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First published online August 14, 2009; 10.1104/pp.109.145409 Plant Physiology 151:893-904 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Expression of Pyrococcus furiosus Superoxide Reductase in Arabidopsis Enhances Heat Tolerance1,[C],[W],[OA]Department of Plant Biology (Y.J.I., W.F.B.) and Department of Microbiology (M.J., A.L., R.K., A.M.G.), North Carolina State University, Raleigh, North Carolina 27695
Plants produce reactive oxygen species (ROS) in response to environmental stresses sending signaling cues, which, if uncontrolled, result in cell death. Like other aerobic organisms, plants have ROS-scavenging enzymes, such as superoxide dismutase (SOD), which removes superoxide anion radical (O2–) and prevents the production and buildup of toxic free radicals. However, increasing the expression of cytosolic SODs is complex, and increasing their production in vivo has proven to be challenging. To avoid problems with endogenous regulation of gene expression, we expressed a gene from the archaeal hyperthermophile Pyrococcus furiosus that reduces O2–. P. furiosus uses superoxide reductase (SOR) rather than SOD to remove superoxide. SOR is a thermostable enzyme that reduces O2– in a one-electron reduction without producing oxygen. We show that P. furiosus SOR can be produced as a functional enzyme in planta and that plants producing SOR have enhanced tolerance to heat, light, and chemically induced ROS. Stress tolerance in the SOR-producing plants correlates positively with a delayed increase in ROS-sensitive transcripts and a decrease in ascorbate peroxidase activity. The SOR plants provide a good model system to study the impact of cytosolic ROS on downstream signaling in plant growth and development. Furthermore, this work demonstrates that this synthetic approach for reducing cytosolic ROS holds promise as a means for improving stress tolerance in crop plants.
Reactive oxygen species (ROS) such as singlet oxygen (1O2), superoxide anion radical (O2–), hydrogen peroxide (H2O2), and hydroxyl radical (OH·) are produced as part of normal metabolism by organisms living in aerobic environments (Grene, 2002  ; Mittler, 2002; Apel and Hirt, 2004; Gapper and Dolan, 2006; Halliwell, 2006; Moller et al., 2007). While an increase in ROS can result in cell death, it is now well accepted that ROS also can function as signaling molecules (Foyer and Noctor, 2003, 2005a, 2005b; Mittler et al., 2004; Bailey-Serres and Mittler, 2006; Mittler, 2006; Kim et al., 2008). In this work, we have focused on the cytosolic O2– signal. Cytosolic O2– is normally metabolized by superoxide dismutase (SOD) to produce H2O2 and oxygen. H2O2 can elicit additional signals, and oxygen can serve as a substrate for further ROS production.
Several previous studies have indicated that increasing endogenous SODs enhances stress tolerance (McKersie et al., 1993
Altering cytosolic SOD also affects stress tolerance. Recently, new insights into the regulation of the copper/zinc (Cu/Zn) SOD were revealed through microRNA studies (Sunkar et al., 2006
Our approach has been to use a heterologous system to constitutively dampen cytosolic O2– signaling and reduce ROS toxicity. To avoid endogenous regulatory mechanisms, we selected superoxide reductase (SOR), an enzyme found in anaerobic microorganisms that reduces superoxide in a one-electron reduction reaction. Pyrococcus furiosus normally lives in anaerobic hydrothermal vents (Fiala and Stetter, 1986
Employing SOR to remove O2– has many advantages compared with SOD. First, in contrast to plant SODs, P. furiosus SOR reduces O2– without producing O2, thus lowering the potential for further ROS generation (Jenney et al., 1999
Previously, we have shown that P. furiosus SOR can be expressed in tobacco (Nicotiana tabacum) cells (NT-1) and that it will produce a functional enzyme (Im et al., 2005
Generation of Transgenic Arabidopsis Expressing P. furiosus SOR P. furiosus SOR was expressed in Arabidopsis plants as a GFP fusion under the control of the cauliflower mosaic virus 35S promoter using the Gateway vector construct pK7WGF2. We selected four independent homozygous lines (GFP-SOR1, -3, -8, and -9) and one line transformed with the 35S promoter containing only the GFP for further characterization. Expression of the GFP-SOR transgene was confirmed by reverse transcription (RT)-PCR using internal GFP forward and SOR reverse primers (Fig. 1A ). With these primers, no transcript was detected in either the wild type or the GFP line. The full-length GFP-SOR (42-kD) protein was detectable in a soluble protein fraction using antibodies raised against P. furiosus SOR (Fig. 1B), and the GFP fluorescence is readily detectable in the cytosol of both root and leaf cells (Supplemental Fig. S1, A and B).
