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First published online October 2, 2003; 10.1104/pp.103.025916 Plant Physiology 133:1148-1157 (2003) © 2003 American Society of Plant Biologists Seed 1-Cysteine Peroxiredoxin Antioxidants Are Not Involved in Dormancy, But Contribute to Inhibition of Germination during Stress1Division of Cell and Molecular Biology, Department of Biology, University of Oslo, P.O. Box 1031 Blindern, N–0315 Oslo, Norway
Peroxiredoxins (Prx) are thiol-dependent antioxidants containing one (1-cysteine [-Cys]) or two (2-Cys) conserved Cys residues that protect lipids, enzymes, and DNA against reactive oxygen species. In plants, the 1-Cys Prxs are highly expressed during late seed development, and the expression pattern is dormancy related in mature seeds. We have expressed the Arabidopsis 1-Cys Prx AtPER1 in Escherichia coli and show that this protein has antioxidant activity in vitro and protects E. coli in vivo against the toxic oxidant cumene hydroperoxide. Although some 1-Cys Prxs are targeted to the nucleus, a green fluorescent protein-AtPER1 fusion protein was also localized to the cytoplasm in an onion epidermis subcellular localization assay. It has been proposed that seed Prxs are involved in maintenance of dormancy and/or protect the embryo and aleurone layer surviving desiccation against damage caused by reactive oxygen species. These hypotheses were tested using transgenic Arabidopsis lines overexpressing the barley (Hordeum vulgare) 1-Cys PER1 protein and lines with reduced levels of AtPER1 due to antisensing or RNA interference. We found no correlation between Prx levels and the duration of the after-ripening period required before germination. Thus, Prxs are unlikely to contribute to maintenance of dormancy. RNA interference lines almost devoid of AtPER1 protein developed and germinated normally under standard growth room conditions. However, seeds from lines overexpressing PER1 were less inclined to germinate than wild-type seeds in the presence of NaCl, mannitol, and methyl viologen, suggesting that Prx can sense harsh environmental surroundings and play a part in the inhibition of germination under unfavorable conditions.
Peroxiredoxin (Prx) antioxidants, first identified in yeast (thiol-specific antioxidant; Kim et al., 1988  ), can be divided into three classes: typical 2-Cys Prx, atypical 2-Cys Prx, and 1-Cys Prx. Prxs can reduce H2O2, alkyl hydroperoxides, and hydroxyl radicals (Lim et al., 1993; Chae et al., 1994; Netto and Stadtman, 1996; Baier and Dietz, 1997) and have in vitro been shown to have antioxidant activity through protection of lipids, enzymes, and DNA against radical attack. In plants, antioxidant activity has been demonstrated for the 2-Cys BAS1 (Baier and Dietz, 1997) and the 1-Cys PER1 (Stacy et al., 1996) of barley (Hordeum vulgare). Transgenic plants overexpressing the rice (Oryza sativa) 1-Cys Prx (R1C-Prx) exhibit higher resistance to oxidative stress, suggesting antioxidant activity (Lee et al., 2000) in accordance with the function of 1-Cys homologs in other organisms (Shichi and Demar, 1990; Netto and Stadtman, 1996; Peshenko et al., 1996).
The plant 1-Cys Prx genes pBS128 from brome grass (Goldmark et al., 1992
Due to this expression pattern, Stacy et al. (1996
A function in desiccation tolerance during late stages of seed development has also been suggested (Stacy et al., 1996
Recently, a new member of the 1-Cys Prx group, XvPer1, was isolated from the resurrection plant Xerophyta viscosa (Mowla et al., 2002 In this study, we have investigated the antioxidant activity and subcellular localization of AtPER1. We have also generated transgenic Arabidopsis lines with increased and decreased levels of 1-Cys Prxs to investigate their importance in maintenance of dormancy. Seeds of such lines were also used to study germination frequencies in the presence of osmotic and oxidative stress that inhibit germination.
