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Keywords:

  • abiotic stress;
  • purple acid phosphatases (PAP);
  • reactive oxygen species (ROS) scavenging;
  • soybean;
  • stress tolerance

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • • 
    The primary biochemical reaction of purple acid phosphatases (PAP) is to catalyze the hydrolysis of phosphate esters and anhydrides. However, the soybean GmPAP3 gene expression is induced by NaCl, osmotic, and oxidative treatments, indicating a possible role of PAP in abiotic stress responses.
  • • 
    Confocal and electron microscopic studies demonstrated that GmPAP3 protein is mainly localized in mitochondria, a primary site for reactive oxygen species (ROS) production.
  • • 
    When subjected to NaCl and polyethylene glycol (PEG) treatments, ectopic expression of GmPAP3 in transgenic tobacco BY-2 cells mimicked the protective effects exhibited by the antioxidant ascorbic acid: increase in the percentage of cells with active mitochondria; reduction in the percentage of dead cells; and reduced accumulation of ROS. In addition, when GmPAP3 transgenic Arabidopsis thaliana seedlings were subjected to NaCl, PEG, and paraquat (PQ) treatments, the percentage of root elongation was significantly higher than the wild type. Furthermore, PQ-induced lipid peroxidation in these transgenic seedlings was also reduced.
  • • 
    In summary, the mitochondrial localized GmPAP3 may play a role in stress tolerance by enhancing ROS scavenging.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Purple acid phosphatases (PAPs) represent a diverse group of acid phosphatases in animals, microorganisms, and plants (Klabunde & Krebs, 1997; Schenk et al., 2000; Vogel et al., 2001; Olczak et al., 2003). They all possess a binuclear metal center: Fe(III)-Fe(II) in animals and Fe(III)-Zn(II) or Fe(III)-Mn(II) in plants. The primary biochemical reaction of PAPs is to catalyze the hydrolysis of phosphate esters and anhydrides. While the interconvertible redox forms of mammalian PAPs were found to be related to reactive oxygen species (ROS) evolution in phagocytosis and bone resorption (Hayman & Cox, 1994; Klabunde et al., 1995), the roles of plant PAPs remain largely unknown (Olczak et al., 2003).

In mammals, some PAPs were believed to be associated with physiological processes that involve ROS forming and/or scavenging (Klabunde et al., 1995; Kaija et al., 2002). The binuclear metal center of mammalian PAPs allows both evolution and scavenging of ROS through the Fenton-type reaction. In plants, the PAP isolated from kidney bean (KBPAP) was able to reduce Fe(III) to Fe(II) in the presence of ascorbic acid. The redox potential of Fe(II) is so low that it immediately reduces oxygen to water, thereby lowering the concentrations of free radicals (Klabunde et al., 1995). However, the possible role of plant PAPs in ROS metabolism is only implicated (Bozzo et al., 2004; Veljanovski et al., 2006) but remained speculative. No in vivo ROS scavenging function of plant PAPs was reported previously. In this report, we focused on the physiological role of the GmPAP3 gene (and its gene product) in relation to its protection effect towards oxidative damage at the cell and whole plant levels.

We previously showed that the GmPAP3 gene (encoding a putative PAP) from soybean is induced by NaCl and oxidative stresses but not phosphorus deficiency (Liao et al., 2003). Most other PAPs are extracellular enzymes (Kaida et al., 2003; Wasaki et al., 2003), and membrane localization of PAPs was verified only in a few cases (Morita et al., 1998; Nakazato et al., 1998; Veljanovski et al., 2006). However, bioinformatic tools suggested that GmPAP3 may be a novel PAP that localized in mitochondria (Liao et al., 2003), one of the primary sites for the production of ROS (Apel & Hirt, 2004).

Abiotic stresses such as salt and osmotic stresses can disturb important metabolic processes, upsetting the equilibrium between the formation and the scavenging of reactive oxygen species (ROS), and resulting in oxidative stress (Hernandez et al., 1993; Gomez et al., 1999; Mittova et al., 2003, 2004; Chaves & Oliveira, 2004). Therefore, manipulation of ROS scavenging or antioxidant enzymes may be a promising approach to obtain plants with multiple stress tolerance (Bartels, 2001). The inducibility of the GmPAP3 gene expression by salinity, osmotic, and oxidative stresses and the predicted mitochondrial localization of GmPAP3 prompt us to investigate further the possible physiological roles of GmPAP3 under oxidative stress.

