the cytoplasmic compartment (plasmalemma) should have dominated (initial uptake of tracers) or should have contributed substantially to the measured Ps (Tyerman and Steudle, 1984). These results were obtained with solutes exhibiting a fairly large range of permeability and different solubility in lipids (Ps=0.1–3x10-7 m s-1). They strongly suggest a large permeability of the tonoplast. The findings are in line with other results (Kiyosawa and Tazawa, 1977; Tazawa et al., 1996), where no change was found in Lp(Pf), when removing the tonoplast of perfused internodes of Chara. These authors concluded that, in Chara, the plasma membrane rather than the tonoplast is the main barrier of water transport.
Besides compartmentation, unstirred layers may contribute to the overall measured transport parameters tending to reduce Lp, Ps(Pd), and s. Two different types of unstirred layers have to be distinguished, i.e. unstirred layers of the diffusion versus convection type (‘sweep-away’ effects) and purely diffusive unstirred layers (Dainty, 1963; Barry and Diamond, 1984). It has been readily shown that, in the case of measurements of isolated cells with the pressure probe, sweep-away effects are small or negligible (Steudle and Tyerman, 1983; Steudle et al., 1980; Steudle, 1993). This is so because the amounts of water moved across the membrane are very small (as discussed in detail for Chara by Hertel and Steudle, 1997). External diffusive layers should also have been small in the presence of a vigorously stirred medium as in the present experiments. However, diffusional unstirred layers inside the internodes could be as large as a few hundred micrometers (at maximum) which is not negligible. So, there may be a contribution of these internal unstirred layers of the diffusive type to the overall measured values of transport coefficients (Ps and s). Cytoplasmic streaming, the cylindrical geometry, and the fact that H2O2 is rapidly diffusing would reduce the effects of internal unstirred layers. The diffusion coefficient of H2O2 at 20 °C is D=1.3x10-9 m2 s-1 (see Meyer, 1966, and references therein). Assuming a permeability of Ps=3.6x10-6 m s-1, the resistance of the membrane to the solute flow (i.e. 1/Ps) would be equivalent to a diffusional resistance of a plane water layer of as large as 
=360 µm (1/Ps=/D). This is similar to the radius of the internodes (500 µm). Therefore, the internal equilibration of H2O2 gradients should be fairly rapid as compared with membrane permeation even for the giant internodes of Chara. Accordingly, there are indications from osmotic experiments with solutes having a diffusion coefficient similar to that of H2O2 (e.g. monohydric alcohols, acetone, formamide), that membrane permeation rather than internal diffusion limits transport. First, solute phases could be nicely fitted by a single exponential which would not be true in the presence of a limitation by diffusion within the cell. During internal diffusion, the thickness of the unstirred layer would increase with time during the solute phase (see preceding paragraph). Second, the Ps of different solutes used to date with Chara internodes ranged over nearly two orders of magnitude. If uptake were controlled by diffusion, Ps values should have been similar, because the diffusion coefficients of these solutes are similar. So, for these reasons it is safe to conclude that, although there may be some influence of internal unstirred layers on the absolute values of the Ps and s of H2O2, membrane permeation and the subsequent degradation of the substrate in the cell should have dominated the processes measured.
The numerical simulation yielded predictions which were in line with the experimental results. When the effect of the chemical reaction was taken into account, it could be verified that H2O2 rapidly permeated the cell membrane. At high external concentrations, membrane permeation dominated the overall rate of the process. At low concentrations, the chemical reaction contributed to as much as 44% to the overall rate (Table 1). The latter case may be found in cells where concentrations are barely millimolar. Hence, when H2O2 is going to be exported from one cell compartment into another (e.g. from the cytosol to the wall space) or is concentrated in a tissue (as during oxidative bursts in the presence of pathogens) this requires a balance between production and degradation on one hand and transport on the other. When H2O2 uses water channels (or when some of the water channels are in fact H2O2 channels), the balance could be affected on the transport side to allow either rapid export (open H2O2 channels) or concentration of hydrogen peroxide (closed H2O2 channels) in a given compartment.