AtPER1 Has Antioxidant Activity in Vitro and in Vivo
Antioxidant activity of a purified and concentrated AtPER1 fusion protein, produced using the PinPoint system (Promega, Madison, WI), was tested in a DNA protection assay, i.e. DNA was exposed to a mixed function oxidation assay where ROS is generated due to the presence of Fe3+ (Stacy et al., 1996
Antioxidant activity of AtPER1 was also tested in vivo. The AtPER1 PinPoint fusion protein was used to produce polyclonal antibodies against AtPER1 (see "Materials and Methods"; Fig. 2A, lane 3). The inframe AtPER1 cDNA construct (PPAtPER1) and the cDNA cloned out of frame in the PinPoint vector system (nonsense construct) were introduced into Escherichia coli JM109 cells. Protein isolated from bacteria harboring the nonsense construct confirmed that this construct did not express a full-length AtPER1 protein. The polyclonal antiserum does, however, recognize the PinPoint tag (Fig. 2A, 14 kD). In bacteria with the PPAtPER1 construct, AtPER1 antiserum detected a protein of the expected size (Fig. 2A, 38 kD), corresponding to the AtPER1 fusion protein purified by means of the N-terminal PinPoint tag (Fig. 2A, lane 3).
The E. coli cells were tested for survival in the presence of the toxic oxidant cumene hydroperoxide in an assay previously used to show in vivo antioxidant activity of the 2-Cys Prx of barley, BAS1 (Baier and Dietz, 1997
Plant 1-Cys Prxs have been suggested to contain a bipartite NLS at the 3' end (Stacy et al., 1999
To investigate the relationship between 1-Cys Prx level and maintenance of dormancy, we generated transgenic Arabidopsis plants that overexpressed the barley 1-Cys Prx PER1 (pPCV002 35S::Per1) and plants with down-regulation of the AtPER1 level using an AtPER1-antisense construct (pKOH110 35S::AtPER1; Fig. 4). The expression levels of PER1 in independent 35S::Per1 transformants were tested by immunodetection with PER1 antiserum (Stacy et al., 1999
Siliques were harvested at the same stage of maturity, i.e. the first three brown, dried, and just opened siliques were collected. At the point of harvest, these seeds are completely dormant. Seeds from such siliques were sown every 3rd d after harvest, and germination frequencies were scored 8 d after sowing. In the wt C24 ecotype, germination frequencies are slowly increasing with the number of days of after-ripening. After 12 to 15 d, germination frequencies begin to increase more rapidly, and 60% germination is reached at 18 d of after-ripening (Fig. 5B). After 33 d, more than 95% of the seeds germinate. The pPCV002 35S::Per1 line 1 showed a similar germination pattern as wt seeds, whereas pPCV002 35S::Per1 line 2 needed longer time of after-ripening to reach a similar germination frequency (Fig. 5B). However, after 33 d, 88% of the line 2 seeds also germinated. The PER1 levels are not significantly different in the two lines (Fig. 5A). Thus, differences in germination profiles cannot be attributed to barley 1-Cys Prx but can possibly be attributed to different genomic insertion positions of the T-DNAs in the two 35S::Per1 lines. The variation in germination frequency between seeds of individual wt plants or transgenic plants of the same line, reflected by the SD, can be attributed to environmental variance. To test whether a reduction in AtPER1 level reduced dormancy, lines transformed with the antisense AtPER1 construct was used. mRNA was isolated from mature seeds of different lines and the level of mRNA investigated by northern hybridization. The lines 3 to 7 showed from 10% to 90% reduction in mRNA level compared with wt seeds (Fig. 6A). Germination tests were performed in the same manner as for the pPCV002 35S::Per1 lines. Average germination frequencies after 8 d of imbibition of seeds sown 18 d after harvest (Fig. 6B) was lower than that of wt seeds for two lines with transcript levels of 74% and 82%, however, only significant for one line. For three lines, among them line 3 with a transcript reduction of 90%, germination frequencies were comparable with wt (about 60%). After 24 d of after-ripening, there was a variation in germination frequencies from 65% to 95% (Fig. 6B). However, the frequencies did not correlate to the level of AtPER1 in the seeds.