To understand the cellular functions of the GmPAP3 protein, we used a gain-of-function approach in transgenic BY-2 cells to show that ectopic expression of GmPAP3 can partially alleviate oxidative damage caused by salinity and osmotic stresses, probably via ROS scavenging. In planta experiments using transgenic Arabidopsis thaliana provided further supportive evidence.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Gene expression study

To study the gene expression patterns of GmPAP3 under NaCl and polyethylene glycol (PEG) treatments, surface-sterilized soybean (Glycine max L. Merr. cv. Union) seeds were first germinated on filter paper containing modified Hoagland's solution (Li et al., 2006). One-week-old seedlings were transferred to a hydroponic system containing the same culture medium. After opening of the first trifoliates, seedlings were transferred to modified Hoagland's solution containing no other supplements (untreated) or with 125 mm NaCl or 5% PEG. The youngest, fully expanded trifoliates were then collected for total RNA extraction after 48 h. To study the gene expression pattern of GmPAP3 under PQ treatment, surface-sterilized seeds were germinated in silicon sand containing half Hoagland's solution (Liao et al., 2003). After germination, 10-d-old seedlings were transferred to a hydroponic system containing the same culture medium. After equilibration for 24 d, 10 mm paraquat (PQ) solution was sprayed on both surfaces of trifoliate leaves, and leaf samples were collected after 4 h. Paraquat was previously shown to induce the production of ROS (Tsang et al., 1991; Jiang & Zhang, 2002).

Northern blot analysis was performed as described by Sambrook & Russell (2001). Antisense single-stranded DNA probes were labeled with digoxygenin (DIG) (Roche, Mannheim, Germany) (Finckh et al., 1991). Since GmPAP3 was cloned into the pBluescript II KS (+) vector, the T3 (5′-AATTAACCCTCACTAAAGGG-3′) and T7 (5′-GTAATACGACTCACTATAGGGC-3′) promoter primers were used for synthesizing the PCR probe.

Establishment of transgenic tobacco (Nicotiana tabacum) BY-2 cell lines and transgenic A. thaliana

Recombinant constructs containing GmPAP3 or GmPAP3-T7 under the control of the constitutive Cauliflower Mosaic Virus 35S promoter were cloned into a binary vector (Brears et al., 1993) and introduced into Agrobacterium tumefaciens (strain LBA4404 and GV3101 for BY-2 cells and A. thaliana, respectively). The constructs were transformed into BY-2 cells (Nagata et al., 1992) (GmPAP3 and GmPAP3-T7) or A. thaliana (GmPAP3) using a cocultivation method (An, 1985) or a vacuum infiltration protocol (Bechtold & Pelletier, 1993), respectively. After selecting the transformants on antibiotic-containing media, PCR screening using gene-specific primers was performed to verify the successful integration of the transgenes into the genomes (data not shown); and northern blot analysis was performed to confirm the expression of the transgenes in the transgenic cell lines and plant lines (data not shown). For transgenic A. thaliana, seeds of T3 homozygous lines with a single T-DNA insert were obtained and used in subsequent physiological studies.

Confocal microscopic studies

The subcellular localization of the GmPAP3 protein was analyzed by confocal microscopy using transgenic BY-2 cells expressing the GmPAP3-T7 fusion construct. The location of the T7 tag was visualized by immunolabeling with the FITC-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA, USA). BY-2 cell fixation and confocal immunofluorescence were carried out as described by Jiang & Rogers (1998) with minor modifications. Cells were prestained with the mitochondria marker MitoTracker® Orange (Molecular Probes, Eugene, OR, USA) before the fixation and immunolabeling process. All confocal images were collected using the Bio-Rad Radiance 2100 system. Laser excitation (MitoTracker® Orange: 543 nm Green HeNe; FITC: 488 nm argon) was followed by signal detection using specific filters (MitoTracker® Orange: HQ 590/70; FITC: HQ 515/30). Colocalization of FITC (pseudocolored green) and MitoTracker® Orange signals (pseudocolored magenta) was examined by superimposing images. Overlapping signals were indicated by the yellow color. Using the program ImageJ1.34n (http://rsb.info.nih.gov/ij/), the pixel area occupied by yellow divided by the pixel area occupied by green (total) was calculated. Images from at least 10 different cells from the double-labeling experiment were analyzed.

Electron microscopic studies

Embedding and electron microscopy were performed as described by Tse et al. (2006) with slight modifications. Samples were fixed in a primary fixative solution contain 0.25% (v/v) glutaraldehyde and 1.5% (v/w) paraformaldehyde in 50 mm phosphate buffer, pH 7.4, for 15 min at room temperature before incubating at 4°C for an additional 16 h. After washing with phosphate buffer at room temperature, cells were dehydrated in an ethanol series and then embedded in Lowicryl (HM20) resin. Ultrathin sections were then prepared from these blocks using Ultra Cut S (Leica, Wetzlar, Germany). The GmPAP3 specific antibody was used as the primary antibody followed by detection using the gold-conjugated anti-rabbit secondary antibody. Immunolabeled sections were then poststained with 4% uranyl acetate and examined using a transmission electron microscope (JEM-1200EXII, JEOL, Tokyo, Japan).