Other important predictions from the model and from the numerical simulations are that the time-course of the internal concentration can be fitted by a single exponential to a good approximation, and that the rate constant (ks*) of this exponential curve is dependent on external concentration. Predictions have been verified in experiments. Values of rate constants decreased with increasing external concentration and converged to the value that represents the permeation of solute only. This means that in order to separate the transport step from the chemical reaction, the concentration dependence has to be analysed. The fact that there is a simple exponential rate should not be misunderstood in terms of either a simple diffusional mechanism for the uptake or by a first order chemical reaction. The results show that even in the simplest case of a decomposition and permeation process, a rather complex type of kinetics may result. A numerical analysis, in combination with experimental results, provides a powerful tool to get a deeper insight into the processes.
The absolute value for the permeability of hydrogen peroxide (Ps) has been worked out from values of ks* extrapolated to high external concentrations. The value of Ps was 3.6x10-6 m s-1, which is smaller by a factor of two than the diffusional permeability of water (Pd) for the same species and similar to that of rapidly permeating small organic solutes (Henzler and Steudle, 1995; Table 2). The polarity of H2O2 is similar to that of water and its van-der-Waals volume (a measure of the volume required by a molecule) larger by about a factor of about 1.5 (in 10-6 m3 mol-1: VH2O=15.5;VH2O2=10.7; calculated according to Bondi, 1964). Therefore, one would expect that H2O2 is not able to permeate the membrane rapidly. In order to move across the bilayer, its polarity would be too high. The molecule would be too big to move across highly selective water channels. However, the data presented in this paper show that permeability of H2O2 is high and similar to that of low molecular weight solutes (monohydric alcohols, acetone) which largely move across the bilayer by solubility mechanism (Table 2). The permeability of H2O2 is also somewhat lower than the diffusional permeability of water (as measured in diffusion experiments with heavy water; Henzler and Steudle, 1995; Table 2). Hence, it is assumed that H2O2 crosses the membrane largely via water channels (aquaporins). This assumption is based on the fact that the conformation of H2O2 is that of a stretched molecule with a mean diameter of about 0.25 nm and a mean length of about 0.28 nm (calculated from geometrical data: 
O–OH=94.8°, HO–OH=111.5°, dOH=0.095 nm, dOO=0.147 nm, dH=0.12 nm; Hollemann et al., 1995, p. 533). The H2O2 molecule may fit into the pores of water channels (width: 0.2–0.4 nm; Steudle and Henzler, 1995). The assumption is supported by the observation that the passage of H2O2 is strongly inhibited by mercurials (see next paragraph).
At first sight, the relatively high absolute value of the reflection coefficient of hydrogen peroxide (s=0.33±0.12; Table 2) seems to contradict the assumption that H2O2 crosses the membrane via water channels. However, this finding can be explained when it is assumed that the population of water channels in the Chara internode consists of some narrow individuals which allow the passage of H2O2 and others which do not. According to the composite transport model, the latter would then cause the overall value of the reflection coefficient to be rather high, whereas the former would tend to keep it low (Steudle and Henzler, 1995). Thus, the difference in the transport pattern for heavy water (s=0.0034 and Pd=7.7x10-6 m s-1) and for H2O2 (s=0.33 and Ps=3.6x10-6 m s-1) would be understandable. This interpretation is in line with the experiments which show that the channel blocker HgCl2 caused a decrease of Lp(Pd) and of Ps and a decrease of the overall reflection coefficient (Fig. 7). To date, these findings can not completely rule out the possibility that the channels which permit the passage of H2O2 are highly selective for this substrate and do not enable the passage of water. However, considering the polarity and sizes of H2O2 and H2O, this possibility seems to be unlikely. Alternatively, the population of water channels could be fairly homogenous and HDO and H2O2 would just differ in their reflection coefficients because of the bigger size of the H2O2 molecule. This latter explanation is also unlikely because from the small differences in permeability coefficients one would expect a much smaller difference in reflection coefficients. More experiments are necessary to answer the question as to whether there are two functionally different types of channels present or just one population of water channels with somewhat variable diameters as the present results suggest. The problem can be solved functionally by measuring Lp, Ps, and s with a pressure probe before and after closure of channels. This approach is powerful because water and solute transport and their coupling can be measured simultaneously in one experiment which gives a much deeper insight into the processes. A molecular characterization of water channels versus H2O2 channels in Chara would be helpful as well, but is not yet available. An extended functional analysis requires an estimate of the contribution of the lipid array to the overall transport and of the reflection coefficient of H2O2 for the lipid array. Because closure of water channels (HgCl2, at external concentrations of some 10 µM) causes a stress to which the oxidative stress by H2O2 is then added, these experiments are difficult to perform. The use of less harmful treatments other than HgCl2 would be helpful.
