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Originally published In Press as doi:10.1074/jbc.M603761200 on November 14, 2006

J. Biol. Chem., Vol. 282, Issue 2, 1183-1192, January 12, 2007
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Specific Aquaporins Facilitate the Diffusion of Hydrogen Peroxide across Membranes*

Gerd P. Bienert{ddagger}, Anders L. B. Møller{ddagger}, Kim A. Kristiansen§, Alexander Schulz§, Ian M. Møller{ddagger}, Jan K. Schjoerring{ddagger}, and Thomas P. Jahn{ddagger}1

From the Departments of {ddagger}Agricultural Sciences and §Plant Biology, Faculty of Life Science, Copenhagen University, DK-1871 Frederiksberg C, Denmark

Received for publication, April 19, 2006 , and in revised form, October 31, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hydrogen peroxide (H2O2)2 belongs to the group of reactive oxygen species (ROS). ROS are generated in a number of key metabolic processes in cells like the electron transport chain in the inner mitochondrial membrane (1) and, specific for plants, the chloroplast thylakoid membrane (2).

Because ROS can potentially damage proteins, lipids, and nucleic acids, cells have a number of ROS-scavenging systems that are able to remove these molecules and to maintain a relatively low and constant ROS concentration (3). However, ROS are also intermediates in various signal transduction pathways and have been shown to initiate responses to various stresses and disorders (for recent reviews, see Refs. 4 and 5). Arabidopsis mutants lacking an NADPH oxidase were not able to respond adequately to potassium deficiency (6) and were impaired in stomatal closure (7), providing genetic evidence for a role of NADPH oxidase in signaling.

ROS are interconvertible molecules including singlet oxygen, superoxide, hydroxyl radical, and H2O2.H2O2 has a distinctive set of features compared with other ROS. (i) It is not charged, (ii) it is not a radical, (iii) it possesses an intermediate oxidation number, (iv) it is relatively stable under physiological conditions, and (v) catalase can disproportionate it into water and molecular oxygen without the expense of reduction equivalents.

Although substantial progress has been made regarding the formation and scavenging of ROS, little is known about their transport from the site of origin to the place of action or detoxification. Recently three studies from mammalian systems have provided evidence that H2O2, in addition to the well studied role in intracellular signaling, is also used as an intercellular signal molecule (8-10). This implies that a necessary step within these signal transduction pathways is the transport of H2O2 or ROS across at least two membranes: (i) the plasma membrane of the H2O2-producing cell and (ii) the plasma membrane of the H2O2 signal-perceiving cell.

The role of biological membranes is to act as barriers between organelles and cells and to separate compartments for different metabolic pathways. Only small and non-polar molecules easily cross the hydrophobic membrane lipid bilayer by simple diffusion. The transport of larger, polar or charged molecules depends on transporters and channels to decrease the activation energy needed for passive diffusion. Expression of aquaporins in yeast secretory vesicles lowered the activation energy for diffusion of water from between 46 and 55 to 17 kJ mol-1 (11).

H2O2 has a permanent dipole moment of 2.26 x 10-18 electrostatic unit (12), very similar to that of water (1.85 x 10-18 electrostatic unit). Consequently simple passive diffusion of H2O2 across the lipid bilayer should be limited as for water. Recent studies have demonstrated that some membranes are indeed poorly permeable to H2O2 (13-16); this together with scavenging processes explains the formation of H2O2 gradients across membranes (17). Differences in permeability could either be explained by different membrane lipid compositions or by diffusion-facilitating channel proteins or a combination of both (for a review, see Ref. 18).

The striking chemical similarity between water and H2O2 points to aquaporins as likely candidates for H2O2 permeation. Aquaporins form a large family of membrane proteins with members in all kingdoms of life and are known as efficient water channels (19, 20). H2O2 has almost the same size, dielectric properties, and capacity to form hydrogen bonds as does water (12). Exactly those properties are the main factors determining the diffusion through major intrinsic proteins including aqua-porins. Henzler and Steudle (21) showed that mercury, an aqua-porin blocker, repressed H2O2 accumulation in internodal cells of the algae Chara corallina. The authors therefore suggested that some aquaporins in Chara served as peroxoporins.