Extraction of mitochondrial protein

Mitochondria-enriched protein fraction was extracted by differential centrifugation as described by Douce et al. (1987) with slight modifications. Plant material was gently homogenized in two volumes of ice-cold extraction buffer (0.25 m sucrose, 5 mm EDTA, 1 mm EGTA, 1 mm dithioerythritol, 0.1% BSA, 0.6% PVPP in 10 mm HEPES-Tris, pH 7.4). The homogenate was filtered and squeezed through Miracloth and the mitochondria were immediately separated from the cytoplasmic fraction by centrifugation at 10 000 g for 10 min. The resulting crude mitochondrial pellet was resuspended in medium I (0.25 m sucrose, 5 mm EDTA, 1 mm EGTA, 0.1% BSA in 10 mm HEPES-Tris, pH 7.4) and centrifuged at 600 g for 5 min to remove nuclei and heavy cell debris. This washing procedure was repeated twice. Washed mitochondria were resuspended in medium II (0.25 m sucrose, 30 µm EGTA in 10 mm HEPES-Tris, pH 7.4) and stored on ice.

Antibodies and western blot analysis

Primary antibody (polyclonal) targeting the GmPAP3 protein was obtained from a commercial service (Invitrogen, Custom Antibody) by injecting a synthetic peptide (‘N’-SFVLHNQYWGHNRR-‘C’) into rabbits. Unpurified serum was used in western blot analysis and immunolabeling. The antibody for the T7 tag was commercially available (Novagen, Madison, WI, USA). Anti-rabbit (for GmPAP3 antibody) secondary antibody conjugated to an alkaline phosphatase was used to recognize the primary antibodies.

For western blot analysis, the proteins were electrophoretically separated on a SDS-polyacrylamide gel (4% stacking; 10% resolving) as described by Laemmli (1970) before being transferred to an activated PVDF membrane. The transfer, blocking, and detection (solutions provided in the Aurora™ Western Blot Chemiluminescent Detection System; ICN, Solon, OH, USA) steps were performed according to the manufacturer's instructions.

Analysis of mitochondria integrity

Cells were treated with 200 mm NaCl or 2% PEG for 1 h before staining with 10 µg ml−1 rhodamine 123 (Rh123) (Molecular Probes) for 1 h. The signal of Rh123 was excited by green HeNe laser at 543 nm. The filter set HQ590/70 was used and confocal images were collected by the Bio-Rad Radiance 2100 system. Ten to 25 cells were counted for each sample for statistical analysis. The experiment was repeated three times.

Cell viability assay

Cells were treated with 200 mm NaCl or 2% PEG for 24 h before staining with 0.4% trypan blue (Sigma, St Louis, MO, USA). Stained cells were observed under a light microscope. Around 200 cells were counted for each sample. The experiment was repeated twice.

Detection of ROS

The chemical probe dichlorodihydrofluorescein diacetate (H2DCFDA) (Molecular Probes) has been used extensively as a noninvasive, in vivo measure of intracellular ROS (Allan & Fluhr, 1997; Maxwell et al., 1999). Cells were prestained with 10 µm H2DCFDA for 30 min before treatment with 200 mm NaCl or 2% PEG for 1 h. The signals of H2DCFDA were obtained by excitation with the 488 nm argon laser and detected with the HQ 515/30 filter set. The fluorescence intensity of H2DCFDA was estimated (Sukumvanich et al., 2004) using the program ImageJ1.34n. Quantitative analysis was done by tracing the entire cell, and the total fluorescence intensity measured (in pixels) was then divided by the area of the cell to obtain the average pixel fluorescence intensity. Background fluorescence intensity was measured in the same field and subtracted. Ten to 20 cells were analyzed for each sample in order to perform statistical analysis. The experiment was repeated twice.

For the experiments involving ferric chelator, 1,2-dimethyl-3-hydroxypyrid-4-one, which can bind Fe(III) specifically (Hayman & Cox, 1994), was employed. Cells were pretreated with 2.5 mm 1,2-dimethyl-3-hydroxypyrid-4-one for 5 min before staining with H2DCFDA and treatment with NaCl or PEG. The experiment was repeated twice.

Root elongation assay under stress treatments

Seeds of transgenic lines (GmPAP3 and the empty vector) and their untransformed parent Columbia-0 (Col-0) were sown on vertical MS plates containing 3% sucrose and 0.9% (w/v) agar. Five-day-old (PQ) or 7-d-old (NaCl and PEG) seedlings were transferred onto MS agar plates without other supplements (untreated), with 150 mm NaCl, with 15% PEG 6000, or with 1 µm PQ. The root length of each individual seedling before (by marking on the plates) and 7 d after treatment was recorded and the percentage root elongation was calculated accordingly. Forty-eight seedlings were measured for each treatment. The experiments were repeated twice. For the PEG treatment, the PEG-infused agar plates were prepared as described by Verslues et al. (2006).