For the first time, parameters of an enzyme kinetics have been evaluated directly from a series of pressure relaxation curves. The Michaelis constant (kM) obtained from the effect of external concentration on P were 28–153 mM. This is of an order of magnitude which is similar to that reported in the literature for other systems. For example, the kM of catalase extracted from pea leaves (Pisum sativum L.) was 190 mM (del Rio et al., 1976). For Mycobacterium avium, Wayne and Diaz found two different catalases which exhibited values of kM of about 5 mM and 150 mM, respectively (Wayne and Diaz, 1982). The reason for the big relative errors in these parameters found in this paper may be that different cells exhibited different absolute amounts of catalase either due to size or age of cells. It can be seen from Table 1 that referring vmax to unit cell volume reduced the relative error by over 15%. It should be noted that the physiologically relevant parameter ß(vmax/(kMPs)) which characterizes the relative contributions of solute degradation and permeation to overall solute flow has an even smaller relative error. One may expect that the content of catalase in the internodes would be dependent on age. Perhaps, older cells have a lower capability to degrade hydrogen peroxide than younger. However, to date, it has not yet been possible to characterize the effect of age on the catalase content of Chara internodes.
In conclusion, it has been shown that the measured kinetics of a simple system where transport of a substrate (H2O2) into a cell is followed by its chemical decomposition, quantitatively agrees with model predictions. From the kinetics, transport properties of the solute have been derived (permeability and reflection coefficients of the plasma membrane for H2O2) as well as the Michaelis constant and the maximum rate of decomposition. Measured osmotic pressure relaxation curves quantitatively agreed with predictions derived from a two-compartment model. The two processes (permeation and degradation) could be quantitatively separated. The model may be used for other systems such as during osmoregulation when osmotic processes (water relations) interfere with metabolism. The permeability of the plasma membrane for H2O2 was fairly high at relatively high values of the reflection coefficient for this solute. This and the similarity of the chemical structure of the solute to that of water suggested that H2O2 is transported across water channels, but that the bigger solute uses somewhat wider aquaporins of the water channel population. To date, the data do not allow the conclusion that there are special H2O2 channels which are not used by water. Since water channels in Chara can be affected by different triggers (temperature, high concentration, and heavy metals), they may allow the regulation of H2O2 passage depending on conditions. Currently, the role of water channels during the permeation of hydrogen peroxide is studied in greater detail to answer the question whether or not some of the different water channels could be ‘peroxoporins’ rather than aquaporins.
