Introduction

Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) that is generated during diverse metabolic processes, and has an important role in biological systems. As an oxidant it directly affects intracellular redox systems, can initiate lipid peroxidation, degrade nucleic acids and inactivate enzymes, and, therefore, has the potential to affect physiological functions and cause cell damage or even death (Halliwell and Gutteridge 1999). At relatively low concentrations, H2O2 appears to perform a function in signal transduction processes within biological systems. It can influence gene expression and activate or modify redox-sensitive transcription factors, and thereby trigger tolerance to environmental stress (Orozco-Cárdenas et al. 2001; Laloi et al. 2004; Miller and Mittler 2006). Hydrogen peroxide has been implied to act as an intercellular messenger through activation and/or modulation of cellular antioxidants and disease resistance genes in plants (Levine et al. 1994; Chappell et al. 1997; Hu et al. 2005), and during xylem differentiation and lignification processes (Bienert et al. 2006). Signal transduction processes are only effective if rapid transport of the intercellular messenger molecule across the plasma membrane can be achieved. Movement of H2O2 across the plasma membrane of the signal-producing as well as the signal-perceiving cell by diffusion is slow, and many membranes are poorly permeable to H2O2 (Bienert et al. 2006). Efficient transport of H2O2 across plasma membranes could be greatly increased by specific channel proteins. Indeed, experimental evidence suggests that certain aquaporins (AQPs) act as peroxoporins and thus facilitate the diffusion of H2O2 across biological membranes (Henzler and Steudle 2000; Bienert et al. 2007; Dynowski et al. 2008).

Aquaporins are membrane channels that belong to the major intrinsic protein (MIP) super family. These integral membrane proteins have highly conserved regions, and facilitate the transport not only of water but also other substrates, such as glycerol, ammonia, boric acid, carbon dioxide, nitric oxide, and H2O2 (Tyerman et al. 2002; Wu and Beitz 2007). Arabidopsis thaliana has 35 AQP isoforms which can be divided into four distinct groups based on sequence homology and subcellular location (Chaumont et al. 2005; Maurel 2007). A recent study investigated the involvement of several AQPs of Arabidopsis in H2O2 transport, and confirmed that several tonoplast intrinsic proteins (TIP1;1, TIP1;2, and TIP2;3) are permeable for H2O2 in yeast cells (Bienert et al. 2007; Dynowski et al. 2008). Despite the fact that the importance of an intercellular messenger like H2O2 to be quickly transported across plasma membranes is unquestionable, reports demonstrating the permeability of aquaporins of the plasma membrane intrinsic protein (PIP) family toward H2O2 are severely limited.

This study aims to analyze the H2O2-regulated expression patterns of plasma membrane AQPs of A. thaliana (AtPIPs), and to determine their possible involvement in H2O2 transport. The AtPIPs are divided into two subgroups, AtPIP1 and AtPIP2 with 5 and 8 isoforms, respectively (Johanson et al. 2001). Heterologous expression studies of plant aquaporins revealed that PIP2 is mainly involved in water transport, whereas PIP1 facilitates the diffusion of small electrolytes and gases as well as water (Kaldenhoff and Fischer 2006). Here, we examine H2O2-responsive expression patterns of thirteen AtPIP genes in Arabidopsis, and determine the permeability of AtPIPs to H2O2 in yeast cells.

Materials and methods

Plant materials and growth conditions

Seeds of A. thaliana (Columbia ecotype) were placed on water saturated rock wool and held at 4°C in darkness for 3 days for stratification. The seeds were germinated in a growth chamber at 23°C with 12 h-day/12 h-night cycle. Light was provided at an irradiance of 100 μmol m−2 s−1 by fluorescence tubes (FL40EX-D 40W, Eaglite). Three days after germination, the seedlings were transferred to an aerated hydroponic solution (Cooper 1975). To determine the effect of H2O2 on AtPIPs expression, three-week-old Arabidopsis plants were placed into distilled water (control) or exposed to exogenous H2O2 concentrations of 1 mM for 0.5, 1, 2, or 4 h.

Quantitative real-time RT-PCR

The expression patterns of endogenous AtPIPs in response to H2O2 were examined by quantitative real-time RT-PCR. Total RNA was extracted from roots and leaves using an RNeasy extraction kit (Qiagen). Real-time RT-PCR was performed in a Rotor-Gene 2000 (Corbett Research) using a QuantiTect SYBR Green RT-PCR kit (Qiagen), essentially as described by Jang et al. (2004). Three independent experiments with subsequent RNA extractions were used as replicates. The expression levels of AtPIPs at each time point after H2O2 treatment were compared with that of the untreated-control sample. The expression level of actin was used as a reference.