Because the antisense construct only to a limited extent lowered AtPER1 expression levels, an RNA interference (RNAi) construct (pPCV002 AtPER1RNAi) was made. Two copies of the AtPER1 cDNA were cloned in inverse orientations on each side of a stuffer fragment (Fig. 4). Transcription from the 35S promoter would make possible the generation of a double-stranded AtPER1 cDNA intended for down-regulation of the endogenous AtPER1 expression by RNAi (Chuang and Meyerowitz, 2000
No obvious phenotype, e.g. embryo lethality or morphological changes, was detected in the primary RNAi transformants or their progeny plants that also had drastically reduced AtPER1 levels (data not shown). The seeds developed normally, and the dormancy level correlated closely to that of seeds from wt plants grown and harvested at the same time as the RNAi lines (data not shown). In after-ripened seeds, the germination frequencies were similar, i.e. between 95% and 98%. The number of abnormal germinating seedlings was not significantly different either between AtPER1RNAi lines and the wt or between the RNAi lines and 35S::Per1-overexpressing lines.
Only one of our tests indicated a role for AtPER1. Mature, after-ripened, thus non-dormant, AtPER1RNAi seeds were germinated under ionic osmotic stress (10–175 mM NaCl) and nonionic osmotic stress (10–500 mM mannitol), which both are known to inhibit germination (see Espelund et al., 1995
To further investigate the influence of seed 1-Cys Prx levels under conditions shown to inhibit germination, we compared germination frequencies between seeds from wt and 35S::Per1 lines (Fig. 5A). Germination frequencies were scored on 10 to 175 mM NaCl, 10 to 500 mM mannitol, and 5 to 500 µM MV, a compound known to create superoxide anions and H2O2. On the lowest concentrations of NaCl used (10–50 mM), germination frequencies of wt and 35S::Per1 lines were almost similar, but on 100 mM NaCl the 35S::Per1 lines germinated to a lower extent than wt seeds (Fig. 8A), i.e. after 8 d, 90% of wt seeds but only 50% to 60% of the 35S::Per1 seeds germinated. Germination of the latter was also slower, because after 4 d, almost all of the wt seeds that germinated had germinated, whereas hardly any of the 35S::Per1 seeds had started to germinate (Fig. 8B). One hundred and twenty-five and 175 mM NaCl drastically impaired germination of wt seeds, and transgenic seeds did not germinate at all (Fig. 8A).
Concentrations of 10 to 300 mM mannitol had only minor effect on germination of wt, whereas germination in 35S::Per1 lines showed lower total germination frequencies after 8 d with increasing concentrations (Fig. 8C). Germination was also slower (Fig. 8D shows this for 200 mM mannitol). At concentrations of 400 and 500 mM mannitol, wt germination was also critically reduced (Fig. 8C). Germination on the lowest concentration (5 µM) of MV did not result in any difference between wt and 35S::Per1 seeds (Fig. 8E). At 50 to 200 µM MV, germination of wt and transgenic seeds were gradually delayed and also much lower after 8 d. Wt seeds could, however, withstand higher concentrations than 35S::Per1 seeds (Fig. 8, E and F). Four hundred and 500 µM MV dramatically impaired germination of wt and 35S::Per1 seeds (Fig. 8E). In summary, the 35S::Per1 lines were slower and less inclined to germinate when exposed to osmotic and oxidative stress. To investigate the fate of the AtPER1 protein during imbibition with and without stress, we determined protein levels of wt and 35S::Per1 seeds imbibed on 400 and 200 mM mannitol or on water, using western blotting (Fig. 8G). In wt seeds and 35S::Per1 seeds, AtPER1 levels correlate inversely to germination frequency, i.e. the level is highest in seeds imbibed on 400 mM mannitol and lowest in seeds germinated on water for 8 d. On water, with a germination frequency of near to 100%, only traces of AtPER1 protein was seen in the wt—with double the amount of total protein and 10 times longer exposure time that in Figure 8G, a faint AtPER1 protein band could be detected (not shown). It should also be noted that compared with wt, the AtPER1 level is higher in both 35S::Per1 lines (Fig. 8G), which have significantly lower germination frequencies than wt on mannitol (Fig. 8C). To investigate whether the AtPER1 protein level was reflected in the AtPER1 transcript level, mRNA from seeds imbibed on water and on 400 mM mannitol was isolated and subjected to northern hybridization with an AtPER1 probe (Fig. 8H). Although the AtPER1 transcript is hardly visible in the mRNA from seeds germinated on water, the AtPER1 transcript level is high in seeds imbibed on mannitol, and considerably higher in 35S::Per1 seeds than in wt, consistent with the lower germination frequencies of the 35S::Per1 seeds.
