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First published online March 7, 2008; 10.1105/tpc.107.052647 The Plant Cell 20:786-802 (2008) © 2008 American Society of Plant Biologists Modulation of Nitrosative Stress by S-Nitrosoglutathione Reductase Is Critical for Thermotolerance and Plant Growth in Arabidopsis [W]
a Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721 2 Address correspondence to vierling{at}u.arizona.edu.
Nitric oxide (NO) is a key signaling molecule in plants. This analysis of Arabidopsis thaliana HOT5 (sensitive to hot temperatures), which is required for thermotolerance, uncovers a role of NO in thermotolerance and plant development. HOT5 encodes S-nitrosoglutathione reductase (GSNOR), which metabolizes the NO adduct S-nitrosoglutathione. Two hot5 missense alleles and two T-DNA insertion, protein null alleles were characterized. The missense alleles cannot acclimate to heat as dark-grown seedlings but grow normally and can heat-acclimate in the light. The null alleles cannot heat-acclimate as light-grown plants and have other phenotypes, including failure to grow on nutrient plates, increased reproductive shoots, and reduced fertility. The fertility defect of hot5 is due to both reduced stamen elongation and male and female fertilization defects. The hot5 null alleles show increased nitrate and nitroso species levels, and the heat sensitivity of both missense and null alleles is associated with increased NO species. Heat sensitivity is enhanced in wild-type and mutant plants by NO donors, and the heat sensitivity of hot5 mutants can be rescued by an NO scavenger. An NO-overproducing mutant is also defective in thermotolerance. Together, our results expand the importance of GSNOR-regulated NO homeostasis to abiotic stress and plant development.
Nitric oxide (NO) is a short-lived, endogenously produced radical that acts as a signaling molecule in all higher organisms (Lamattina et al., 2003  ; Wendehenne et al., 2004; Delledonne, 2005; Crawford, 2006; Besson-Bard et al., 2008). Despite its deceivingly simple structure, the rich chemistry of NO in biological systems gives rise to multiple secondary and tertiary reaction products, greatly complicating our mechanistic understanding of NO-related effects (Stamler and Hausladen, 1998; Mancardi et al., 2004; Ridnour et al., 2004). Directly and via its various chemical transformations, NO not only accomplishes signaling functions but also acts as a redox modulator with both antioxidant (by quenching other radical reactions) and pro-oxidant (through the production of reactive nitrogen species; RNS) properties. In addition to effects on redox status, the formation of RNS leads to nitrosation, nitrosylation, and nitration reactions with other molecules. Most of the regulatory effects of NO are thought to be mediated through posttranslational protein modifications, including heme nitrosylation, Tyr nitration, Cys nitrosation, and even glutathiolation (Lindermayr et al., 2005; Aracena-Parks et al., 2006; Wang et al., 2006b; West et al., 2006; Zaninotto et al., 2006).
In plants, NO is believed to be produced via two different enzymatic pathways (Guo et al., 2003
NO-derived RNS readily react with the major cellular antioxidant GSH to form S-nitrosoglutathione (GSNO). The main reaction of GSNO in biological systems involves the transfer of the NO group to other cellular thiols to form longer-lived nitrosothiols (SNOs), an exemplary transnitrosation reaction. Endogenous GSNO has been proposed to be a significant player in NO regulatory mechanisms, particularly in the nitrosation of protein thiols, a process termed S-nitrosylation (Ji et al., 1999
It is now recognized that an evolutionarily conserved, GSH-dependent formaldehyde dehydrogenase (FALDH), a type III alcohol dehydrogenase, has activity as a GSNO reductase (GSNOR) (Jensen et al., 1998
In plants, there have been limited studies of GSNOR either from the perspective of its formaldehyde-detoxifying activity (Uotila and Koivusalo, 1979
Information about the phenotypes associated with a loss of GSNOR function is scarce. A T-DNA insertion mutant of the single copy GSNOR gene in Arabidopsis was recently isolated (designated gsnor1-3) (Feechan et al., 2005 We now report that GSNOR activity is necessary for the acclimation of plants to high temperature and for normal development and fertility under optimal growth conditions. Our results demonstrate that GSNOR has an important role in the homeostasis of NO and its metabolites, affecting not only abiotic stress but also plant developmental processes.