The accumulating evidence prompted us to perform a comprehensive screen testing 24 aquaporin isoforms from plants and mammals in five yeast strains differing in sensitivity to oxidative stress. Aquaporin-mediated H2O2 transport was further investigated in a fluorescence assay with intact yeast cells using an intracellular ROS-sensitive fluorescent dye. Our data provide the first molecular genetic evidence that the three aqua-porins hAQP8, AtTIP1;1, and AtTIP1;2 facilitate the diffusion of H2O2 across membranes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains and Growth Assay—A wild type (wt) Saccharomyces cerevisiae strain (BY4741) and four deletion mutants in the same genetic background were used (Table 1). The double deletion mutant {Delta}fps1,{Delta}yfl054c was generated by crossing haploid, single deletion mutants Y15675 [GenBank] ({Delta}yfl054c) and Y01531 ({Delta}yll043w) (Euroscarf) and subsequent sporulation according to standard procedures. To complement the lack of the leu2 gene that was used to delete tsa2 to generate the {Delta}tsa1,2 mutant (22), wild type, {Delta}skn7, {Delta}yap1, and {Delta}fps1,{Delta}yfl054c were transformed with the centromeric vector pRS305 carrying the leu2 gene. All yeast strains were transformed with either an empty pYES2 (Invitrogen) as control or derivates of pYES2 carrying cDNAs for 24 different aquaporin homologues (Table 2). Yeast cells were grown on synthetic medium containing 2% galactose (SG), 50 mM succinic acid/Tris base, pH 5.5, 0.7% yeast nitrogen base without amino acids (Difco), 0.3% methionine (M), and 0.3% histidine (H). Yeast cells were diluted in sterile water to an A600 of 0.01, and 10 µl were spotted on solid SG-HM medium containing various concentrations of H2O2 (J. T. Baker Inc.) as indicated. After 6 days of incubation, differences in growth and survival were recorded. For Ag+ treatments, cells were spotted on solid SG-HM medium supplemented with either no or 0.1 mM H2O2 and various concentrations of AgNO3 (Merck). After 6 days of incubation, differences in growth and survival were recorded.


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  		TABLE 1
List of Saccharomyces cerevisiae strains used in this study Yeast strains used in this study differ in a number of essential oxidative defense enzymes or the presence of endogenous aquaporin homologues. Impaired oxidative defense should result in increased sensitivity to hydrogen peroxide in the growth medium. MIP, major intrinsic protein.

 


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  		TABLE 2
Aquaporins used in this study

 
Catalase Activity Assay—wt yeast transformed with pYES2 or pYES2 containing AtTIP1; 1, hAQP8, or hAQP1 were grown in yeast peptone galactose (YPG) washed twice in chilled distilled water, vortexed four times for 1 min with glass beads in a chilled extraction buffer (50 mM K2HPO4, pH 7.0), and centrifuged at 8000 x g at 4 °C for 15 min. The supernatants were used for the catalase activity assay. Catalase activity was determined as the decomposition of H2O2 measured as a decrease in absorbance at 240 nm (23). The reaction mixture contained 10 µg of yeast protein extract and 20 mM H2O2 corresponding to an absorbance A240 of 0.5–0.6. Specific catalase activity (µmol min-1 mg of protein-1) was calculated from {Delta}A240 {epsilon}(H2O2)-1 mg-1 where {Delta}A240 is the change in A240 over 60 s and {epsilon}(H2O2) is the millimolar extinction coefficient of H2O2 at 240 nm, 0.0436 mM-1 cm-1.

Fluorescence Assay—Yeast strain {Delta}yap1 transformed with pYES2 or pYES2 containing AtTIP1; 1, hAQP8, or hAQP1 were precultured for 2 days on solid synthetic medium. Two-milliliter liquid cultures were inoculated and supplemented with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA, Molecular Probes) from a 5 mM stock in Me2SO or ethanol to a final concentration of 1 µM. Cells were grown in darkness overnight to an A600 of 1.6, washed five times in 20 mM HEPES (pH 7.0), and finally suspended in HEPES buffer at an A600 of 1.4. Fluorescence of 2 ml of yeast suspension was followed over time with a spectrofluorometer (MOS-250, BioLogic) at excitation/emission of 492/527 nm and a temperature of 20 °C. For the calculation of the activation energy (Ea), temperatures were set to 10, 15, 20, 25, and 30 °C. Primary data were collected using BioKine software. For Ag+ inhibition assays, yeast cells were preincubated for 45 min in 30 µM AgNO3 in the dark and washed twice with HEPES buffer before measuring fluorescence.

Spheroplast Swelling Assay—{Delta}yap1 transformed with pYES2 or pYES2 containing AtTIP1;1, hAQP1,or hAQP8 were grown in 10 ml of YPG (complex medium) for 16 h at 30 °C. After centrifugation, cells were washed in 50 ml of 5 mM KH2PO4 (pH 7.5), resuspended in 10 ml of 5 mM KH2PO4 plus 20 µl of 2-mercaptoethanol (98%), and incubated for 30 min at 30 °C. Cells were centrifuged and resuspended in 2.4 M sorbitol, 5 mM KH2PO4 (pH 7.5) plus 60 units of lyticase (Sigma)/ml of buffer at an A600 of 1.0. The cells were incubated for 45 min at 30 °C. Following centrifugation, spheroplasts were washed once and finally resuspended in 10 mM Tris/MES, pH 6.0, 1 mM EDTA, 0.5 M sorbitol, and 0.4 M K2SO4 at an A475 of 2.0. Kinetics of spheroplast swelling were measured essentially as described previously (24). Volume changes were recorded at 10 °C as light scattering at an angle of 90° and 475 nm using a fast kinetics instrument (SFM-300, BioLogic) equipped with a spectrofluorometer (MOS-250, BioLogic). For Ag+ or H2O2 treatments, the final spheroplast suspensions were treated with AgNO3 at 30 µM for 10 min or 0.1 versus 0.75 mM H2O2 for 30 min before conducting the swelling assay. All data presented are averages of at least 12 trace recordings. The rate constant of the decrease of scattered light intensity is proportional to the water permeability coefficient (25, 26). Rate constants were calculated by fitting the curves using non-linear regression as described earlier (27). One-exponential functions were used for control spheroplasts, and two-exponential functions were used for aquaporin-expressing spheroplasts (27). The fitting curves were also used to calculate the half-maximal swelling time (t1/2).