Detection of lipid peroxides

FOX assay was used to determine lipid peroxides as described by Sattler et al. (2004) to indicate the extent of oxidative damage in Arabidopsis seedlings under PQ treatments. Cellular contents of 12 seedlings were extracted with 400 µl of methanol : dichloromethane (1 : 1 v/v) containing 0.05% (w/v) butylated hydroxytoluene and 50 µl of 150 mm acetic acid. Lipids were partitioned into the organic phase by adding 300 µl of water, vortexing and centrifugation at 3750 g. The lipid extracts were incubated at room temperature with FOX solution (Pierce, Rockford, IL, USA) for 30 min. Immediately after incubation, the absorbance was measured at 595 nm by a microplate reader. A standard curve was constructed using hydrogen peroxide as suggested in the manufacturer's protocol. The reactivity of 18:2-derived lipid hydroperoxides (LOOHs) with the FOX reagent is nearly identical to hydrogen peroxide (DeLong et al., 2002).

Statistical analysis

Data were analyzed using the SPSS (version 12.0) statistical package. Samples exhibiting significant differences (P < 0.01) were indicated.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

GmPAP3 is induced by salinity, osmotic and oxidative stresses

Since NaCl will also lead to ‘physiological drought’, we tested the effect of osmotic stress on the GmPAP3 gene expression by treating soybean plants with PEG. The expression of GmPAP3 under NaCl, PEG, and PQ treatments was studied (Fig. 1). All stresses led to an increase in the steady-state mRNA levels of GmPAP3, indicating that the GmPAP3 gene expression is coregulated by salinity, osmotic, and oxidative stresses.

image

Figure 1. Northern blot analysis of GmPAP3 under salinity, osmotic, and oxidative stresses. NaCl, 125 mm NaCl; PEG, 5% PEG (polyethylene glycol); PQ, 10 mm paraquat. Ethidium bromide staining of rRNA was used as the reference for total RNA quantitation. 10 µg of total RNA was loaded onto each lane.

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A major portion of GmPAP3 proteins in BY-2 cells is mitochondrial localized

To confirm the subcellular localization of the GmPAP3 protein, we performed detailed analysis at the cellular level using transgenic tobacco BY-2 cells through confocal and electron microscopic studies and western blot analysis of the mitochondria-enriched protein fraction.

The GmPAP3-T7 gene construct was expressed under a constitutive promoter. The subcellular localization of GmPAP3-T7 fusion proteins were tracked by fluorescein isothiocyanate (FITC) (pseudocolored in green) which located the T7 tag and visualized as punctated fluorescent signals (Fig. 2b). Mitochondria were stained with the dye MitoTracker® Orange (pseudocolored in magenta) (Fig. 2d). The degree of colocalization between T7 tag and MitoTracker® dye (Fig. 2f) was quantitated (Table 1) (see the ‘Materials and Methods’ section). In all cases tested, the majority of GmPAP3-T7 fusion proteins (> 60%) were found in mitochondria. As a negative control, the wild-type BY-2 cells did not give signal in the FITC channel (Fig. 2a) while the MitoTracker® dye staining (Fig. 2c) was similar to that of the transgenic cell lines.

image

Figure 2. Confocal microscopic studies of the subcellular localization of GmPAP3. A transgenic BY-2 cell line (1535-2) expressing the GmPAP3-T7 fusion protein was used. The T7 tag was tracked by the T7 antibody and then labeled with the FITC-conjugated secondary antibody (a, b). MitoTracker® Orange was used as a marker to locate mitochondria (c, d). The signal of FITC (pseudocolored in green) and MitoTracker® Orange (pseudocolored in magenta) were visualized by confocal scanning microscopy, and the structures of the corresponding transgenic BY-2 cells were observed using differential interference contrast (DIC) (g, h) and the merged images were also shown (e, f). One typical spot with overlapping signals was indicated by an arrow. Bar, 10 µm. Quantitative analysis is shown in Table 1.

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Table 1.  Estimation of the percentage of mitochondrial-localized GmPAP3 proteins
GmPAP3-T7 transgenic cell lines% colocalizationNumber of cells analyzed
  1. Experimental details are given in Fig. 2. Quantification of the extent of colocalization between the signals of FITC and MitoTracker® Orange was performed from one direction only (i.e. asking how much of the FITC signals colocalized with the MitoTracker® Orange signals, but not the other way round). Three independent cell lines were studied. Percentage colocalization is expressed as means ± SD for the number of cells analyzed.