The Thermotolerance-Defective Mutant hot5 Encodes GSNOR We identified an Arabidopsis thermotolerance-defective mutant, hot5-1, in a screen of ethyl methanesulfonate–mutagenized seedlings using a hypocotyl elongation assay that was described previously (Hong and Vierling, 2000 ). Dark-grown, 2.5-d-old hot5-1 seedlings are completely blocked in hypocotyl elongation after 150 min of 45°C heat treatment, even following a pretreatment at 38°C, which allows wild-type seedlings to survive (Figure 1B
). The hot5-1 mutant was backcrossed to the wild-type ecotype Columbia (Col) for standard genetic analysis. F2 backcrossed lines showed that the thermotolerance-defective phenotype segregated as a single recessive trait (data not shown). Using established map-based cloning methods (see Methods), the hot5-1 mutation was located toward the bottom of chromosome 5, between BAC clones F6B6 and MLN1. We sequenced all annotated genes in the mapped region using genomic DNA from hot5-1 mutant plants. Sequence analysis revealed a single G-to-A mutation, resulting in a Glu-to-Lys substitution at amino acid 283 in the seventh exon of the GSNOR gene (At5g43940) (Figure 1A). Glu-283 is 100% conserved in GSNOR from plants and other organisms, including bacteria and human (see Supplemental Figure 1 online).
To confirm that GSNOR is indeed the gene responsible for the observed hot5-1 phenotype, we isolated additional alleles of the GSNOR gene. A second missense mutation (hot5-3) was isolated from available Tilling lines (Col erecta background) (Till et al., 2003 ). The hot5-3 mutation leads to the substitution of a conserved amino acid also in exon 7 (G288R), five amino acids from hot5-1 (Figure 1A; see Supplemental Figure 1 online). Two T-DNA insertion alleles were also obtained, hot5-2 (Col background), which is located in exon 1 and is identical to gsnor1-3 reported by Feechan et al. (2005), and hot5-4 (Wassilewskija [Ws] background) in exon 4 (Figure 1A). The hot5-1, hot5-2, and hot5-3 mutant alleles were backcrossed to the wild-type Col ecotype, and the hot5-4 allele was backcrossed to the Ws ecotype, two times to remove background mutations.
The hot5 mutants were tested for their ability to acquire heat tolerance in comparison with the null mutant of Heat-Shock Protein101 (Hsp101; hot1-3), which has an established heat-sensitive phenotype (Hong and Vierling, 2001
GSNOR Is Not Heat Induced, and HSPs Are Normally Expressed in Mutant Plants
We further confirmed previous observations of the ubiquitous expression of GSNOR throughout the plant (Martínez et al., 1996 ; Dolferus et al., 1997) by protein gel blot analysis. Samples were isolated from mature seeds, 2.5-d-old dark-grown hypocotyls, and different organs of mature plants. GSNOR protein was present in all organs tested, including dried seeds (see Supplemental Figure 2C online). These data indicate that the loss of GSNOR activity could affect phenotypes through the plant life cycle.
We next measured the effect of the hot5 mutations on GSNOR enzyme activity in total plant extracts (Figure 2B). In leaves of 25-d-old plants, the GSNO reduction activity of wild-type plants was similar to values reported previously (Feechan et al., 2005 We also measured activity in 2.5-d-old dark-grown seedlings of wild-type Col and the two missense alleles (Figure 2B). Expressed per milligram of total protein, GSNOR activity was actually higher for all seedling samples than in leaves, but this appears to reflect the higher levels of GSNOR protein per milligram of total protein in seedlings versus leaves (see Supplemental Figure 2A online). Surprisingly, the activity in missense mutant, dark-grown seedlings, expressed as a percentage of wild-type values, was similar to the activity seen in 25-d-old plants (33.7% of wild-type values for hot5-1 and 64.8% of wild-type values for hot5-3). We also measured changes in GSNOR activity after heat stress in the wild type and GSNOR missense mutants. We found no statistically significant change in GSNOR activity under heat-stress conditions in the wild type or mutants, or when protein extraction was performed plus or minus DTT, or when seedlings were grown for 2.5 d in the light instead of the dark (data not shown). The reason that dark-grown seedlings of hot5-1 and hot5-3 have a heat-stress phenotype in the dark, despite having apparently reasonable GSNOR activity at this stage, is not obvious. Possible explanations for this result are that higher GSNOR activity is required in the dark for proper growth after heat stress, that in light-grown seedlings other factors are present that compensate for the reduced GSNOR activity, or that the missense alleles of GSNOR have altered regulation in the dark that is not preserved by our extraction and measuring conditions.