Bioimaging—Dye-loaded yeast cells were placed on Novakemi AB 10-well microscope slides (Menzel) covered with poly-L-lysine allowing the yeast cells to attach. Excess cells were washed off with double distilled H2O. The slides were placed in a confocal microscope (Leica TCS SP2, Leica Microsystems) and monitored using a dipping lens (63x and numerical aperture of 0.9). Time lapse experiments in xyt mode with a line average of 2 and intervals of approximately 3 s between each frame were carried out. Total experiment time was about 5 min (93 frames). ROS development was monitored during experiments where the yeast cells were treated with 0.1 mM H2O2 using the 488 nm laser line with 3% laser power. To treat the yeast cells with H2O2, a peristaltic pump (P-3, GE Healthcare) was connected with the microscope stage to facilitate suction. H2O2 solution was added after about 30 s (10 frames).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Aquaporins Increase the Sensitivity of Yeast Cells to Externally Supplied H2O2—To test whether aquaporins are permeable to H2O2, five different yeast strains (S. cerevisiae, Table 1) were transformed with 24 different aquaporins from human, rat, Arabidopsis, and wheat (Table 2) or with an empty vector as control. Four of the different yeast strains differed in their sensitivity toward oxidative stress and, consequently, to externally supplied H2O2. The fifth yeast strain, {Delta}fps1,{Delta}yfl054c, used lacked all functional endogenous aquaporin homologues. Yeast strains were plated on synthetic medium with various concentrations of H2O2. Growth was recorded after 6 days at 30 °C.

In the absence of any heterologous aquaporin, addition of H2O2 decreased growth and survival of the different yeast strains to various degrees. wt, {Delta}tsa1,2, and {Delta}fps1,{Delta}yfl054c were able to grow in the presence of 0.75 mM H2O2, whereas growth of {Delta}skn7 and {Delta}yap1 was completely repressed at 0.75 mM H2O2 (Fig. 1). Stability of H2O2 during the growth period was tested by preincubating H2O2-containing medium for 6 days at 30 °C prior to the growth assay. Very similar growth responses were recorded with and without preincubation of the medium (data not shown).

Transformation with the majority of aquaporins did not significantly change the sensitivity of yeast cells toward increasing concentrations of H2O2 in the medium. Results from hAQP1 are shown as an example (Fig. 1). However, expression of hAQP8 and AtTIP1;1 markedly reduced growth and cell survival on medium containing H2O2 (Fig. 1). The same was seen when expressing AtTIP1;2 (data not shown). Following expression of hAQP8 and AtTIP1;1, growth of all yeast strains tested was repressed at a 2–3 times lower concentration of H2O2 compared with the empty vector controls. The strain {Delta}yap1 was again most sensitive followed by {Delta}skn7, {Delta}tsa1,2, and wt. Although for example the wt transformed with hAQP8 was able to grow on medium containing up to 0.5 mM H2O2, growth of {Delta}yap1 when transformed with hAQP8 was completely repressed at 0.3 mM H2O2. Transformants of {Delta}fps1,{Delta}yfl054c showed growth and cell survival comparable to the corresponding wild type transformants. Growth was repressed by addition of H2O2 at very similar concentrations. Fps1p is known to play a role in osmotic adaptation (28).

Increased sensitivity of hAQP8- and AtTIP1-expressing yeasts toward H2O2 in the medium could be the result of a decreased capacity to scavenge H2O2 inside the cells. Catalase is the most abundant ROS-scavenging enzyme; it directly decomposes H2O2 to water and molecular oxygen. To investigate whether heterologous expression of aquaporins resulted in a decreased scavenging capacity of the yeast cells, we measured catalase activity as the breakdown of H2O2 by diluted extracts from yeast cells transformed with either the empty vector pYES or pYES containing hAQP1, hAQP8, or AtTIP1;1 (Table 3). Expression of all three aquaporins resulted in either no change or an increase rather than a decrease of H2O2 scavenging capacity in the yeast extracts. Pretreatment with H2O2 caused a further increase in catalase activity. Therefore the increased sensitivity toward H2O2 in the external medium was not caused by a decrease in H2O2 scavenging of aquaporin-expressing yeasts. The reduced growth of yeast expressing AtTIP1;1 or hAQP8 was rather due to increased oxidative stress as the result of increased uptake of H2O2 from the external medium.


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  		TABLE 3
Specific catalase activity in extracts from yeast cells transformed with hAQP8, hAQP1, AtTIP1;1, or pYES as a control Shown is specific catalase activity (µmol min-1 mg-1) in yeast extracts from cells grown in YPG (n = 4) (A) and cells grown in YPG and supplemented with 0.1 mM H2O2 6 h before protein extraction (n = 2) (B). C, as in A but boiled for 10 min prior to the assay. Results are presented with ±S.E. with n = 4 for A and C and n = 2 for B. Activity calculated from the assay in the absence of yeast extract was calculated to -0.14 ± 0.55.