GFP-SOR plants (hereafter denoted as SOR plants) have no morphological differences compared with the wild type and grow similarly under normal growth conditions (8 h of light/16 h of dark; Fig. 1C). The number and size of rosettes are comparable in wild-type and SOR plants over their life cycle. Under short-day conditions, flowering in the SOR plants is usually delayed by 2 to 4 d compared with wild-type and GFP plants (data not shown). Delays in flowering are more pronounced (4–6 d) when plants are grown under continuous light (Supplemental Fig. S2). Continuous light leads to increased ROS and favors the transition from the vegetative to the reproductive phase in Arabidopsis (Gapper and Dolan, 2006 ). Delays in transition to reproductive growth indicate that flowering in SOR plants is less sensitive to continuous light.
SOR activity was quantified using a standard SOD assay that will measure both SOD and class II SOR activity (Im et al., 2005
To obtain ample material for the assay, we used leaves from mature plants. Endogenous SOD present in the plant extract also decreases O2– levels, as seen in the basal activity in the wild-type leaves (Table I
). Although we cannot distinguish between the SOD and SOR activities with this assay, because P. furiosus SOR is more heat stable than plant SODs, we could enrich for SOR activity by heat treating the plant extracts prior to assaying, as shown in Table I. Using this same protocol, we detected from 3.5- to 4-fold higher SOR/SOD activity in heat-treated samples of young seedlings from the two SOR lines compared with controls (Supplemental Table S1). Further evidence of P. furiosus SOR activity in the plant extracts is given in Supplemental Figure S3. The assay is based on the study from Jenney et al. (1999)
Because chromophores in the extracts from green tissue interfered with the in vitro SOR/SOD assay, we assayed root tissue. Basal activity was higher in root samples not only because of a lack of chromophore but also because we did not dialyze the samples overnight prior to the assay. Importantly, roots expressing SOR had 26% to 56% more SOR/SOD activity (Table II ). The reproducibly higher activities in the roots of the SOR lines in addition to the increased activity in leaves and seedlings after heat treatment (Table I; Supplemental Table S1) make a compelling argument that extracts from the SOR plants have greater ability to reduce O2– than control plant extracts.
Because of the endogenous SOD activity, however, it was important to try to purify the recombinant SOR from the plant extracts so that we could confirm its function. To this end, we immunoprecipitated the recombinant SOR from leaf extracts with antibodies raised against P. furiosus SOR (Supplemental Fig. S4). To demonstrate the function of the purified recombinant SOR protein, leaf extracts were heat treated, the recombinant SOR protein was immunoprecipitated, and activity was assayed using a more sensitive tetrazolium salt (WST-1 [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]) to monitor O2–-mediated reduction (Dojindo Molecular Technologies; Fig. 2 ). The immunoprecipitant from the SOR transgenic plants had SOR activity, and the wild-type control had none. All of the above results support our thesis that P. furiosus SOR is produced in Arabidopsis as a functional enzyme.
SOR-Expressing Arabidopsis Seeds and Seedlings Have Increased Heat Stability
Heat stress can affect plant metabolism and major physiological processes by generating ROS (Larkindale and Knight, 2002
Previous work has shown that thermotolerance can vary with the growth stage (Hong and Vierling, 2000 ; Hong et al., 2003; Clarke et al., 2004; Larkindale et al., 2005); therefore, we investigated acquired thermotolerance with a hypocotyl elongation assay of young seedlings described by Hong and Vierling (2000). Hypocotyl elongation was measured and analyzed before and after treatment. The 2.5-d dark-grown SOR seedlings tended to have the same or slightly lower hypocotyl length for the first 2.5 d compared with wild-type and GFP seedlings (Fig. 3B). After heat treatment, hypocotyl length increased slightly more in the SOR lines such that the ratio of growth after treatment to total growth tended to be slightly (1.3- to 1.5-fold) greater compared with the wild type (Fig. 3B), but there was no dramatic change in acquired thermotolerance using the hypocotyl elongation assay. Vegetative-stage plants also were tested. In these experiments, 10-d-old seedlings growing under a short-day light regime were analyzed either for basal thermotolerance by heating directly to 45°C for 2 h or for acquired thermotolerance by acclimation at 38°C for 1.5 h and then incubating 2 h at 45°C. The percentage of seedlings that survived was determined 7 d after heat treatments. All the SOR lines showed a higher survival compared with wild-type plants. The survival for the SOR lines was approximately 50% to 75% for basal thermotolerance and 35% for acquired thermotolerance (Fig. 4 ). In contrast, none of the wild-type seedlings survived the basal thermotolerance assay. These results indicate that producing the P. furiosus-derived ROS-scavenging enzyme (SOR) in light-grown plants significantly enhanced their tolerance of heat stresses. Furthermore, the effect of SOR was more evident when assessing basal thermotolerance.