Because HSP expression is known to be an important component of acquired thermotolerance, we also assayed the accumulation of different HSPs by protein blot analysis in the hot5 mutants (Figure 2A). All of the hot5 alleles showed wild-type levels of Hsp101, which is essential for heat tolerance (Hong and Vierling, 2000
hot5 Null Mutants Have Pleiotropic Phenotypes
When hot5-2 was grown under long-day conditions (16 h of light), the mutant was less vigorous and had a decreased number of rosette leaves, leaves were pale green and distorted, and bolts were shorter compared with plants grown under 8 or 12 h of light (data not shown). Indeed, under long days, the chlorophyll content of hot5-2 was only 62% of that of wild-type plants (see Supplemental Figure 3A online). After bolting, the hot5 null mutants were highly branched and semidwarf under all light conditions (Figure 3C). The roots of hot5 -2 were also reduced in length compared with those of wild-type plants (see Supplemental Figure 3B online), but this difference is consistent with the reduced growth of the rosette, and its relationship to the reduced root length reported previously for GSNOR antisense plants is not clear (Espunya et al., 2006 ). The plants were also long-lived, continuing to produce leaves for as long as 25 d after wild-type plants had senesced. The most dramatic phenotype of the hot5 null mutants was reduced fertility. Leaf numbers before bolting were not altered in hot5-2 compared with the wild type under either long or short days. Under 12 h of light, the hot5 homozygous null mutants produced many flowers and siliques but set very few seeds per plant. The mutants showed normal floral organ formation, with a wild-type number of sepals and petals and normal pistil formation. However, petals of mutant flowers were somewhat shriveled and smaller than wild-type petals, and stamens did not elongate normally, although pollen was produced at wild-type levels (Figure 3D). Most flowers in the hot5 null mutants could not produce seeds; consequently, the siliques did not elongate normally (Figure 3E). There is no doubt that the failure of the hot5-2 and hot5-4 stamens to elongate properly contributes to the severely reduced fertility of these mutants. To determine whether the pollen and stigma of hot5-2 function normally for fertilization, we performed manual self-pollination and reciprocal test crosses between hot5-2 and wild-type plants (Table 1 ). Self-pollination of hot5-2 produced only 17.4 ± 6.2 (SD) seeds/silique, in contrast with 73.2 ± 5.3 seeds/silique for the wild type. In the reciprocal crosses, 23.1 ± 8.2 seeds/silique were generated using hot5-2 as the female with wild-type pollen, and 42.4 ± 7.7 seeds/silique were generated with hot5-2 pollen and wild-type females. These data indicate that in addition to reduced anther length, loss of HOT5 function compromises both the male and female functions required for fertilization and/or seed development.
GSNOR Affects Intracellular NO/Nitrosation Levels By metabolizing GSNO, a cytoplasmic reservoir of NO and a nitrosating species, GSNOR potentially modulates cellular NO status. To determine whether the absence of GSNOR indeed affects NO/nitroso levels, and how this is further affected by elevated temperature, we examined endogenous NO production using the NO-sensitive fluorescent dye 4-amino-5-methylamino-2',7'-difluorescein diacetate (DAF-FM DA) (Arnaud et al., 2006 ). For staining, protoplasts were prepared from leaves of 25-d-old wild-type and hot5-2 mutant plants either before or after heat stress. NO-dependent fluorescence signals were dramatically higher in the cytosol and chloroplasts of hot5-2 protoplasts compared with wild-type protoplasts from untreated leaves; in fact, no significant DAF-FM DA staining was observed in wild-type plants (Figure 4A
). The same high levels of DAF-FM DA staining were also observed in protoplasts of hot5-4 (data not shown). Despite the severe effect of heat on the viability of hot5-2 and hot5-4 leaf tissues, heat treatment led to only a minor increase in NO-related fluorescence in the wild type, and no apparent change was seen in the mutant when heat stress was performed prior to protoplast isolation. We were unable to visualize intact cells when protoplasts were heat stressed after isolation and stained, so we could not test for rapid or transient heat-induced changes in DAF-FM DA staining in protoplasts.