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Notes |
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1 To whom correspondence should be addressed. Fax: +49 921 552564. E-mail: tobias.henzler{at}uni\|[hyphen]\|bayreuth.de
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Abbreviations |
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A, surface area; Ci/o, internal/external concentration; Ci
, internal concentration at steady-state; JV/s, volume/solute flow; kM, Michaelis constant; ks, rate constant of permeation; ks*, overall rate constant; s*, normalized rate constant; kcat, rate constant of decomposition; Lp, hydraulic conductivity; dni(s/cat), change of moles inside (due to permeation/decomposition); P(0//min), cell turgor pressure (at time=0/steady-state/pressure minimum); P/max, steady-state/maximal pressure difference; Ps, permeability coefficient; s, reflection coefficient; t(min), time (at pressure minimum); , normalized time; vmax, maximal velocity of decomposition; V, volume; 
w, molar volume of water; x(o), normalized (external) concentration of solute. |
References |
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TopAbstractIntroductionTheoryMaterials and methodsResultsDiscussionReferences |
|---|
Apostol I, Heinstein FH, Low PS.1989. Rapid stimulation of an oxidative burst during elicitation of cultured plant cells. Role in defense and signal transduction. Plant Physiology 90, 109–116.
Asada K.1992. Ascorbate peroxidase—a hydrogen peroxide-scavenging enzyme in plants. Physiologia Plantarum 85, 235–241.
Baker CJ, Orlandi EW.1995. Active oxygen in plant pathogenesis. Annual Review of Phytopathology 33, 299–321.[Web of Science]
Barry PH, Diamond JM.1984. Effects of unstirred layers on membrane phenomena. Physiological Review 64, 763–872.
Bondi A.1964. Van der Waals volumes and radii. Journal of Physical Chemistry 68, 441–451.
Dainty J.1963. Water relations of plant cells. Advances in Botanical Research 1, 279–326.
Dainty J, Ginzburg BZ.1964. The permeability of the protoplast of Chara australis and Nitella translucens to methanol, ethanol and iso-propanol. Biochimica et Biophysica Acta 79, 122–128.[Medline]
Foyer CH, Descourvières P, Kunert KJ.1994. Protection against oxygen radicals: an important defense mechanism studied in transgenic plants. Plant, Cell and Environment 17, 507–523.
Henzler T, Steudle E.1995. Reversible closing of water channels in Chara internodes provides evidence for a composite transport model of the plasma membrane. Journal of Experimental Botany 46, 199–209.
Hertel A, Steudle E.1997. The function of water channels in Chara: the temperature dependence of water and solute flow provides evidence for composite membrane transport and for a slippage of small organic solutes across water channels. Planta 202, 324–335.
Hollemann AF, Wiberg E, Wiberg N.1995. Lehrbuch der Anorganischen Chemie. Berlin, New York: Walter de Gruyter.
Ishikawa T, Takeda T, Shigeoka S, Hirayama O, Mitsunaga T.1993. Hydrogen peroxide generation in organelles of Euglena gracilis. Phytochemistry 33, 1297–1299.
Kiyosawa K, Tazawa M.1977. Hydraulic conductivity of tonoplast-free Chara cells. Journal of Membrane Biology 37, 157–166.
Meyer RJ.1966. Sauerstoff, Lieferung 7, Wasserstoffperoxid, Systemnummer 3. In: Gmelins Handbuch der anorganischen Chemie. Verlag Chemie, Weinheim/Bergstraße, Germany.
Peng M, Kuc J.1992. Peroxidase-generated hydrogen peroxide as a source of antifungal activity in vitro and on tobacco leaf disks. Phytopathology 82, 696–699.
van Rensburg CEJ, van Staden AM, Anderson R, van Rensburg EJ.1992. Hypochlorous acid potentiates hydrogen peroxide-mediated DNA-strand breaks in human mononuclear leucocytes. Mutation Research 265, 255–261.[Web of Science][Medline]
del Rio LA, Ortega MG, López AL Gorgé JL.1976. A more sensitive modification of the catalase assay with the Clark oxygen electrode. Analytical Biochemistry 80, 409–415.
Rüdinger M, Hierling P, Steudle E.1992. Osmotic biosensors. How to use a characean internode to determine the alcohol content of beer. Botanica Acta 105, 3–12.
Schomburg D, Salzmann M, Stephan D. (eds) 1994. Enzyme handbook 7. Berlin, Heidelberg: Springer-Verlag.