One of the critical intracellular sites of oxidative damage in plants is the chloroplast, where heat-induced disruption of electron transport can take place. Heat stress results in photoinhibition and photobleaching of chlorophyll as well as an increase in ROS (H2O2) in the cytosol (Willekens et al., 1995 ). Even though SOR is localized in the cytosol, we reasoned that reducing ROS in the cytosol might enhance survival in response to chloroplast-generated ROS (Koussevitzky et al., 2008). To measure the impact of SOR on chlorophyll biosynthesis and stability, 2.5-d dark-grown seedlings were heat treated at 48°C for 30 min and were then exposed to light. Dramatic differences were evident in the SOR lines, as shown in the photograph in Supplemental Figure S5. When chlorophyll was extracted and the levels were measured, heat-treated wild-type seedlings had only 7% of the chlorophyll of non-heat-treated control seedlings, while heat-treated SOR seedlings had 48% to 100% of the control chlorophyll (Table III
). The fact that dark-grown SOR lines had increased heat tolerance under high light indicates that the effects of SOR went beyond the cytosol.
P. furiosus SOR Increases Plant Tolerance to Chemically Induced ROS in Vivo Methyl viologen (paraquat), an effective electron acceptor that generates O2–, was used to generate ROS. Seeds were grown in medium with different concentrations of paraquat (0, 0.25, 0.5, and 1 µM) and maintained under continuous light. As indicated in Figure 5 , SOR seedlings were more tolerant of chemically generated ROS. SOR3 and SOR9 germinated and survived 1.5- to 2.5-fold higher on 0.25 µM paraquat and 2- to 4-fold higher on 0.5 µM paraquat compared with wild-type and GFP lines after 14 d under continuous light. The SOR lines even germinated on 1 µM paraquat, unlike wild-type and GFP seeds. These data provide evidence that even though SOR is produced in the cytosol, it may increase the capacity of the plants to detoxify ROS generated from other organelles, such as the chloroplast or mitochondria. These data also help to explain the resilience of the SOR plants after heat and light stress shown in Supplemental Figure S5 and Table III.
Response to Heat Stress Is Delayed in SOR Seedlings
To further investigate how the SOR plants were coping with heat-induced ROS, we analyzed in more detail the response of light-grown seedlings (8 h of light/16 h of dark) to heat stress. For these studies, we performed biochemical analyses of cytosolic ascorbate peroxidase (APX) activity (Mittler and Zilinskas, 1991
The fact that APX activity does not increase in SOR plants indicates that APX activity is not limiting in this system and suggests that basal H2O2 is not elevated (Fig. 6
, zero time point). In contrast, the GFP line has higher APX activity. This is consistent with reports that GFP can generate H2O2 (Haseloff and Amos, 1995
Panchuk et al. (2002) reported that APX activity decreased in response to acute heat stress. We found that APX activity decreased in all lines (wild-type, GFP, and SOR plants) in a similar manner in response to heat stress. Importantly, SOR plants were always more resilient than GFP or wild-type plants and yet had lower APX activity, indicating that APX activity was not a major factor contributing to heat stress tolerance in SOR plants. To determine how other ROS- and heat stress-sensitive proteins were affected in the SOR seedlings, we monitored the relative abundance of HSP70 (a heat shock protein), BiP (an endoplasmic reticulum [ER] chaperone), and calreticulin (CRT; an ER chaperone and calcium-binding protein). As shown in Figure 7 , similar trends are seen in protein patterns for both wild-type and SOR seedlings; however, for BiP and HSP70, changes in protein abundance appear to be less in SOR seedlings. That is, induction of HSP70 and BiP are delayed in response to heat stress in SOR seedlings. CRT decreased rather than increased with heat stress, and loss of CRT protein was faster in SOR seedlings compared with wild-type seedlings.