 


Figure 1
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  		FIGURE 1.
Yeast growth and survival test on medium containing H2O2. 10 µl of various yeast strains (wt, {Delta}tsa1,2, {Delta}skn7, {Delta}yap1, and {Delta}fps1,{Delta}yfl054c) transformed with the empty vector pYES2 (control (c)), hAQP8, hAQP1,or AtTIP1;1 at an A600 of 0.01 were spotted on agar plates containing various concentrations of H2O2. Growth was recorded after 6 days at 30 °C. All data were duplicated in at least two independent experiments with consistent results.

 
Silver Ions Abolish the Aquaporin-induced H2O2 Sensitivity of Yeast Cells—Ag+ has been introduced as an aquaporin inhibitor (29). Ag+ acts by binding to cysteine or histidine residues in proteins. Both hAQP8 and AtTIP1;1 contain cysteine residues in their pore region that have been identified as targets for inhibition by mercury when water permeability was investigated in oocyte swelling assays (30, 31). Ag+ is also a biocide with various negative effects on organisms. In the absence of H2O2, increasing concentrations of AgNO3 (up to 30 µM) resulted in a substantial decrease in growth and viability of cells irrespective of whether cells were transformed with empty vector, hAQP8, or AtTIP1;1 (data for {Delta}skn7, Fig. 2A). Toxicity became evident at about 6 µM Ag+, a concentration level that Niemietz and Tyerman (29) found most effective for inhibition of aquaporins. The control yeast (pYES2) showed a very similar growth repression at increasing Ag+ concentrations in the absence and presence of H2O2 (Fig. 2, A and B). Conversely yeast expressing hAQP8 and AtTIP1;1 were only able to grow in the presence of 6 µM Ag+ and higher (Fig. 2B). The results suggested that Ag+ blocks the diffusion of H2O2 through these aquaporins.


Figure 2
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  		FIGURE 2.
Yeast growth and survival on medium containing various concentrations of AgNO3 in the presence or absence of H2O2. {Delta}skn7 transformed with the empty vector pYES2 (control (c)), hAQP8,or AtTIP1;1 were spotted at an A600 of 0.01 on medium containing various concentrations of AgNO3 as indicated. A, without (w/o) addition of H2O2. B, plus 0.1 mM H2O2. Growth was recorded after 6 days. Data were duplicated in two independent experiments.

 
Interestingly the presence of 0.1 mM H2O2 slightly alleviated the growth repression induced by Ag+ in all yeast transformants tested (Fig. 2B). This may be due to a direct effect of H2O2 on Ag+. An alternative explanation is that a moderate concentration of H2O2 causes a stress-alert response in the yeast cells and consequently improves growth in the presence of the biocide Ag+.

Water Transport and Swelling of Yeast Spheroplasts—To confirm that the heterologous aquaporins were functionally expressed at the yeast plasma membrane and to investigate the effect of Ag+ and H2O2 on aquaporin function, we performed a water transport assay. Yeast spheroplasts were prepared and divided into three fractions, one of which was preincubated with Ag+, another was preincubated with H2O2, and the third was untreated control. After exposing the spheroplasts to hypoosmotic conditions (transfer from 400 to 200 mM K2SO4), fast initial swelling was observed with spheroplasts expressing AtTIP1;1, hAQP8, and hAQP1 compared with the empty vector control (Fig. 3). Pretreatment with 6 µM Ag+ had only marginal effects on swelling kinetics of aquaporin-expressing spheroplasts (data not shown). This may be due to the incubation with the reducing agent 2-mercaptoethanol during spheroplast preparation. At higher concentration (30 µM Ag+), both aqua-porin-expressing and control spheroplasts did not reach the same final volume as in the absence of Ag+ (data not shown), indicating some non-aquaporin-specific, toxic effects. However, pretreatment with 30 µM Ag+ substantially decreased the rate constant for AQP1- and TIP1;1-expressing spheroplasts but not for AQP8-expressing and control spheroplasts (Fig. 3B). Ag+ pretreatment also increased the time needed to reach half-maximal swelling (t1/2). This was seen for all aquaporin-expressing spheroplasts but not for the control (Fig. 3C). Together the results support the view that Ag+ acts as an aqua-porin blocker to inhibit water flux. Pretreatment with H2O2 did not change the swelling kinetics of the spheroplast regardless of the presence of the various aquaporins (Fig. 3, B and C).


Figure 3
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  		FIGURE 3.
Aquaporin-mediated osmotic water transport. Spheroplasts from yeast cells ({Delta}yap1) transformed with hAQP8, hAQP1, AtTIP1;1, or pYES2 as control were suspended in 0.5 M sorbitol plus 0.4 M potassium sulfate at an A475 of 2.0 and mixed in a fast kinetics instrument with an equal volume of 0.5 M sorbitol at 10 °C. The kinetics presented are averages of 12–15 trace recordings each over a time period of 6 s. The assay was performed either without further addition (A) or after preincubation in 0.75 mM H2O2 or 30 µM AgNO3 for 10 min. Based on the swelling kinetics the rate constants (k) and the half-maximal swelling time (t1/2) was calculated for all treatments, B and C, respectively.