1535-166 ± 6%29
1535-265 ± 4%31
1535-367 ± 5%27

To further confirm the above results, western blot analysis was performed using the antibody against the GmPAP3 protein generated for this study (see the ‘Materials and Methods’ section). Signals were observed only in the mitochondria-enriched protein fraction of the transgenic cell lines, but not the wild-type BY-2 cells (Fig. 3a). A major band of the right size was observed. In all cell lines tested (wild-type and transformed), the soluble protein fraction gave no detectable western blot signal using the same antibody (data not shown). These results provided biochemical evidence to support the mitochondrial localization of the GmPAP3 protein and verified the specificity of the antibody. Western blot experiments were also conducted for proteins from the native host soybean and similar results were obtained (data not shown). Transmission electron microscopic analysis was then performed using the GmPAP3-specific antibody (Fig. 3b). Most gold particles observed were confined to mitochondria.

image

Figure 3. Western blot analysis and electron microscopic studies of GmPAP3. (a) Western blot analysis of the mitochondria-enriched protein fraction from different cell lines was performed. WT, untransformed wild-type BY-2 cells; 1535-1 and 1535-2, two independent transgenic cell lines expressing the GmPAP3-T7 fusion protein. No GmPAP3 signal was obtained in the soluble protein fraction (data not shown). (b) Immunoelectron-microscopic study of GmPAP3 was conducted using the two independent GmPAP3-T7 transgenic cell lines (1535-1, 1535-2). GmPAP3-specific antibody was employed in both western blot analysis and immunoelectron-microscopic study. Arrowheads indicated the location of gold particles that were found mostly in mitochondria. Bar, 200 nm.

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Ectopic expression of GmPAP3 mimicked the effects of ascorbic acid to alleviate the oxidative damage induced by salinity and osmotic stresses in transgenic BY-2 cells

To test if GmPAP3 plays a protective role toward abiotic stresses, gain-of-function tests were conducted using the GmPAP3 transgenic BY-2 cell lines. Two parameters were used to examine the detrimental effects caused by salinity (200 mm NaCl) and osmotic (2% PEG) stresses in BY-2 cells: loss of mitochondrial activities (indicated by diffused signals of rhodamine 123 (Rh123) staining) (Wu, 1987; Petit, 1991) under short-term treatment (Fig. 4, Table 2); and cell death (indicated by trypan blue staining) (Hou & Lin, 1996) under prolonged treatment (Fig. 5, Table 3).

image

Figure 4. Mitochondrial membrane integrity under salinity and osmotic stresses. Wild-type (WT) BY-2 cells (a–c) and GmPAP3 transgenic BY-2 cell lines 20 and 29 (g–i and j–l, respectively) without ascorbic acid supplements, and wild-type BY-2 cells with 10 mm ascorbic acid supplements (+Asc) (d–f) were pretreated in a cell culture medium without stress (a, d, g, j), with 200 mm NaCl (b, e, h, k), or with 2% PEG (polyethylene glycol) for 1 h (c, f, i, l) before staining with 10 µg ml−1 Rh123 for another hour. The signal of Rh123 was observed using a confocal laser scanning microscope. For each line 10–25 cells were counted. Bar, 50 µm. Quantitative analysis is shown in Table 2.

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Table 2.  Quantitative analysis on the effects of salinity and osmotic stresses on mitochondria integrity
Cell lines% cells with intact mitochondria
ControlNaClPEG
  • PEG, polyethylene glycol.

  • Experimental details are given in Fig. 4. Quantitation of the percentage of cells with intact mitochondria was estimated by the uptake of the fluorescent dye Rh123. The percentage is presented as the mean value of three experiments (10–25 cells for each sample) ± SD.

  • **

    , statistically different (P < 0.01) from the wild-type BY-2 cells under the same treatment, based on one-way analysis of variance followed by Tukey test.

Wild-type BY-210045 ± 648 ± 3
Wild-type BY-2 +10 mm ascorbic acid10086 ± 3**82 ± 4**
GmPAP3 transgenic cell line 2010081 ± 4**81 ± 6**
GmPAP3 transgenic cell line 2910080 ± 5**80 ± 7**
image

Figure 5. Cell viability under salinity and osmotic stresses. Wild-type (WT) BY-2 cells (a–c) and GmPAP3 transgenic BY-2 cell lines 20 and 29 (g–i and j–l, respectively) without ascorbic acid supplements and wild-type BY-2 cells with 10 mm ascorbic acid supplements (+Asc) (d–f) were pretreated for 24 h in a cell culture medium without stress (a, d, g, j), with 200 mm NaCl (b, e, h, k), or with 2% PEG (polyethylene glycol) (c, f, i, l). Treated cells were stained with 0.4% trypan blue. Nonviable cells were stained. For each sample 200 cells were counted. Bar, 50 µm. Quantitative analysis is shown in Table 3.

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Table 3.  Quantitative analysis on the effects of salinity and osmotic stresses on cell survival
Cell lines% of viable cells
Untreated200 mm NaCl2% PEG
  • PEG, polyethylene glycol.