A very sensitive measure of changes in oxidative status is the induction of ROS-sensitive transcripts such as Zat12. Zat12 is a H2O2-sensitive transcription factor (Rizhsky et al., 2004 ; Davletova et al., 2005b). As predicted, heat stress (48°C for 1 h) increased the Zat12 transcript in wild-type and all of the SOR seedlings (Fig. 8
). The transcript levels of H2O2-scavenging cytosolic enzymes, ascorbate peroxidases (APX1 and APX2), were monitored. We did not detect altered expression of APX1 basal levels between wild-type and SOR seedlings or between control and heat-treated samples. APX2, a cytosolic APX, is normally hard to detect but increases readily with heat and high-light stress (Rossel et al., 2002; Davletova et al., 2005b; Bechtold et al., 2008). As anticipated, APX2 increased in response to stress in wild-type and GFP lines; however, in the most highly expressing SOR lines, SOR3, -8, and -9, there was a decrease in APX2 transcript (Fig. 8). Furthermore, the chloroplast stromal APX transcript was less abundant in SOR8 and -9 after 1 h of heat stress (Fig. 8). These data suggested that either the levels of these transcripts and/or endogenous ROS scavenging enzymes were adequate and other compensatory pathways were induced or SOR seedlings did not produce as much H2O2 under these conditions.
More quantitative measurements of transcript levels of known heat-induced (Hsp101) and oxidation-inducible genes, including Zat12, APX1, Catalase1 (Cat1), and Cat2, were conducted during time-course experiments using wild-type and SOR seedlings. Transcript levels of each gene monitored are expressed as the fold change compared with the level of expression in the wild-type zero time point. Quantitative RT-PCR analysis of wild-type and SOR seedlings confirmed that Hsp101, Zat12, APX1, Cat1, and Cat2 are all induced in response to heat stress (Fig. 9 ). Hsp101 is a well-characterized, heat stress response transcript (Queitsch et al., 2000 ). In the SOR lines, there was a delay in the increase of this transcript. The response of Hsp101 at the 2-h time point showed 30% to 50% less induction in the SOR lines. These data are consistent with delayed production of heat stress response proteins (HSP70 and BiP) and oxidation-induced transcripts (Zat12) in the heat-tolerant SOR lines. All H2O2-scavenging enzymes, including catalase and APX, show similar profiles in wild-type and SOR lines, indicating a normal, although in some instances delayed, ability for these enzymes to sense and respond to oxidative stress at the transcriptional level.
Importantly, basal levels of all of these ROS- and stress-induced transcripts were not elevated in SOR plants under nonstressed conditions. Purified, recombinant P. furiosus SOR produces H2O2 when it reduces superoxide in vitro (Grunden et al., 2005 ). If this occurred when P. furiosus SOR was produced in the cytosol of Arabidopsis plants, one would anticipate that the endogenous catalase or perdoxidase activity would increase to compensate for an increase in H2O2 production. However, there was no significant difference in the basal APX activity of the seedlings under nonstressed conditions, as shown in Figure 6. Nor were there differences in catalase activity or H2O2 using the in vitro assay (Table IV
). There also were no significant increases in the ratio NAD/NADH (7.9 ± 1.1 for the wild type, 4.8 ± 1.2 for GFP, 6.4 ± 0.5 for SOR3, and 5.0 ± 0.1 for SOR9) or NADP/NADPH (1.0 ± 0.2 for the wild type, 0.6 ± 0.4 for GFP, 0.6 ± 0.3 for SOR3, and 0.8 ± 0.1 for SOR9), which implies that, as was found with E. coli expressing SOR (Molina-Heredia et al., 2006), there was no increased demand on cellular reductant in SOR plants. Taken together, these data suggest that in planta either SOR is not very active under nonstressed conditions, and therefore excess H2O2 is not being produced, or SOR is functional all the time and endogenous enzymes are capable of reducing the H2O2 produced. A third explanation also is quite possible. When produced in planta, SOR might form a complex that reduces superoxide completely to water without producing H2O2, as was reported when P. furiosus SOR was produced in E. coli (Molina-Heredia et al., 2006).