 
Fluorescence Measurements—Growth inhibition following supplementation of the growth medium with H2O2 is a rather slow and indirect approach. To more directly follow H2O2 transport across the yeast plasma membrane, we developed a fluorescence-based assay. Yeast cells were grown in the presence of the dye CM-H2DCFDA, which has been used to monitor the level of ROS in various systems (3). In its acetylated form, the dye can diffuse into yeast cells. Deacetylation traps the fluorochrome inside the cells and makes it susceptible to oxidation by ROS. After culture overnight, yeast cells were washed five times, and fluorescence was monitored. To test whether all yeast transformants took up the dye, cells loaded with CM-H2DCFDA were upon washing made permeable with 1% Triton, and the supernatants were exposed to H2O2. In all cases, an increase in fluorescence was detected (data not shown).

In the absence of H2O2 in the buffer solution, none of the dye-loaded yeast cells ({Delta}yap1 transformed with either AtTIP1;1, hAQP1, hAQP8, or pYES) showed a significant increase in fluorescence even after 1800 s (not shown). Addition of 0.1 mM H2O2 to control or hAQP1-transformed yeast cells only marginally increased fluorescence (Fig. 4, A and B). However, addition of H2O2 to yeast cells expressing AtTIP1;1 and hAQP8 increased fluorescence significantly (Fig. 4). The same was seen for AtTIP1;2 (data not shown). When cells were removed from the assay medium by centrifugation, no fluorescence could be detected after addition of H2O2 to the assay supernatants (Fig. 5A). However, after resuspension with fresh assay buffer, yeast cells transformed with hAQP8 and AtTIP1;1 showed the increase in fluorescence upon addition of H2O2 (Fig. 5B) as in Fig. 4A. Removal of the cells by centrifugation after completion of the assay (addition of H2O2) also reduced the fluorescence signal from the assay medium (not shown). Together this demonstrates that the increase in fluorescence was not due to increased leakage of the fluorescent dye from the cells expressing hAQP8 and AtTIP1;1 but was rather caused by increased uptake of H2O2 into the cells. Fig. 4B shows the average relative increase of fluorescence during the first 100 s after addition of H2O2. Expression of AtTIP1;1 led to a higher increase in fluorescence than expression of hAQP8. The Arrhenius plot suggested a reduction in the activation energy (Ea) for the diffusion of H2O2 from 58 to about 42 kJ mol-1 (Fig. 4C). Preincubation of yeast cells for 45 min with 30 µM AgNO3 almost completely abolished the fluorescence increase upon addition of H2O2 (Fig. 4, A and B). In an in vitro control experiment, preincubation of CM-H2DCFDA with 30 µM AgNO3 produced an almost identical fluorescence signal upon addition of peroxidase, esterase, and 0.1 mM H2O2 as the dye in the absence of Ag+ (data not shown). Thus, the reduction of the fluorescence increase upon addition of AgNO3 in vivo was not due to a direct effect of AgNO3 on the dye.

Some metal ions (e.g. iron and copper ions) can react with H2O2 to produce hydroxyl radicals. To investigate whether Ag+ could perform a similar reaction, 100 mM H2O2 was pre-treated with 100 mM FeCl2, CuSO4, or AgNO3 overnight and subsequently used in the fluorescence assay. Incubation with either iron or copper ions led to a visible gas development (Fig. 6A), whereas incubation with Ag+ did not. After overnight incubation, H2O2 pretreated with iron or copper ions did not result in an increase in fluorescence when added to dye-loaded yeast cells expressing AtTIP1;1. However, H2O2 pretreated with Ag+ was still able to evoke an increase in fluorescence (Fig. 6B). Preincubation of cells with 30 µM Ag+ as used in Fig. 4, A and B, led to a slower fluorescence increase (Fig. 6B). The results demonstrate that Ag+ does not react with H2O2 even at 100 mM (for both H2O2 and Ag+) and that a preincubation of cells with Ag+ is needed to achieve inhibition of AtTIP1;1.


Figure 4
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  		FIGURE 4.
Aquaporin-mediated H2O2 transport across yeast membranes as measured using the ROS-sensitive fluorescent dye CM-H2DCFDA. Fluorescence (excitation, 492 nm; emission, 527 nm) of CM-H2DCFDA-loaded

{Delta}yap1 yeast cells expressing hAQP8, AtTIP1;1,or hAQP1 was recorded over time. {Delta}yap1 transformed with the empty vector pYES2 was used as control. A, the assay was performed either without further addition or after preincubation with 30 µM AgNO3. B, bar diagram showing the average increase in fluorescence after 100 s. White bars, control (n = 15); black bars, after addition of H2O2 (n = 15); gray bars, after preincubation with 30 µM AgNO3 (n = 2). C, Arrhenius plot. The natural logarithm (ln) of the rate of H2O2 flux into {Delta}yap1 transformed with pYES2 (control) or AtTIP1;1, measured as the increase in fluorescence of CM-H2DCFDA within the first 20 s, is plotted against the reciprocal of temperature in Kelvin (n = 4). The activation energies (Ea) were calculated from the linear regressions. Values are shown ±S.E.