Gene cloning and yeast transformation

The coding regions of AtPIPs were first cloned into a pET-22b(+) vector (Novagen) to tag the protein with 6 histidine residues at the C-terminal end for later protein expression analysis. The insert was then cut out of the vector by enzymatic digestion, and finally ligated into pYES2 (Invitrogen), which was used as an expression vector for Saccharomyces cerevisiae. Two yeast strains were used in this study; the JC0176 mutant strain in which two AQPs (aqy1 and aqy2) have been deleted (Carbrey et al. 2001; Meyrial et al. 2001) and the Δskn7 mutant strain which has an impaired oxidative stress defense response and is sensitive to H2O2 (Bienert et al. 2007). The yeast strains were transformed with AtPIPs using the lithium acetate/single stranded carrier DNA/PEG method (Gietz and Schiestl 2007). Transformants were selected by colony PCR after 4 days incubation at 30°C on synthetic drop-out (SD) medium (Sigma) containing uracil and histidine as selective markers.

Analysis of RNA and protein expression in yeast

RNA expression of AtPIPs in yeast was analyzed by RT-PCR and protein expression by western blotting. The expression of AtPIPs was induced by growing the yeast cells at 30°C on SD medium containing 2% galactose (SG) as a carbon source. For RNA extraction, the yeast cells were disrupted mechanically with 0.5 mm glass beads using a mini bead-beater (BioSpec Products Inc.) at 4°C with five 1 min cycles and subsequent 1 min cooling intervals on ice. Total RNA was extracted using an RNeasy extraction kit (Qiagen), and the expression of AtPIPs in yeast was determined via RT-PCR using an Omniscript RT kit (Qiagen) with gene specific primers. RNA transcripts were loaded onto a 1% agarose gel and visualized after staining with ethidium bromide. In addition, the expression of His-tagged AtPIPs in yeast was analyzed by western blot analysis. For protein extraction, the yeast cells were suspended in protein sample buffer (45 mM Tris, pH 6.8, 10% glycerol, 1% sodium dodecyl sulfate, 50 mM dithiothreitol, 0.01% bromophenol blue). To denature highly hydrophobic proteins, the suspension was incubated at 55°C for 1 h, which prevents precipitation and multimerization of proteins with multiple transmembrane domains (Sambrook and Russell 2001). The proteins were then separated by 12% SDS-PAGE and the gel was transferred to an Immobilon-P membrane (Millipore) using a tank-blotting procedure (Qiagen). Immunodetection with penta-His HRP conjugates was used for the detection of AQP according to the manufacturer’s protocol (Qiagen). After the final washing step, the membrane was incubated in WEST-ZOL plus (iNtRON Biotechnology), and the proteins on the membrane were visualized by chemiluminescence (ECL, Amersham Pharmacia Biotech).

Growth and survival assay of yeast cells in response to H2O2

The sensitivity of AtPIP transformed yeast cells to H2O2 was examined by a growth and survival assay on the two mutant yeast strains. For the JC0176 mutant yeast strain in which the two endogenous AQPs (aqy1 and aqy2) have been deleted (Meyrial et al. 2001), transgenic yeast cells harbouring pYES2:AtPIP or an empty pYES2 vector (control) were grown at 30°C in liquid SG, and the OD600 was adjusted to 1.0. The cells were then serially diluted, and 10 μL of cells were spotted on solid SG medium without or with H2O2. For the Δskn7 mutant yeast strain, previously used by Bienert et al. (2007), transformants harbouring pYES2:AtPIP or an empty pYES2 vector (control) were pre-cultured on solid SG medium for 2 days, and fresh yeast cells were diluted in 0.5 ml sterile water and then streaked on solid SG medium without or with H2O2. The plates were incubated at 30°C for 3–5 days, and the growth and survival of the cells was recorded.

Results and discussion

The effect of H2O2 on the endogenous expression levels of AtPIPs in leaves and roots of A. thaliana