It has been proposed that one function of seed 1-Cys Prxs is protection against ROS (Stacy et al., 1996 ), because this is an important aspect of desiccation tolerance (Leprince et al., 1994). Protection against ROS is also needed during imbibition of mature dry seeds when respiration is resumed. Previously, antioxidant activity of the barley PER1 has been shown in vitro in a mixed function oxidation assay (Stacy et al., 1996). Using the same assay, we here show in vitro activity of the AtPER1 fusion protein (Fig. 1). However, the protection level of AtPER1 appears lower than previously shown for PER1 (Stacy et al., 1996). On the other hand, the in vivo protection demonstrated by enhanced survival of E. coli bacteria subjected to cumene hydroperoxide (Fig. 2) is similar to that shown for the 2-Cys barley BAS1 protein in the same assay (Baier and Dietz, 1997).
In support of a protective function, Lee et al. (2000
In the promoters of Per1 and AtPER1, a putative antioxidant-responsive element has been identified (Haslekås et al., 1998
Where in the cells does AtPER1 exert its function? In mammals, Prx isoforms have been found localized to the cytosol, to the nucleus, and to mitochondria and peroxisomes (Wood et al., 2003b
In the onion epidermis cell transient expression assay, our GFP-AtPER1 fusion protein was localized to the cytoplasm and to the nucleus (Fig. 3). The signal distribution of GFP-AtPER1 was similar to that of GFP, which does not contain a NLS. The partial nuclear localization of GFP is caused by bidirectional diffusion through the nuclear pore complex (von Arnim et al., 1998
Most seeds possess some degree of dormancy at the completion of seed development, but so far, no genes exclusively controlling dormancy have been isolated (Koornneef et al., 2002 To thoroughly test the hypothesis on 1-Cys Prx involvement in maintenance of dormancy, we have performed germination trials on Arabidopsis seeds with reduced or increased level of 1-Cys PRXs, i.e. lines with high expression of the barley PER1, elevating the total level of 1-Cys Prx in the seeds, and antisense lines with 10% to 90% reduction in AtPER1 levels. Instead of bulk-harvested seeds, we have used seeds harvested at precisely the same stage of maturity. Neither for the five antisense 35S::AtPER1 lines nor for the 35S::Per1 lines was there any correlation between 1-Cys Prx level and the length of the after-ripening period required for germination (Figs. 5 and 6). Thus, our results do not support 1-Cys Prx involvement in maintenance of dormancy.