 


Figure 5
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  		FIGURE 5.
H2O2-dependent fluorescence increase in cultures of yeast expressing AtTIP1;1 and hAQP8 is associated with the cells and does not result from increased leakage of the dye from aquaporin-expressing yeast cells to the medium. CM-H2DCFDA-loaded {Delta}yap1 yeast transformed with AtTIP1;1, hAQP8, hAQP1, or pYES2 (control) were preincubated in the assay medium and subsequently pelleted by centrifugation. Cells were resus-pended in fresh assay medium, and both the supernatants (A) and the resus-pended cells (B) were monitored to measure fluorescence after addition of 0.1 mM H2O2.

 
The Uptake of H2O2 by Single Yeast Cells Can Be Followed by Confocal Microscopy—Yeast cells transformed with either pYES2 or pYES2 containing hAQP1, hAQP8,or AtTIP1;1 were preloaded with CM-H2DCFDA, washed, and attached to poly-L-lysine-coated microscopy slides. CM-H2DCFDA was developed as a dye with an improved retention within the cytoplasm of cells compared with previous derivatives (32). We observed that not all cells fluoresced when loaded with CM-H2DCFDA overnight (Fig. 7A). In the fluorescing cells, however, the signal was clearly detected in the cytoplasm and the region closely associated with the nucleus but not in vacuoles (Fig. 7, A and B). Single cells from all transformants with comparable fluorescence signal were observed to quantify fluorescence changes upon addition of 0.1 mM H2O2. Fluorescence increased severalfold in cells transformed with hAQP8 and AtTIP1;1 but not in cells transformed with hAQP1 or the empty vector pYES2 (Fig. 7 C).


Figure 6
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  		FIGURE 6.
Effect of iron, copper, and silver ions on the stability of H2O2. A, gas formation in the reaction between 100 mM H2O2 and 100 mM FeCl2 (a), CuSO4 (b), or AgNO3 (c) after 4 h of incubation. B, aliquots of the H2O2 solutions pretreated with the various metal ions from A were added to CM-H2DCFDA-loaded {Delta}yap1 yeast expressing AtTIP1;1, and fluorescence was monitored. As additional control, {Delta}yap1 expressing AtTIP1;1 was pretreated for 30 min with 30 µM AgNO3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present work, a broad screen was conducted to search for potential aquaporin isoforms with the capacity to facilitate diffusion of H2O2. Members of all four plant aquaporin subfamilies as well as aquaporins and aquaglyceroporins from mammals were included in the survey. The study made use of yeast as a simple and well studied heterologous expression system. Oxidative stress response in yeast is well characterized, and various mutants are available that differ in their sensitivity to oxidative stress and, consequently, to externally supplied H2O2. Tsa1p and Tsa2p are thiol peroxidases, which are known to eliminate H2O2 and alkyl hydroperoxides by using thiol-reducing equivalents (33, 34). {Delta}tsa1,2 has been shown earlier to be more sensitive toward H2O2 when compared with wt (24). Yap1p and Skn7p are two transcription factors, each regulating the expression of a number of oxidative stress response genes (35, 36). Both transcription factors activate the expression of defense genes coding for enzymes like catalase, superoxide dismutase, alkyl hydroperoxide reductase, thioredoxins, thiol peroxidases (including Tsa1p and Tsa2p), cytochrome c peroxidase, and heat shock proteins. Accordingly exposing the different strains to increasing concentrations of H2O2 resulted in decreasing growth and cell survival in the order wt > {Delta}tsa1,2 >{Delta}skn7 {approx}{Delta}yap1 with wt being most resistant toward H2O2 (Fig. 1).


Figure 7
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  		FIGURE 7.
Addition of H2O2 to CM-H2DCFDA-loaded yeast cells expressing AtTIP1;1 and hAQP8 results in increased fluorescence in the cytoplasm. A, yeast cells transformed with AtTIP1;1 and preloaded with CM-H2DCFDA visualized in fluorescence mode (left) and Nomarski contrast (right). B, a yeast cell expressing AtTIP1;1 before (left) and after (right) the addition of 0.1 mM H2O2. B is shown in pseudocolor (blue = pixel saturation), Scale bar,2 µm. C, using confocal microscopy, single yeast cells were observed, and fluorescence was monitored over time. Graphs are presented with ±S.E. with n = 5 for all strains. w/o, without.