To determine whether exogenously applied H2O2 affects the expression of AtPIPs in Arabidopsis, the transcript level of each AtPIP was measured via real-time RT-PCR analysis. To compensate for any circadian effects on AtPIP gene expression, the expression levels of AtPIPs at each time point after H2O2 treatment were compared with that of untreated-control samples. A hydrogen peroxide treatment of 1 mM over the course of 4 h did not greatly affect the endogenous expression levels of AtPIP1 in the leaves and roots of Arabidopsis (Fig. 1), although the expression of AtPIP1;3 in the leaves was slightly upregulated, especially during the first hour of H2O2 treatment. Hydrogen peroxide treatment did not affect the expression of AtPIP2 genes in the leaves. By contrast, AtPIP2 genes in the roots were generally downregulated during the course of H2O2 treatment. The expression of AtPIP2;1 and AtPIP2;8 decreased during the first hour of H2O2 treatment and then returned to the pre-treated values. The expression of AtPIP2;2, AtPIP2;3, AtPIP2;4, AtPIP2;5 and AtPIP2;7 decreased during the entire period of H2O2 treatment. GENEVESTIGATOR (http://www.genevestigator.com) analysis of microarray data on the transcript levels of AtPIPs in Arabidopsis also revealed that the expression of AtPIP2;1, AtPIP2;2, AtPIP2;3, AtPIP2;4, AtPIP2;5 and AtPIP2;7 decreased slightly at 1 h of H2O2 treatment (data not shown), which supports the results of our current quantitative real-time RT-PCR analysis. Although we do not presently know the physiological significance of different regulation of AtPIPs expression by H2O2, it is noteworthy that permeability of AtPIPs for H2O2 appears to be exclusive to certain members of the AtPIP2 subfamily only (see below). The transcript levels of each AtPIP in Arabidopsis have been determined in previous report, and the 13 AtPIP genes are classified into three groups based on their expression levels; the highest, intermediate, and low expression groups have the copy numbers in the range of 5,000–7,000 copies, 2,000–5,000 copies, and 500–2,000 copies per nanogram of total RNA, respectively (Jang et al. 2004). The high expression group includes PIP1;1, PIP1;2, and PIP2;7, the intermediate expression group includes PIP1;3, PIP1;5, PIP2;2, and PIP2;3, and the low expression group consists of PIP1;4, PIP2;1, PIP2;4, PIP2;5, PIP2;6, and PIP2;8 (Jang et al., 2004). The AtPIP2;2, AtPIP2;3, AtPIP2;4, AtPIP2;5 and AtPIP2;7, the expression of which is modulated by H2O2 treatment, belong to all three groups, indicating that the absolute transcript levels of AtPIPs are not directly related to H2O2 responsiveness. It seems that Arabidopsis responds mainly to the signalling molecule H2O2 by regulating the expression of AtPIP2 family members. It has been reported that exogenously applied H2O2 to the roots of cucumber, wheat, and maize decreased the hydraulic conductivity (Ktitorova et al. 2002; Lee et al. 2004; Aroca et al. 2005), and this reduction of water transport into the root cells could well be an effect of genetic down-regulation of root AQPs. The expression of AQPs in Arabidopsis is tissue specific, and not only depends on the developmental stage of the plants but can also be affected by environmental signals such as cold, drought, salt, osmotic stress, and ABA (Jang et al. 2004; Kaldenhoff and Fischer 2006). Our current results directly demonstrate that H2O2 is another environmental factor that regulates the expression of AQPs in plants.

Fig. 1
figure 1

The effect of H2O2 on the endogenous expression levels of AtPIPs in the leaves and roots of A. thaliana. Hydrogen peroxide (1 mM) was applied for 0.5, 1, 2, and 4 h, and the expression levels of each AQPs were plotted relative to non-treated control plants. Actin was used as a reference to show that equal amounts of RNA were present in the samples. Values are means ± SE (n = 6). Asterisks above the columns indicate values that are statistically different from control values (P < 0.05). The numbers 1;1, 1;2, 1;3, etc., in the figure represent AtPIP1;1, AtPIP1;2, AtPIP1;3, etc., respectively

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AtPIP expression in yeast cells

To examine whether AtPIPs possess the ability to transport H2O2 across the plasma membrane, each AtPIP gene was expressed in the JC0176 mutant yeast strain in which the two endogenous AQPs (aqy1 and aqy2) have been deleted (Meyrial et al. 2001). We first determined the expression of each AtPIP gene in the yeast cells at both mRNA and protein levels. Yeast cells expressing each AtPIP were investigated for the expression levels of AtPIPs, and Supplementary Fig. S1 shows the results of representative experiments including AtPIP1;1, 1;5, 2;1, 2;7, and 2;8. When the yeast cells transformed with each AtPIP gene from Arabidopsis were grown in synthetic drop-out medium containing the inducer galactose (gal), the expression levels of AtPIPs transcripts were similar to each other (Supplementary Fig. S1a). In contrast, when the yeast cells transformed with AtPIP2;1 were grown in a medium containing glucose (glu) instead of galactose, no AtPIP2;1 transcript was detected in the cells. The absence of PIP2;1 expression in the medium with glucose as a carbon source clearly indicates that the Arabidopsis AtPIP genes are induced in the yeast cells by the addition of galactose. With the observation that the transcript levels of AtPIP genes are similar to each other, we then investigated the expression of AtPIP at protein level in the yeast cells. The total proteins in the cellular extract were separated by 12% SDS-PAGE, and the expression levels of AtPIP proteins were determined by western analysis with an anti-His antibody. Yeast cells expressing either AtPIP1;4, 2;1, or 2;6 were selected for H2O2-insensitive cells and yeast cells expressing either AtPIP2;2, 2;4, or 2;5 were selected for H2O2-sensitive cells, and the expression levels of AtPIP proteins were analyzed by western analysis. The protein levels of AtPIP in the yeast cells are comparable with each other (Supplementary Fig. S1b), further indicating that the Arabidopsis AtPIPs are expressed in a similar amount in the yeast cells. Since the His-tag was fused in-frame at the C-terminal end of the protein, these results also indicate that the full-length AtPIPs are successfully translated in the yeast cells.