All aerobic organisms have multiple, interacting pathways for reducing ROS. Plants, as sessile organisms, predictably have developed plasticity in their ability to remove ROS and, as a result, present a challenge to biologists attempting to identify key regulatory factors in ROS-mediated signaling and responses. We constitutively expressed the SOR gene from P. furiosus in plants to enhance ROS scavenging and potentially reduce basal ROS. Such a synthetic system, in which O2– is rapidly reduced, should in theory decrease severe responses to stress and enhance survival.
Heat stress leads to the production of ROS and oxidative damage in cells, and many ROS-mediated heat stress responses have been characterized (Mittler, 2006
Paradoxically, cytosolic APX genes, specifically APX2, which is heat and high-light inducible (Rossel et al., 2002
The data also make a compelling argument that stress tolerance of the SOR seedlings does not result from an increase in HSP70 or BiP. The promoter region of HSP70A contains independent cis-elements that can be activated by heat and ROS (H2O2 and 1O2; Shao et al., 2007
While it is well accepted that ROS affect plant growth and development, underlying mechanisms controlling ROS are not well understood (Gapper and Dolan, 2006 In summary, we have shown that P. furiosus SOR can be produced as a functional protein in Arabidopsis. We also demonstrate that expressing P. furiosus SOR delays the response to heat stress and enhances survival under conditions known to produce increased ROS. Future genomic and metabolomic analyses will be required to understand the impact of SOR on basal plant metabolism and to fully characterize the effects on downstream events mediated by cytosolic O2– in planta.
Generation and Selection of SOR Transgenic Plants
The gene encoding Pyrococcus furiosus SOR (accession no. AE010234) was cloned into pK7WGF2 (Functional Genomics Division, Department of Plant Systems Biology, Ghent University, Ghent, Belgium; Im et al., 2005
RNA was isolated using the RNeasy kit (Qiagen), with an additional DNase I treatment to remove contaminating genomic DNA. RT was carried out to generate cDNA using Omniscript reverse transcriptase enzyme (Qiagen). GFP-fused SOR transcripts were detected by PCR as described by Im et al. (2005)
Total protein extract was obtained from plants frozen in liquid N2 or seedlings grown as described by Weigel and Glazebrook (2002)
Samples were ground with liquid nitrogen and lysed as described previously (Im et al., 2005
Unless otherwise indicated, the standard SOR/SOD assay was performed as described by Im et al. (2005)
To confirm activity of the recombinant protein, SOR was immunoprecipitated from the heat-treated (80°C for 15 min) leaf extracts with P. furiosus SOR antibodies using protein A-Sepharose beads as described previously (Shank et al., 2001
A ferrous ammonium sulfate/xylenol orange (FOX) method was used to quantify H2O2 in plant extracts (Wolff, 1994
APX activity was determined as described previously (Nakano and Asada, 1981
Arabidopsis seeds were surface sterilized as described by Weigel and Glazebrook (2002)
To test seed basal thermotolerance, stratified seeds were treated at 45°C for 5 h and germination was evaluated 2 d later following the protocol of Larkindale et al. (2005)
Seedlings were ground with liquid nitrogen and extracted with 80% (v/v) acetone by shaking until the leaves became breach. The chlorophyll content was quantified spectrophotometrically based on A663 as described (Burke et al., 2000
The following materials are available in the online version of this article.
We thank Dr. Becky Boston (North Carolina State University) for kindly providing the HSP70, BiP, and CRT antibodies. We also thank former undergraduate students, Caroline Smith, Leslie Hewes, Carla Pistole, and Casey Lowder, for technical help. Received July 27, 2009; accepted August 7, 2009; published August 14, 2009.
1 This work was supported by a grant from the National Aeronautics and Space Administration Institute for Advanced Concepts to A.M.G. and W.F.B., by the U.S. Department of Agriculture (Cooperative State Research, Education, and Extension Service grant no. 35318–05024 to A.M.G., W.F.B., and Mary M. Peet), and by the North Carolina Agricultural Research Service. 
2 Present address: Syngenta Biotechnology, Inc., 3054 East Cornwallis Road, Research Triangle Park, NC 27709. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Wendy F. Boss (wendy_boss{at}ncsu.edu).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.145409 * Corresponding author; e-mail wendy_boss{at}ncsu.edu.
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