 
The genome of S. cerevisiae contains four open reading frames for major intrinsic proteins: aqy1, aqy2, and fps1 and its homologue yfl054c. Open reading frames aqy1 and aqy2 are represented by two alleles, called aqy1-1/aqy1-2 and aqy2-1/aqy2-2, respectively. Open reading frames aqy1-2 and aqy2-2 are present in most laboratory strains including BY4741 and have been shown to be non-functional (24, 37, 39). Therefore, deletion of fps1 and yfl054c produced a yeast strain devoid of any functional aquaporin. This mutant was used to test whether the endogenous yeast aquaporins Fps1p and YFL054cp could facilitate diffusion of H2O2. Wild type and {Delta}fps1,{Delta}yfl054c displayed very similar sensitivity toward H2O2 that was independent of the expression of hAQP8 and AtTIP1 (Fig. 1). Notably expression of hAQP8 and AtTIP1 increased sensitivity to H2O2 as in other yeast strains. Based on this result it can be ruled out that the increased sensitivity toward H2O2 was due to oligomerization with yeast endogenous aquaporins that potentially could have changed the transport capacity of the heterologous genes under investigation. Hetero-oligomerization has been shown to change transport capacity of PIP1 isoforms from plants (40).

The cellular concentration of H2O2 is the result of both diffusion across the plasma membrane and scavenging inside the cells. To test whether a decrease in ROS scavenging of aqua-porin-expressing yeast cells could be the reason for growth repression in the presence of H2O2, we used mutants defective in various key regulators of oxidative stress response in yeast. {Delta}skn7 and {Delta}yap1 were generally more sensitive to H2O2, and the expression of hAQP8 and AtTIP1 additionally increased sensitivity (Fig. 1). We also measured catalase activity as the breakdown of H2O2 from extracts of the various yeast transformants (Table 3). The results showed that expression of aqua-porins did not lead to a reduction of catalase activity. Thus, it is concluded that hAQP8 and AtTIP1 increased the capacity for the diffusion of H2O2 into yeast cells.

The activation energy (Ea) required for H2O2 to cross the yeast plasma membrane in the absence of heterologous aqua-porins was 58 kJ mol-1 (Fig. 4C). Mathai and Sitaramam (41) calculated the Ea for H2O2 in artificial liposomes to be between 26 and 39 kJ mol-1 depending on the lipid species. The specific composition of yeast membranes may explain a higher Ea. Our values for H2O2 compare well with values calculated for water diffusion into yeast secretory vesicles (11), membranes that probably more closely match the yeast plasma membrane in composition. For yeast cells expressing AtTIP1;1, a reduction of the Ea of about 25% for passive diffusion of H2O2 across the membrane was calculated (Fig. 4).

Recent work suggested that water channels could be regulated via oxidative gating (42-44). H2O2 lowered the hydraulic conductivity of whole roots as well as single cells of maize and Chara. Two alternative gating mechanisms were proposed: (i) hydroxyl radical or other ROS directly oxidize and thereby inhibit AQPs or (ii) H2O2 acts as a signaling molecule activating a pathway that ultimately leads to channel closure. In plasma membranes isolated from pea seedlings, addition of reducing agents dithiothreitol and tributylphosphine reduced the rate constant of vesicle swelling while increasing the content of reduced SH groups in the plasma membrane proteins (44). Addition of the protein-oxidizing agent diamide had the opposite effects (44). These results suggest a direct modification and regulation by reversible oxidation of aquaporins. Our results suggest that selected aquaporins channel H2O2 and that hAQP1 was not inhibited by H2O2 when analyzed in a spheroplast swelling assay (Fig. 3). The possibility that other aquaporins are gated by H2O2 or other ROS, however, cannot be excluded. More research is needed to investigate the role of redox regulation of aquaporins in vivo.

The aromatic Arg constriction region represented by Phe58, His182, Cys191, and Arg197 in hAQP1 is known as one selectivity filter for aquaporin homologues. Substitutions within this region have been shown to be at least in part responsible for the substrate specificity in a number of aquaporins (45-47). Interestingly hAQP8, AtTIP1;1, and AtTIP1;2 share three substitutions in this critical region (F58H, H182I, and C191G), suggesting that indeed these substitutions may classify a new subgroup of H2O2-channeling aquaporins. However, TIP2;1 possesses the same substitutions as hAQP8 but failed to increase sensitivity of yeast on medium containing H2O2 (data not shown). TIP2;1 is known to be functionally expressed at the yeast plasma membrane (45). Therefore, additional structural features must be important for diffusion of H2O2 through aquaporins.

Here yeast was used as a heterologous expression system. The validity of this system requires that at least some aquaporin molecules are functionally expressed at the yeast plasma membrane. This was clearly the case for hAQP1, hAQP8, and AtTIP1 as demonstrated by increased water permeability of the yeast plasma membrane upon expression of the respective heterologous genes (Fig. 3). However, other potential H2O2-transporting aquaporins may have escaped identification due to lack of functional expression at the plasma membrane. In addition, the plasma membrane of yeast may be more permeable for H2O2 than membranes of homologous systems. Therefore expression of other aquaporins with relatively lower capacity to channel H2O2 may not have increased the uptake of H2O2 above that based on simple diffusion across the lipid bilayer of the yeast plasma membrane.