Sensitivity of AtPIP-expressing yeast cells to H2O2

To examine whether AtPIPs possess the ability to transport H2O2 across the plasma membrane, the growth and survival of two mutant yeast strains expressing each AtPIP gene was analyzed on solid SG medium containing 0.1–1 mM H2O2. Similar growth patterns were observed at different H2O2 concentrations, and the results showing most striking differences (1 mM H2O2 for JC0176 strain and 0.5 mM H2O2 for Δskn7 strain) are presented in Figs. 2 and 3. We first employed the JC0176 strain in which two native AQPs (aqy1 and aqy2) have been deleted (Carbrey et al. 2001; Meyrial et al. 2001). The yeast cells harbouring AtPIPs or an empty pYES2 expression vector grew well on SG medium without H2O2 with no noticeable difference (Fig. 2a). Similarly, the yeast cells harbouring any member of the AtPIP1 subfamily grew well on SG medium containing 1 mM H2O2 and thus did not show increased sensitivity towards H2O2. However, the growth and survival of yeast cells expressing AtPIP2;2, AtPIP2;4, AtPIP2;5, or AtPIP2;7 was markedly reduced on H2O2 containing medium when compared to the empty vector control (Fig. 2a), whereas the yeast cells harbouring AtPIP2;1, AtPIP2;3, AtPIP2;6, or AtPIP2;8 grew well in the presence of 1 mM H2O2. This increase in sensitivity to H2O2 disappeared completely when the cells were grown on SD medium containing glucose as a carbon source (Fig. 2b), indicating that the decreased growth of the yeast cells is due to the expression of AtPIPs in the yeast cells. These results indicate that AtPIP2;2, AtPIP2;4, AtPIP2;5, and AtPIP2;7 are permeable for H2O2. Similar results were observed in the analysis of the second mutant yeast strain (Δskn7) that has a defective oxidative stress defense response and is sensitive to H2O2 (Bienert et al. 2007).The yeast cells harbouring any member of the AtPIP1 subfamily did not show any increased sensitivity towards H2O2, whereas the growth and survival of yeast cells expressing AtPIP2;2, AtPIP2;4, AtPIP2;5, or AtPIP2;7 was markedly reduced on H2O2 containing medium when compared to the empty vector control (Fig. 3). Increased sensitivity of these AtPIP2-expressing yeast cells towards H2O2 in the medium could be the result of a decreased capacity to scavenge H2O2 inside the mutant yeast cells. This increase in sensitivity to H2O2 disappeared completely when the cells were grown on SD medium containing glucose as a carbon source (data not shown), indicating that the decreased growth of the yeast cells is due to the expression of AtPIPs in the yeast cells. These results further support the above observation that AtPIP2;2, AtPIP2;4, AtPIP2;5, and AtPIP2;7 are permeable for H2O2.

Fig. 2
figure 2

The effect of H2O2 on the growth and survival of JC0176 mutant yeast cells expressing AtPIPs. a Yeast was transformed with either an empty pYES2 vector (control) or an AtPIP gene, and 10 μL of serially diluted yeast cells were spotted on galactose medium (SG) without or with 1 mM H2O2. b As control experiment, transgenic yeast cells were spotted on glucose medium (SD) without or with 1 mM H2O2. The plates were photographed after 5 days incubation at 30°C. The experiments were repeated at least three times with consistent results

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Fig. 3
figure 3

The effect of H2O2 on the growth and survival of Δskn7 mutant yeast cells expressing AtPIPs. Yeast cells transformed with either an empty pYES2 vector (control) or an AtPIP gene were pre-cultured on galactose medium (SG), and fresh yeast cells were streaked on SG medium without or with 0.5 mM H2O2. The plates were photographed after 3 days incubation at 30°C. The experiments were repeated at least three times with consistent results