  • Experimental details are given in Fig. 5. Quantitation of the percentage of dead cells was estimated by trypan blue staining. The percentage is presented as the mean value of 200 cells ± SD.

  • **

    , statistically different (P < 0.01) from the wild-type BY-2 cells under the same treatment, based on one-way anova followed by the Tukey test.

  • a,b

    different letters indicate two separate sets of experiments.

Wild type BY-2a99 ± 348 ± 967 ± 8
Wild type BY-2 +10 mm ascorbic acida98 ± 693 ± 8**95 ± 9**
Wild type BY-2b93 ± 1142 ± 760 ± 13
GmPAP3 transgenic cell line 20b95 ± 680 ± 17**96 ± 6**
GmPAP3 transgenic cell line 29b98 ± 386 ± 18**92 ± 13**

In untreated BY-2 cells, discrete signals of Rh123 were observed, indicating the presence of active mitochondrial membrane uptake activities (Fig. 4a). There were no dead cells that could be stained by trypan blue (Fig. 5a). Both salinity and osmotic stresses reduced the percentage of cells possessing active mitochondria (Fig. 4b,c) and increased the percentage of dead cells (Fig. 5b,c). Addition of the antioxidant ascorbic acid to the wild-type BY-2 cells under the same stress conditions imposed a significant protective effect (Figs 4e,f and 5e,f), indicating that the NaCl- and PEG-induced oxidative damage was the immediate cause of their detrimental effects in BY-2 cells. Ectopic expression of GmPAP3 in BY-2 cells mimicked the effect of ascorbic acid and protected the cells from salinity (Figs 4h,k and 5h,k) and osmotic stresses (Figs 4i,l and 5i,l), supporting the notion that GmPAP3 may function to alleviate oxidative damages. On the other hand, expression of the yellow fluorescent protein in transgenic BY-2 cells did not lead to any protective effects (data not shown).

Since oxidative damage and tolerance are tightly linked to the formation and scavenging of ROS, we monitored the accumulation of cellular hydrogen peroxide using a fluorescent dye (see the ‘Materials and Methods’ section) under the stress treatments described earlier (Fig. 6, Table 4). In parallel with the degree of stress-induced cellular damage (see earlier discussion), hydrogen peroxide was accumulated in the BY-2 cells under both NaCl and PEG treatments (Fig. 6b,c), but not in untreated cells (Fig. 6a). The concentration of hydrogen peroxide under both stress conditions was lowered by the addition of ascorbic acid (Fig. 6e,f) or ectopic expression of GmPAP3 (Fig. 6h,i,k,l).

image

Figure 6. Cellular reactive oxygen species (ROS) production under salinity and osmotic stresses. Wild-type (WT) BY-2 cells (a–c) and GmPAP3 transgenic BY-2 cell lines 20 and 29 (g–i and j–l, respectively) without ascorbic acid supplements and wild-type BY-2 cells with 10 mm ascorbic acid supplements (+Asc) (d–f) were prestained with H2DCFDA for 30 min before being placed in a cell culture medium without stress (a, d, g, j), with 200 mm NaCl (b, e, h, k), or with 2% PEG (polyethylene glycol) for 1 h (c, f, i, l). The signals of H2DCFDA were observed using a confocal laser scanning microscope. For each line 10–20 cells were counted. Bar, 50 µm. Quantitative analysis is shown in Table 4.

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Table 4.  Quantitative analysis of the effects of salinity and osmotic stresses on reactive oxygen species (ROS) accumulation
Cell linesSignal intensity of H2DCFDA (pixels cm−2)
Untreated200 mm NaCl2% PEG
  • PEG, polyethylene glycol.

  • Experimental details are given in Fig. 6. Numerical data represent the mean value of 10–20 cells ± SD.

  • **

    , statistically different (P < 0.01) from the wild-type BY-2 cells under the same treatment, based on one-way anova followed by the Tukey test.

  • a,b

    different letters indicate two separate sets of experiments.

Wild-type BY-2a201 ± 36533 ± 77570 ± 49
Wild-type BY-2 +10 mm ascorbic acida197 ± 37199 ± 47**190 ± 31**
Wild-type BY-2b180 ± 24505 ± 35565 ± 38
GmPAP3 transgenic cell line 20b194 ± 23234 ± 45**210 ± 65**
GmPAP3 transgenic cell line 29b196 ± 32223 ± 38**225 ± 58**

The ROS scavenging effects of GmPAP3 depended on the presence of ferric ions

The GmPAP3 protein contains all consensus amino acid residues for metal (Fe(III) and Mn(II)/Zn(II)) binding at the active site (Schenk et al., 1999; Liao et al., 2003). We further tested if the presence of ferric ion is important for ROS scavenging activities in the GmPAP3 transgenic BY-2 cell lines. In the presence of ferric chelator, the ROS contents in all lines (transgenic or wild-type) were similar (Table 5). Ferric chelator also aggravated salinity stress in soybean suspension cells (supplementary material, Table S1).