Feechan et al. (2005) reported that the hot5-2 mutant (named gsnor1-3 by this group) has increased SNO species compared with the wild type, and increased SNO levels were also reported for plants in which GSNOR was reduced using an antisense strategy (Rustérucci et al., 2007). To confirm this observation and to determine the effects of heat stress, we quantified total nitroso species in leaves from wild-type and hot5 null plants using gas-phase chemiluminescence (Feelisch et al., 2002) (Figure 4B). The hot5 null mutants were found to have approximately double the amount of nitroso species compared with the wild type, consistent with previous reports. Heat stress did not significantly change nitroso species levels in either the mutant or the wild type. Unexpectedly, nitrate levels were also markedly higher in the hot5 null mutants than in the wild type (Figure 4C), despite the fact that all plants had been grown at the same time on the same soil. The increase in nitrate content appears to be correlated with the increase in nitroso species concentration, suggesting a link between protein nitrosation and the nitrate assimilation pathway. Collectively, these results suggest that hot5 null mutants have an increased basal NO tone, which translates into a higher level of nitrosative stress. They further indicate that GSNOR is likely required to prevent excessive nitrosation of intracellular targets and that the effects of heat stress are minor compared with the effects of GSNOR mutation.
Endogenous NO Status Affects Heat Tolerance
Results of SNP and CPTIO treatments were also quantified by the measurement of chlorophyll content over time after heat stress in leaf discs from 25-d-old plants (Figure 5B). Four days after heat treatment, buffer-treated hot5-2 retained only  30% of chlorophyll and SNP-treated hot5-2 was fully bleached. By contrast, wild-type leaf discs retained high levels of chlorophyll in buffer alone, and when treated with SNP they retained 60% of their chlorophyll after 4 d. Treatment with CPTIO dramatically rescued the hot5-2 chlorophyll loss, with 75% of initial chlorophyll content remaining at 4 d after heat treatment. To show that the effect of CPTIO was specific to the hot5 mutant and not just a general effect of NO scavenging, we also tested the ability of CPTIO to rescue the heat sensitivity of the Hsp101 null mutant, hot1-3. In contrast with hot5-2, the thermotolerance defect of hot1-3 was not rescued by the NO scavenger, indicating that the heat-sensitive defect of hot5 is unique and distinct from the defect in the hot1-3 mutant (Figure 5B). We next determined whether the phenotypes observed for the wild type and hot5-2 in the presence of the exogenous NO scavenger or NO donor correlated with cellular NO status. Protoplasts were isolated at 2 h after heat treatment from leaf discs exposed to CPTIO or SNP. Treatment with CPTIO dramatically decreased the level of DAF-FM DA fluorescence in hot5-2 (Figure 5C) compared with buffer alone (Figure 4A). In addition, the DAF-FM DA fluorescence in the wild type was significantly increased by SNP treatment compared with buffer alone (Figure 4A). Thus, the heat-sensitive phenotype and NO/nitrosation levels are correlated. To confirm that excess NO or metabolites could also explain the thermotolerance defect of the weak hot5 missense mutations, we examined the effect of treatment with the NO donors and scavenger on the hypocotyl elongation of heat-treated, dark-grown hot5-1 seedlings (Figure 5D). Treatment of seedlings with these agents just before heat stress produced quantitative differences in subsequent elongation in the dark, consistent with the results with hot5-2 leaf discs. CPTIO very clearly enhanced the thermotolerance of hot5-1 seedlings, while SNP, but not KCN, increased the heat sensitivity of wild-type and hot5-1 seedlings, and addition of CPTIO with SNP reversed this effect. DETA/NO treatment also impaired the heat tolerance of both the wild type and hot5-1, although in addition it reduced hypocotyl growth at room temperature. In total, these data demonstrate the involvement of excess NO and/or nitrosative stress in the heat-sensitive phenotype of the missense mutations, confirming that the control of endogenous NO status is critical for survival of heat stress.