Catalases form non-membrane channels permitting the passage of water and hydrogen peroxide. Catalases are ubiquitous antioxidant enzymes disproportionating H2O2 to water and oxygen with an extraordinarily high turnover (48). Within each monomer, the active heme site is deeply buried inside the protein, and the substrate H2O2 gains access to the active site through the so-called main channel, a 30–50-Å-long path, in a proposed partly single file (49). The amino acids lining the narrow channel create an environment to favor H2O2 while excluding other small solutes from the hidden active site. Simulation experiments showed that besides H2O2 also water readily enters the channel (50). Only the constriction region constituting the selectivity filter just before the active center favors H2O2 compared with water. The well described example of catalase demonstrates that there is a mechanism to discriminate H2O2 from water despite their close similarity. Our data and the finding that H2O2 transport was specific for hAQP8 and AtTIP1 suggest that a similar selectivity exist in aquaporins. However, water remains the common substrate. It will be relevant to investigate whether aquaporins can change their selectivity by changes in their conformation.

In mammals, AQP8 is present in the plasma membrane (51) and has recently been localized to the inner mitochondrial membrane (26). AQP8 may have a function in releasing H2O2 from the mitochondrial matrix in situations when the electron transport chain is highly reduced, conditions known to generate ROS (1). For AQP8 knock-out mice, however, no phenotype has yet been reported that could demonstrate a unique and important role of AQP8 in respiratory function (52, 53). In the plasma membrane, AQP8 and other potential H2O2-channeling aquaporins may play a role in cell-to-cell signaling.

In plants, down-regulation of TIP1;1 by RNA interference technology in Arabidopsis resulted in a strong and complex phenotype of early senescence and death (54). F1 mutants were classified according to the level of expression of the RNA interference construct, which correlated well with the severity of the phenotype. The various classes of mutants were analyzed compared with wt for water status and metabolites as well as global expression by microarray analysis. Affected transcripts were grouped in categories of defense, redox control, signaling, and carbon metabolism followed by others. Notably the water status seemed to be unaffected by down-regulation of TIP1;1.At the metabolic level, mutants accumulated starch and showed changed sugar content. Based on their results, Ma et al. (54) suggested that TIP1;1 may have a role in vesicle trafficking to the central vacuole and vesicle-based metabolite routing in the cell. Thus, they suggested a unique function of TIP1;1. Our data offer a new explanation for the severe phenotype described for these mutant plants. Genes within the functional categories of defense, redox control, and signaling directly relate to ROS and oxidative stress, indicating that an imbalance in ROS and consequently oxidative stress preceded transcriptional regulation. The disturbance in carbon metabolism and storage potentially relates to a cross-talk between soluble sugars and ROS as demonstrated in numerous metabolic and stress transduction pathways (for a review, see Ref. 55) and by the direct redox modification of key enzymes of carbon fixation and starch metabolism (for a review, see Ref. 56). TIP1 is highly expressed in cells surrounding developing vascular tissues in maize roots (57). High levels of ROS including H2O2 have been detected in the same tissues and have been implicated in cell wall loosening and elongation growth (58). TIP1;1 may have a function in the regulation of the level of H2O2 during xylem formation, which is likely to have a strong impact on the development of the whole plant via impaired long distance transport. Further analysis of plants with changed expression of TIP1;1 should help to resolve these potential roles.

In its dual role as an indispensable signal molecule and a potential threat for biological components (38), H2O2 plays the double role as "Dr. Jekyll and Mr. Hyde." With the current study, we provide evidence for a new membrane transport mechanism for this paradox player. The possibility to specifically increase diffusion of H2O2 via aquaporins potentially allows a fine regulation of membrane permeability. This knowledge will further the understanding of the dual role of H2O2 in biological systems.


   FOOTNOTES
 
* This work was supported by Ministry of Science, Technology and Innovation Grant 23-03-0103 (to T. P. J). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Plant and Soil Science Laboratory, Dept. of Agricultural Sciences, Faculty of Life Science, Copenhagen University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark. Tel.: 45-3528-3484; Fax: 45-3528-3460; E-mail: tpj{at}kvl.dk.

2 The abbreviations used are: H2O2, hydrogen peroxide; AQP, aquaporin; CM-H2DCFDA, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester; ROS, reactive oxygen species; TIP, tonoplast intrinsic protein; wt, wild type; MES, 4-morpholineethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Dong Yan Jin for the generous gift of the yeast {Delta}tsa1,2 strain.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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HvPIP1;6, a Barley (Hordeum vulgare L.) Plasma Membrane Water Channel Particularly Expressed in Growing Compared with Non-Growing Leaf Tissues
Plant Cell Physiol., August 1, 2007; 48(8): 1132 - 1147.
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 Plant Physiol.Home page
G. Miller, N. Suzuki, L. Rizhsky, A. Hegie, S. Koussevitzky, and R. Mittler
Double Mutants Deficient in Cytosolic and Thylakoid Ascorbate Peroxidase Reveal a Complex Mode of Interaction between Reactive Oxygen Species, Plant Development, and Response to Abiotic Stresses
Plant Physiology, August 1, 2007; 144(4): 1777 - 1785.
[Abstract] [Full Text] [PDF]

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 J. Am. Soc. Nephrol.Home page
P.-X. Wang and P. W. Sanders
Immunoglobulin Light Chains Generate Hydrogen Peroxide
J. Am. Soc. Nephrol., April 1, 2007; 18(4): 1239 - 1245.
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