Dormancy is thought to be beneficial for the seeds because this quality enhances the possibility of survival when the seeds are allowed to germinate, e.g. the seeds do not germinate during non-favorable conditions as when they fall to the ground in the autumn. Similarly, other non-favorable conditions like oxidative or osmotic stress might inhibit germination. How can the seeds sense such unfavorable conditions? Answers to this question may come from mutant lines with reduced or enhanced sensitivity to NaCl and mannitol during germination and from Quantitative Trait Locis (QTLs) mapping close to genes involved in abscisic acid (ABA) responses, biosynthesis, or modulation (Quesada et al., 2002
The AtPER1 promoter contains an ABA-responsive element (Aalen, 1999 Is there a threshold level of 1 Cys Prx that is critical? The marginal effect of lowering the AtPER1 level suggests that other genes are also involved in sensing harsh conditions and to a considerable extent can compensate for the absence of AtPER1. On the other hand, the results using the 35S::Per1 lines imply that higher levels of 1-Cys Prx increase the sensitivity of imbibed seeds with regard to unfavorable conditions. Although a role for AtPER1 in the maintenance of dormancy seems unlikely, we suggest that the antioxidant function of 1-Cys Prx is employed to sense and/or react to seed environmental conditions, thus preventing germination to take place under unfavorable conditions. It would therefore be of interest to compare the germination ability of different Arabidopsis ecotypes under such conditions and to investigate whether putative differences are correlated to AtPER1 levels.
Purification of the AtPER1 Fusion Protein for Antiserum Production AtPER1 cDNA was amplified by reverse transcriptase-PCR using the gene-specific primers 5'-CGG GAT CCG GTA AAA TGC CAG GGA TC-3' and 5'-GGA ATT CGT AGG CTT TTG TTA TTA TCT TTT C-3', containing BamHI site and EcoRI sites. The AtPER1 cDNA digested with EcoRI, blunted, and then digested by BamHI was ligated into the BamHI and SmaI sites of PinPoint Xa3 vector (Promega), generating the construct PPAtPER1. Sequencing confirmed the cDNA sequence to be in the correct reading frame following the polypeptide tag. AtPER1 fusion protein was purified using the PinPoint batch purification protocol (Promega), with 50 mM HEPES and 5 mM biotin as elution buffer. Purified AtPER1 fusion protein was used for antiserum production in two rabbits (AGRI SERA AB, Vännäs, Sweden).
Antioxidant activity of purified AtPER1 fusion protein, concentrated in a Centricon-10 concentrator (Millipore, Bedford, MA), was tested in a DNA cleavage assay as by Stacy et al. (1996
An AtPER1 nonsense construct was made by PCR amplification with the primers 5'-CGG GAT CCG TAA AAT GCC AGG GAT CAC-3' and 5'-GGA ATT CGT AGG CTT TTG TTA TTA TCT TTT C-3', which contain BamHI and EcoRI sites in their 5' ends, respectively. The 5' end primer contained one additional nucleotide that generated an out-of-frame fusion to the PinPoint tag when cloned between the BamHI and EcoRI sites of PinPoint Xa3 vector. The nonsense construct and the PPAtPER1 construct (previous section) were used in an in vivo activity assay in E. coli JM109 according to (Baier and Dietz, 1997
Pelleted E. coli cells expressing the PPAtPER1 and nonsense constructs, dissolved in loading buffer (62 mM Tris-HCl, pH 6.8, 10% [v/v] glycerol, 2% [w/v] SDS, 0.7 M 2-
Isolation of total protein from plant tissue, SDS-PAGE, western blotting, and immunodetection were performed as described by Stacy et al. (1999
For investigation of intracellular localization of AtPER1, the cDNA was amplified by PCR using the primers 5'-GAA GAT CTA TGC CAG GGA TCA CAT A-3' and 5'-GCT CTA GAT CAA GAC CTC TGT GTA C-3', which include the restriction sites BglII and XbaI, respectively. The PCR product was cloned behind the GFP reporter gene between the BglII and XbaI sites of pAVA319 (von Arnim et al., 1998
The pPCV002 35S::Per1 construct was generated by digesting the Per1 cDNA (B15C; Aalen et al., 1994
The pKOH110 35S::AtPER1 antisense construct was generated by digesting the AtPER1 cDNA in pBluescript with EcoRI, blunting, and subsequent XbaI digestion. This fragment was ligated into the SmaI and XbaI sites between a 35S promoter and terminator cloned in pUC18. The 35S-AtPER1-ter cassette was excised using EcoRI and ligated into the EcoRI site of pKOH110 (Meza et al., 2001 The pPCV002 35S::AtPER1 RNAi construct was generated by digesting the AtPER1 cDNA in pBluescript with SmaI and EcoRI, and this fragment was ligated into the respective sites of a pBluescript plasmid harboring a fragment of the GUS (uidA) gene in the EcoRV site of the polylinker. A second AtPER1 cDNA was excised using SmaI and HindIII, and this fragment was ligated between the HindIII and a blunted XhoI site of pBluescript-AtPER1-GUS from the first cloning step. This resulting AtPER1-GUS-AtPER1 construct was digested by ApaI, followed by blunting and XbaI digestion before ligation between the SmaI and XbaI of pPCV002 35S (Fig. 4).