NO Status in the hot5 Missense Mutations Correlates with Heat Sensitivity
Although we were unable to determine the heat sensitivity of the hot5-2 and hot5-4 null alleles as dark-grown seedlings, to determine whether they had the same high DAF-FM DA staining phenotype as the missense alleles when grown in the dark, null mutant seeds were grown in the dark to generate root material (Figure 6C). When stained with DAF-FM DA, these null mutant roots also showed very high levels of fluorescence (Figure 6D). Light-grown seedlings of the same age also had high levels of DAF staining (data not shown). Thus, the missense and null alleles of hot5 share the inability to regulate NO status with dark-grown seedlings, further confirming that this phenotype results from the hot5 mutations.
The NO-Overproducing nox1 Mutant Shows a Thermotolerance Defect Correlated with NO Status
We also tested thermotolerance in the noa1 mutant (formerly nos1), which produces less endogenous NO (Crawford et al., 2006 ), and a nitrate reductase-deficient mutant, nia1/nia, which exhibits minimal nitrate reduction and must be grown on an alternative nitrogen source (Wang et al., 2004). Both 2.5-d-old dark-grown seedlings and 10-d-old light-grown seedlings were indistinguishable from wild-type seedlings in their heat tolerance (see Supplemental Figure 4 online).
By analyzing both missense and null mutations of the gene encoding GSNOR, we have uncovered an important role for this enzyme in modulating cellular NO levels and nitrosation status in plants. Specifically, we demonstrated that GSNOR function is required for acclimation to high temperature and for normal plant growth and fertility. Previous studies supported the conclusion that GSNOR, a type III alcohol dehydrogenase originally associated with the detoxification of formaldehyde (Uotila and Koivusalo, 1979 ; Giese et al., 1994; Martínez et al., 1996; Dixon et al., 1998; Achkor et al., 2003), acts in plants as well as other organisms to metabolize GSNO (Sakamoto et al., 2002; Feechan et al., 2005; Rustérucci et al., 2007). GSNOR is a potentially significant player in the modulation of cellular NO status because it effectively removes GSNO, a compound with NO-generating and thiol-nitrosating (NO+-transferring) potential, from the cellular pool. GSNOR will also act to regulate the availability of GSNO for glutathiolation reactions, in which it acts by modifying other cellular thiols, including those on proteins, to form mixed disulfides (R-SSG). This reaction has the potential to affect the redox status and activity of proteins; in addition, it gives rise to the formation of nitroxyl (NO–), a redox cousin of NO with a biological action profile distinct from that of NO (Fukuto et al., 2005). Thus, the effects we describe on thermotolerance, plant growth, and fertility may be mediated by several different pathways or by multiple mediators acting in concert. Although GSNOR does not directly act on S-nitrosated protein substrates, GSNOR knockout mice, Arabidopsis, and yeast cells all showed increased SNO levels (Liu et al., 2001, 2004; Feechan et al., 2005; Rustérucci et al., 2007). Our studies confirm and extend these results, indicating that GSNO modulates cellular nitrosation status. Consistent with this notion, the nitroso content of leaves from the hot5 mutants was about twice that of wild-type leaves, and the fluorescence signal obtained with the NO probe, DAF-FM DA, was clearly higher in the mutants compared with the wild type. This family of fluorescence probes senses NO utilizing nitrosation chemistry following the oxygen-dependent conversion of NO into RNS and the chemical conversion of the weakly fluorescent precursor into a more highly fluorescent molecule (Rodriguez et al., 2005). Thus, a higher fluorescence signal is not necessarily indicative of the presence of free NO but is an integrated readout of cellular nitrosation chemistry (Rodriguez et al., 2005). In aggregate, GSNOR activity and regulation must be recognized as affecting processes controlled by NO-related pathways in plants. The direct cause of the heat sensitivity of the hot5 mutants is not known. Assessment of the levels of major HSPs indicated that GSNOR mutants were not defective in the production of these protective proteins. The connection of heat sensitivity to excess nitrosation, however, is demonstrated by several observations. First, intense NO-related fluorescence staining was observed in