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This study supports previous reports which suggest that only certain AQPs are selectively permeable for H2O2 (Henzler and Steudle 2000; Bienert et al. 2007; Dynowski et al. 2008). However, contrary to the report by Dynowski et al. (2008) who showed permeability of AtPIP2;1 for H2O2, our analysis demonstrates that AtPIP2;1 is not permeable for H2O2. This discrepancy may arise from the fact that different yeast strains were used in each study. Interestingly, permeability of AtPIPs for H2O2 appeared to be exclusive to certain members of the PIP2 subfamily. It is not understood presently why only certain members of the AtPIP2 subfamily but not the AtPIP1 subfamily are permeable for H2O2 in yeast cells. It has been proposed that hydrogen peroxide can increase the phosphorylation of AQPs and thereby improve functionality (Aroca et al. 2005). Considering that AtPIP2s have a longer C-terminal end than AtPIP1s (Johanson et al. 2001), and that the serine residues in the C-terminus are the primary target for phosphorylation by H2O2-activated signalling cascade (Prak et al. 2008), it is likely that permeability toward H2O2 of certain members of AtPIP2s but not any members of AtPIP1s is the result of an increased AtPIP2 activity through phosphorylation by H2O2.

The effect of aquaporin inhibitors on the sensitivity of yeast cells to H2O2

To test the effect of aquaporin inhibitors on the response of the yeast to H2O2, the growth and survival of the JC0176 yeast strain on medium containing AgNO3, one of the commonly used aquaporin blockers, was investigated. In the absence of H2O2, increasing concentrations of AgNO3 above 10 μM reduced the growth and survival of yeast cells (data not shown), a concentration that is similar to the toxic concentration (6 μM) determined by Bienert et al. (2007). The yeast cells expressing either AtPIP2;2, AtPIP2;4, AtPIP2;5, or AtPIP2;7 as well as the control yeast cells harbouring the empty pYES2 vector showed a very similar growth pattern at 10 μM AgNO3 in both the absence and presence of H2O2 (Fig. 4). These results suggest that AgNO3 does not block the diffusion of H2O2 through these AtPIPs under the current experimental conditions.

Fig. 4
figure 4

The effect of the aquaporin blocker AgNO3 on the growth and survival of JC0176 yeast cells expressing AtPIPs. Yeast was transformed with an empty pYES2 vector (control) or an AtPIP gene, and 10 μL of serially diluted yeast cells were spotted on SG medium with 1 mM H2O2, 10 μM AgNO3, or both 1 mM H2O2 and 10 μM AgNO3. The growth and survival of the yeast cells was recorded after 5 days incubation at 30°C. The experiments were repeated at least two times with consistent results

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The channeling activity of AQPs can be inhibited by mercury or silver ions, which can bind to cysteine residues and thereby block the channel path (Niemietz and Tyerman 2002; Maurel et al. 2008). In the present study, however, the toxicity of H2O2 for JC0176 yeast strain was not noticeably reduced when AgNO3 (Fig. 4) or HgCl2 (data not shown) was added to the medium. An insensitivity of AQPs to channel inhibitors has also been demonstrated previously. An AQP from Nicotiana tabacum, which shows great homology with the AtPIP1 subfamily, appeared to be mercury-insensitive (Biela et al. 1999). Moreover, a membrane AQP from Arabidopsis was also found to be insensitive to mercury (Daniels et al. 1994). Hydrogen peroxide can readily oxidize the sulfhydryl group of cysteine residues in proteins (Rhee et al. 2000), and might affect the activity of AQPs. Therefore, the addition of an AQP blocker to H2O2 containing medium would not have reduced the sensitivity of AtPIP-expressing yeast cells for H2O2 because of analogous effects of both molecules on AQP activity. Since the AtPIPs tested in this study seemed to be marginally sensitive to AQP blockers in yeast cells, it would be of interest to determine whether these AQPs are not inhibited equivalently by channel blockers in Arabidopsis.

Conclusions

The current heterologous analysis of PIP-type aquaporins in two different mutant yeast strains clearly demonstrated that specific isoforms of AtPIPs function as H2O2 transporters in yeast. The heterologous expression of AtPIP2;2, AtPIP2;4, AtPIP2;5 and AtPIP2;7 increased the sensitivity of yeast cells for H2O2, which indicates that these plasma membrane AQPs are permeable for H2O2. Concurrently, the endogenous expression of AtPIP2s that appeared to be permeable for H2O2 in yeast was downregulated in the Arabidopsis roots in response to H2O2. Further investigation is needed to establish the effect of H2O2 on AQP functionality and activity in plants.