Table 5.  Quantitative analysis of the effects of ferric chelator on the reactive oxygen species (ROS) scavenging activities in the GmPAP3 transgenic cell lines
Cell linesSignal intensity of H2DCFDA (pixels cm−2)
Untreated200 mm NaCl200 mm NaCl + ferric chelatorFerric chelator
  • PEG, polyethylene glycol.

  • Numerical data represent the mean value of 10–20 cells ± SD.

  • **

    , statistically different (P < 0.01) from the wild-type BY-2 cells under the same treatment, based on one-way anova followed by the Tukey test.

  • a, b

    different letters indicate two separate sets of experiments.

Wild typea232 ± 74598 ± 71547 ± 54244 ± 55
GmPAP3 transgenic cell line 20a244 ± 77255 ± 62**589 ± 91207 ± 63
GmPAP3 transgenic cell line 29a218 ± 53232 ± 48**531 ± 84210 ± 83
Untreated2% PEG2% PEG + ferric chelatorFerric chelator
Wild typeb229 ± 58512 ± 58576 ± 87187 ± 34
GmPAP3 transgenic cell line 20b204 ± 52210 ± 54**510 ± 47215 ± 64
GmPAP3 transgenic cell line 29b204 ± 33223 ± 33**515 ± 83215 ± 64

Ecotopic expression of GmPAP3 in transgenic A. thaliana alleviated salinity, osmotic, and oxidative stresses in planta

The studies described earlier demonstrated the function of GmPAP3 at the cellular level. Subsequently, we examined if ectopic expression of GmPAP3 can also alleviate salinity, osmotic, and oxidative stresses in planta. GmPAP3 was transformed into A. thaliana and expressed constitutively (see the ‘Materials and Methods’ section). The percentage of root elongation after treatment with NaCl, PEG, or PQ was used as a physiological parameter to measure stress-induced growth inhibition. The GmPAP3 transgenic seedlings exhibited a significantly better root elongation under all stress conditions, compared with the untransformed wild-type Col-0 and the transgenic seedlings containing an empty vector (Fig. 7a–c). The percentage root elongation of GmPAP3 transgenic seedlings was significantly higher. Fresh weight of the same batch of samples was also measured. The data were consistent with the result shown in Fig. 7 (supplementary material, Table S2). Furthermore, the degree of lipid peroxidation induced by PQ was also measured as a biochemical parameter to indicate the extent of oxidative damage (Shalata & Neumann, 2001). The enhancement of oxidative tolerance in A. thaliana by ectopic expression of GmPAP3 was demonstrated by a reduction of PQ-induced lipid peroxidation in young seedlings (Fig. 8).

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Figure 7. Effects of salinity, osmotic, and oxidative stresses on root elongation. The percentage of root elongation after treatment with 150 mm NaCl (a), 15% PEG (polyethylene glycol) (b), and 1 µm PQ (paraquat) (c) was compared among the wild-type Col-0, the empty vector transgenic control (V7) and two independent GmPAP3 transgenic lines (F42 and C25). The y-axis on the left and right of each graph indicates the percentage root elongation of untreated (bar) and stressed (line) seedlings, respectively. The percentage root elongation was calculated as follows: root length after treatment minus root length before treatment divided by root length before treatment. Error bar, SE, n = 48. Data obtained were analyzed by one-way analysis of variance (one-way anova) followed by the Tukey test. **, indicates that the mean difference (compared with wild type) is significant at P < 0.01 level.

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image

Figure 8. Lipid peroxidation in Arabidopsis seedlings under oxidative stress. Seedlings were grown and treated with paraquat (PQ) as described in Fig. 7. Lipid peroxidation was measured by the FOX assay. The reactivity of 18:2-derived lipid hydroperoxide (LOOHs) concentrations were expressed in µmol LOOHs g−1 fresh weight (FW). Error bar, SE, n = 4 (four sets of 12 seedlings for each data point). Data obtained were analyzed by one-way anova test followed by the Tukey test. **, indicates that the mean difference (compared with wild type) is significant at P < 0.01 level. Open and closed bars, untreated control and treated samples, respectively.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Salinity and osmotic stresses are two major environmental constrains to plant growth. However, it should be noted that they are sometimes difficult to distinguish. While experiments using PEG treatment can uncouple osmotic stress from salinity stress, NaCl treatment may lead to salinity and/or osmotic stress.

In plants, both salinity and osmotic challenges are able to induce ROS production in cellular compartments (Smirnoff, 1998; Bartels, 2001; Apel & Hirt, 2004). To prevent damage to essential cellular components, most ROS molecules are scavenged at the site of production: mitochondria (Smirnoff, 1998; Bartels, 2001; Apel & Hirt, 2004) and chloroplast (Foyer et al., 1994; Hernandez et al., 1995; Bartels & Sunkar, 2005).