dark-grown seedlings of the HOT5 missense mutants (hot5-1 and hot5-3), which is where the heat-sensitive phenotype is exhibited, and not in light-grown seedlings, which are not heat-sensitive. Second, decreasing NO levels with the NO-scavenger CPTIO partially rescued the heat-sensitive phenotype of both dark-grown hot5-1 and hot5-3 and light-grown hot5 null mutants. Conversely, increasing NO with the NO donors SNP and DETA-NO increased the heat sensitivity of wild-type seedlings and leaves. Finally, the NO-overproducing nox1/cue1 mutant showed NO-correlated thermotolerance defects. These observations support the hypothesis that elevated levels of GSNO enhance heat sensitivity due to the perturbation of pathways sensitive to reactive oxygen species/RNS, which are likely already under strain due to heat stress. Although both the hot5-1 and hot5-3 missense mutants had reduced GSNOR activity compared with the wild type, it is very interesting that we did not see a significant difference in GSNOR activity in the missense mutants when comparing dark-grown seedlings and 25-d-old plants. The missense mutants accumulated DAF-FM DA–staining species and showed the thermotolerance defect only as dark-grown seedlings, suggesting that GSNOR activity might be lower in the dark than in the light in these mutants. The fact that DAF-FM DA staining was also seen in dark-grown hot5-2 and hot5-4 null mutants further supports the idea that this phenotype results from reduced GSNOR activity. We suggest several possible reasons for the apparent discrepancy between the significant GSNOR activity detected in total extracts of dark-grown missense mutants and their DAF-FM DA staining. First, it is possible that there are overall higher levels of GSNO production/flux in dark-grown seedlings and, therefore, higher GSNOR activity is required in the dark for the removal of these species to enable proper growth after heat stress. It is also possible that in light-grown seedlings other components are present that compensate for the reduced GSNOR activity in the mutants and limit the accumulation of excess nitroso species. Another hypothesis is that the hot5-1 and hot5-3 proteins have altered regulatory properties compared with the wild-type protein, being inactive in the dark, and that our extraction conditions relieve this inactivation (e.g., dissociation of an inhibitor or removal of a labile inhibitory modification). This interesting phenotype of the missense mutants no doubt reflects the complexity of the tissue- and environment-specific regulation of nitroso species in plants.
Our data do not suggest that GSNOR is a regulatory player in thermotolerance; we have no evidence that NO is involved in heat stress signaling. Although there is one previous report that NO levels increased during heat stress (Gould et al., 2003
The general importance of GSNOR in plants is emphasized by its ubiquitous presence throughout the plant. While we have documented the expression of GSNOR by protein gel blot analysis in all organs examined, others have visualized its presence using immunocytochemistry (Barroso et al., 2006
Despite the significant role that GSNOR may play in the regulation of nitrosative stress, studies devoted to understanding the role of this enzyme in plants are quite limited. The focus of studies to date has been on the role of GSNOR in pathogen defense pathways and formaldehyde metabolism (Martínez et al., 1996
The hot5 null mutants have more than one defect that leads to reduced fertility. The stamens do not elongate properly, such that anthers remain below the stigma surface at the time of anthesis (Figure 2D). Furthermore, results of self-pollination and reciprocal crosses to the wild type indicate poor function of both the male and female gametophytes of hot5 null mutants (Table 1). Interestingly, release of NO has been proposed as one signal involved in pollen tube repulsion from the ovule after fertilization (Johnson and Lord, 2006
Consistent with its constitutive expression throughout the plant, HOT5 appears to be required during the entire life of the plant. In addition to the fertility defect, hot5 null mutants had an increased number of flowering stalks, multiple short shoots, and were long-lived plants that continued to produce leaves even after wild-type plants had senesced. These phenotypes may be linked in some way to the reduced fertility. Furthermore, while hot5 null mutant seeds could germinate on plant growth medium plates, all further growth was arrested, although seeds could be germinated in soil to produce mature plants. We were unable to rescue the germination phenotype of the hot5-2 null mutant on plant growth medium, including the NO scavenger CPTIO (100 mM to 1 mM) (data not shown). We also observed that hot5-2 was significantly less vigorous and had reduced chlorophyll when grown under long days (16 h light) (growth conditions used by Feechan et al. [2005]