Schopfer P.1994. Histochemical demonstration and localization of H2O2 in organs of higher plants by tissue printing on nitrocellulose paper. Plant Physiology 104, 1269–1275.[Abstract]
Schopfer P.1996. Hydrogen peroxide-mediated cell-wall stiffening in vitro in maize coleoptiles. Planta 199, 43–49.
Sinha BK, Katki AG, Batist G, Cowan KH, Myers CE.1987. Differential formation of hydroxyl radicals by adriamycin in sensitive and resistant MFC-7 human breast tumor cells: implications for the mechanism of action. Biochemistry 26, 3776–3781.
Steudle E.1993. Pressure probe techniques: basic principles and application to studies of water and solute relations at the cell, tissue, and organ level. In: Smith JAC, Griffith H, eds. Water deficits: plant responses from cell to community. Oxford: Bios Sicentific Publishers, 5–36.
Steudle E, Henzler T.1995. Water channels in plants: do basic concepts of water transport change? Journal of Experimental Botany 46, 1067–1076.
Steudle E, Smith JAC, Lüttge U.1980. Water-relations parameters of individual mesophyll cells of the crassulacean acid metabolism plant Kalanchoë daigremontiana. Plant Physiology 66, 1155–1163.
Steudle E, Tyerman SD.1983. Determination of permeability coefficients, reflection coefficients and hydraulic conductivity of Chara corallina using the pressure probe: effects of solute concentrations. Journal of Membrane Biology 75, 85–96.
Takeda T, Yokota A, Shigeoka S.1995. Resistance of photosynthesis to hydrogen peroxide in algae. Plant Cell Physiology 36, 1089–1095.
Tazawa M, Asai K, Iwasaki N.1996. Characteristics of Hg- and Zn-sensitive water channels in the plasma membrane of Chara cells. Botanica Acta 109, 388–396.
Tyerman SD, Steudle E.1984. Determination of solute permeability in Chara internodes by a turgor minimum method. Plant Physiology 74, 464–468.
Wayne LG, Diaz GA.1982. Serological, taxonomic and kinetic studies of the T and M classes of mycobacterial catalase. Journal of Systematic Bacteriology 32, 296–304.
Wojtaszek P.1997. Oxidative burst: an early plant response to pathogen infection. Biochemical Journal 322, 681–692.
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I. C. Mori and J. I. Schroeder Reactive Oxygen Species Activation of Plant Ca2+ Channels. A Signaling Mechanism in Polar Growth, Hormone Transduction, Stress Signaling, and Hypothetically Mechanotransduction Plant Physiology, June 1, 2004; 135(2): 702 - 708. [Full Text] [PDF]
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V. Mittova, M. Guy, M. Tal, and M. Volokita Salinity up-regulates the antioxidative system in root mitochondria and peroxisomes of the wild salt-tolerant tomato species Lycopersicon pennellii J. Exp. Bot., May 1, 2004; 55(399): 1105 - 1113. [Abstract] [Full Text] [PDF]
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X. Wan, E. Steudle, and W. Hartung Gating of water channels (aquaporins) in cortical cells of young corn roots by mechanical stimuli (pressure pulses): effects of ABA and of HgCl2 J. Exp. Bot., February 1, 2004; 55(396): 411 - 422. [Abstract] [Full Text] [PDF]
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Q. Ye, B. Wiera, and E. Steudle A cohesion/tension mechanism explains the gating of water channels (aquaporins) in Chara internodes by high concentration J. Exp. Bot., February 1, 2004; 55(396): 449 - 461. [Abstract] [Full Text] [PDF]
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S. Suga, S. Komatsu, and M. Maeshima Aquaporin Isoforms Responsive to Salt and Water Stresses and Phytohormones in Radish Seedlings Plant Cell Physiol., October 15, 2002; 43(10): 1229 - 1237. [Abstract] [Full Text] [PDF]
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