The expression of the GmPAP3 gene can be induced by salinity, osmotic, and oxidation stresses (Fig. 1) and a major portion of GmPAP3 proteins is mitochondrial-localized (Figs 2, 3). Confocal microscopic studies showed that 60–70% of the proteins were localized in mitochondria, while electron microscopic results indicated that most gold particles were confined in mitochondria. In confocal studies, permeabilization is required to expose the epitope for immunolabeling, but this step will lead to loss of structural integrity. A compromised condition may result in some inefficiency during permeabilization. The reported percentage of colocalization will be reduced in case not all the cells can effectively take up either the FITC-conjugated antibody or the dye MitoTracker.

The activities and gene expression of most plant PAPs were frequently found to be phosphorus (P)-regulated (induced under P starvation) (Cashikar et al., 1997; del Pozo et al., 1999; Li et al., 2002), consistent with their roles in P metabolism. However, a systematic study of the PAP gene family in A. thaliana showed that some of the gene members are unresponsive to P status (Li et al., 2002), suggesting that some members may be involved in other physiological functions. We are not aware of other mitochondrial-localized plant PAPs in the literature. For instance, Arabidopsis PAPs were encoded by a gene family (close to 30 members). However, none of them has a predicted mitochondrial transit peptide. But we cannot rule out the possible existence of mitochondrial-located PAPs in A. thaliana without experimental verification. Some PAPs do possess signal peptides, but they do not share significant homology to the putative mitochondrial transit peptide of GmPAP3 (data not shown).

In plants, mitochondria are the major sites for ROS production (Apel & Hirt, 2004) as well as the main targets of ROS attack under abiotic stress (Tambussi et al., 2000; Bartoli et al., 2004). ROS can cause the collapse of mitochondrial membrane potential (Pastore et al., 2002) and this explains the dramatic and rapid (within 1 h after treatment) decrease in the uptake of Rh123 by mitochondria when the BY-2 cells were subjected to salinity and osmotic stresses (Fig. 4, Table 2). Under prolonged (24 h) stress treatments, a significant portion of the BY-2 cells did not survive (Fig. 5, Table 3). The loss of mitochondrial activity and cell death occurred in parallel with an accumulation of cellular ROS (Fig. 6, Table 4).

Under stress conditions, treatment with the antioxidant ascorbic acid led to an unambiguous effect on maintaining both mitochondrial activities and cell viability (Figs 4, 5; Tables 2, 3) and caused a reduction in ROS accumulation (Fig. 6, Table 4). These results strongly support the notion that the detrimental effects of salinity and osmotic stresses can be alleviated by ROS scavenging activities. Expression of GmPAP3 (which encodes a PAP protein) mimicked all the protective effects (Figs 4–6; Tables 2–4) exhibited by ascorbic acid, suggesting that the GmPAP3 protein may play a significant physiological role in ROS scavenging.

In plant PAPs, the binuclear metal center is essential for their redox activities (Klabunde et al., 1995, 1996). One metal ion involved is Fe(III) (Klabunde et al., 1995; Schenk et al., 1999). In the presence of ferric chelator, the ROS scavenging effects resulting from the expression of GmPAP3 were diminished (Table 5), indicating that redox reactions of GmPAP3 may be involved in the ROS scavenging activities. At this point, we cannot conclude whether GmPAP3 acts directly on ROS or via other redox intermediates. The question of whether GmPAP3 proteins can directly react with ROS through the Fenton-type reaction will be addressed in future studies.

The protection effects of GmPAP3 were also demonstrated in planta. Using percentage root elongation as a measuring parameter, transgenic A. thaliana expressing GmPAP3 exhibited a higher tolerance towards NaCl, PEG, and PQ treatments (Fig. 7). The protective effects conferred by GmPAP3 are probably related to alleviation of oxidative damage, as PQ-induced lipid peroxidation was also significantly reduced in transgenic lines (Fig. 8).

In summary, this work provides evidence of a mitochondrial-located PAP that can alleviate salinity and osmotic stresses by reducing the accumulation of ROS.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors thank Dr L. Jiang for supplying the wild-type BY-2 cells and Mr W.-K.K. Kwok and Ms S.-W. Tong for their excellent technical assistance. We also appreciate the critical comments by Drs H. Liao and X. Yan. This work was supported by the Hong Kong RGC Earmarked Grant CUHK4434/04M and the Hong Kong UGC AoE Plant & Agricultural Biotechnology Project AoE-B-07/09 (to H-ML).

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  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Table S1 Quantitative analysis on the effects of ferric chelator on the ROS scavenging activities in a soybean suspension cell culture

Table S2 Effects of salinity, osmotic, and oxidative stresses on fresh weight of transgenic Arabidopsis thaliana expressing GmPAP3

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