NO is also reported to delay flowering through effects on both photoperiod and autonomous flowering time determinants (He et al., 2004
In addition to increased total NO adducts and dramatically enhanced NO-related fluorescence staining, an unexpected finding was that the leaves of hot5 mutants have roughly twice the concentration of nitrate compared with wild-type leaves. Thus, the pattern of nitrate content under basal conditions mirrors that of levels of nitroso species, suggesting a link between protein nitrosation and nitrate assimilation. Since the plants used in the biochemical studies were all grown on the same substrate (soil), the higher levels of nitrate in the hot5 null mutants cannot be attributed to differences in nutrient availability. Rather, they must result from differences in uptake and transport or in nitrate consumption along the nitrate assimilation pathway. However, given the complexity of nitrogen metabolism, its multilayered regulation and connection to other metabolic pathways (Stitt et al., 2002
Although the major role for GSNOR is now proposed to be in GSNO metabolism, the enzyme is nevertheless capable of acting in formaldehyde detoxification. In plants, one-carbon (C1) metabolism can generate formaldehyde, which can react spontaneously with GSH to form S-hydroxymethylglutathione. GSNOR acting as a FALDH oxidizes S-hydroxymethylglutathione to S-formylglutathione. It is clear that GSNOR exhibits FALDH activity in vivo in plants and other organisms. The FALDH enzyme in the aerobic soil bacterium Paracoccus denitrificans is critical for methyltrophic growth (Ras et al., 1995 In addition to the heat-stress phenotype of hot5 null mutants, photoperiod-dependent phenotypes suggest that these mutants may be sensitive to other abiotic stresses. However, when either hot5-1 dark-grown hypocotyls or hot5-2 light-grown leaf discs were treated in salt, cold, or high osmotic conditions, the hot5 mutants were not more sensitive than wild-type plants (data not shown). We cannot rule out the possibility that GSNOR regulation of NO status may be important under stress conditions not yet tested. In summary, GSNOR regulates cellular nitrosation levels by metabolizing GSNO, which is a mobile reservoir of NO in plant cells. Therefore, understanding the mechanisms that regulate the activity of GSNOR is a critical aspect of the study of the overall regulation of NO-related signaling and nitrosative stress in plants.
Plant Materials and Thermotolerance Assays Arabidopsis thaliana seeds of the indicated genetic backgrounds and genotypes were surface-sterilized, planted on nutrient medium plates (Haughn and Somerville, 1986 ) containing 0.5% (w/v) sucrose, and kept at 4°C for a minimum of 3 d. Plates were prepared with exactly 10 mL of medium on a leveling table to ensure even heat treatment and were sealed after planting with Parafilm to prevent desiccation. Plants were grown in illuminated growth chambers (100 µmol·m–2·s–1) on a 22/18°C, 12- or 8-h day/night cycle for analysis of growth phenotypes. To obtain mature hot5-2 and hot5-4 plants, heterozygous seeds were sown directly on soil, and all plants were genotyped by PCR to identify the homozygotes. Note that because of the reduced vigor of null mutants under long days, material for all stress and other physiological assays of these mutants was obtained from plants grown under 12 h of light. For thermotolerance assays, 2.5-d-old dark-grown and 10-d-old light-grown seedlings were treated as described (Hong and Vierling, 2000). For the leaf disc assay, discs (5 mm in diameter) were punched from third to fifth fully expanded rosette leaves of 25-d-old soil-grown plants and then floated on 2 mL of 10 mM MES-KOH buffer, pH 6.8, on 12- or 24-well microplates. Plates were incubated at 22°C only, at 38°C for 90 min, or at 38°C for 90 min, followed by 2 h at 22°C and 2.5 h at 45°C. Leaf discs were returned to 22°C under 12 h of light/12 h of dark and photographed 5 d later.