For transformation of Arabidopsis plants, alternative methods were used; a root transformation protocol from Valvekens et al. (1988
Arabidopsis ecotype C24 (wt) and transgenic plants were cultivated in growth chambers at 22°C and 8 h of dark/16 h of light (100 µE m–2). For germination tests, siliques were harvested at a specific stage; the first three brown, dried, and just opened siliques, were collected and after-ripened at room temperature. Seeds were sown on MS-2 plates at harvest and every 3rd d until 27 to 33 d of after-ripening. Germination was scored every 2nd d until 8 d after sowing. For germination analyses during osmotic and oxidative stress, after-ripened seeds were imbibed on paper discs soaked in concentrations ranging from 10 to 175 mM NaCl, 5 to 500 µM MV, or 10 to 500 mM mannitol. Germination was scored every 2nd d until 8 d after sowing. Seeds used in the same experiment were harvested from wt plants and transgenic plants grown at the same time and in the same growth chamber.
mRNA was extracted using magnetic Genoprep mRNA beads (Geno-Vision, Oslo, Norway). Total RNA isolation was performed after a modified protocol from Downing et al. (1992
Hybridizing probes include AtPER1 (Haslekås et al., 1998
We thank Prof. Gunter Reuter (Martin Luther University of Halle, Germany) for the pKEx-35S::HP1-GFP construct; Lars O. Baumbusch (University of Oslo, Norway) for providing the protocol for total RNA isolation from seeds; and Solveig Hauge Engebretsen, Roy Falleth, and Kirsten E. Rakkestad (University of Oslo, Norway) for technical assistance. Received April 23, 2003; returned for revision June 2, 2003; accepted July 29, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.025916.
1 This work was supported by the University of Oslo (grant to C.H.) and by the Research Council of Norway (project no. 140429/130).
2 Present address: Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radiumhospital, Montebello, N–0310 Oslo, Norway.
3 Present address: Department of Genetics, The Norwegian Radiumhospital, Montebello, N–0310 Oslo, Norway.
4 Present address: Department of Molecular Biology, Institute of Medical Microbiology, University of Oslo, National Hospital, N–0027 Oslo, Norway. * Corresponding author; e-mail reidunn.aalen{at}bio.uio.no; fax 47–22854605.
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Wood ZA, Poole LB, Karplus PA (2003a) Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling. Science 300: 650–653 Wood ZA, Schroder E, Harris JR, Poole LB (2003b) Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci 28: 32–40[CrossRef][Web of Science][Medline] This article has been cited by other articles:
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TOP
ABSTRACT
RESULTS
-glucuronidase stuffer fragment; ApR, ampicillin resistance gene; Pg5, truncated promoter of TL-DNA gene 5; Ori pBR322, ori region from pBR322; 