We also tested thermotolerance in the noa1 mutant (formerly nos1), which produces less endogenous NO (Crawford et al., 2006
NO-Related Chemical Treatments The NO donor DETA/NO as well as the NO donor SNP and the NO scavenger CPTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidadazoline-1-oxy-3-oxide] were dissolved in 10 mM PBS, pH 7.4, and used to treat seedlings on plates at final concentrations from 1 mM to 10 µM. KCN (100 mM) and 100 mM SNP plus 100 mM CPTIO were used as negative controls. For the hypocotyl elongation test, seeds were sterilized and plated in rows on 2-mL nutrient medium plates containing 0.5% (w/v) sucrose on 3.5-cm circular plates, which were wrapped in foil. Plates were incubated at 4°C for a minimum of 3 d and then placed in a vertical position at 22°C for 3 d. One hour before heat treatment (38°C for 90 min followed by 2 h at 22°C and then 2.5 h at 45°C), plates were briefly opened under dim green light and treated with the NO-related chemicals. Two milliliters of solution was added on plates, which were placed in a horizontal position for 1 h at 22°C. For heat treatment, the remaining solutions were poured out and the plates were rewrapped in foil. Hypocotyl lengths were measured after an additional 2.5 d in the dark.
Identification of hot5 Mutant Alleles Single knockout mutants of the HOT5 gene were obtained from the GABI (German Plant Genomics Program; 315D11; hot5-2 in the Col background) and FLAG (Versailles Genomic Resource Center; FLAG_298F11; hot5-4 in the Ws background) T-DNA collections using the accession number of HOT5 (At5g43940) in the database (http://signal.salk.edu/). Homozygous mutants were identified by PCR analysis using the recommended primers from each T-DNA collection. While some seeds could be recovered from the null mutants, routine experiments were performed by identifying the homozygous mutants by PCR from among the progeny of the heterozygotes. Tilling analysis (in the Col ecotype, carrying the erecta mutation) was performed on the HOT5 gene, encompassing approximately amino acid residues 96 to 379 (Arabidopsis Tilling Resource; http://tilling.fhcrc.org:9366). The hot5-3 mutant was recovered as a hypocotyl thermotolerance-defective mutant from a total of eight missense mutations analyzed. Both the homozygous hot5-1 and hot5-3 missense mutant alleles were backcrossed to Col wild-type plants, and one homozygous F3 line for each mutation was used for phenotypic analyses. For the T-DNA null mutant alleles, heterozygous hot5-2 or hot5-4 plants were backcrossed to Col or Ws wild-type plants, respectively, and then genotyped to identify wild-type, heterozygous mutant, and homozygous mutant plants. All homozygous plants were finally obtained after two backcrosses.
Purification of the HOT5 Protein
SDS-PAGE and Protein Blot Analysis
Measurement of Nitrate, Nitroso Species, and Chlorophyll Content
Chlorophyll was extracted from individual leaf discs by boiling in 95% methanol. Chlorophyll concentration was normalized to the fresh weight of the leaf discs and calculated as described (Lichtenthaler, 1998
Imaging of NO Status in Arabidopsis Protoplasts Protoplasts were resuspended in 150 µL of 25 µM DAF-FM DA, 0.4 M mannitol, 15 mM MgCl2, and 4 mM MES/KOH, pH 5.7, and allowed to incubate for 15 min at 22°C in the dark. DAF-FM DA treatment of hypocotyls was performed under complete darkness or green dim light to minimize light exposure. Plants were treated at 22°C only, at 38°C for 90 min, or at 38°C for 90 min followed by 2 h at 22°C and then 2 h at 45°C. All samples for NO visualization were isolated at 2 h after heat treatments and compared with room temperature treatments.
Confocal Microscopy
Measurement of HOT5 Enzyme Activity
Accession Numbers
Supplemental Data
We are indebted to Nathan S. Bryan and John Celenza for their assistance during pilot work related to this project, to Maria-Francisca Garcia-Saura for help with the nitrate measurements, and to Katrina Miranda for the synthesis of the DETA NONOate as well as for many helpful discussions concerning NO chemistry. We also thank William Montfort and members of his laboratory for investigations of GSNOR structure and biochemistry that helped inform this work and Ronan Sulpice of the Max Planck Institute for Molecular Plant Physiology (Potsdam/Golm, Germany) for advice on the assay of GSNOR activity in whole plant extracts. This work was supported by USDA National Research Initiative Competitive Grants Program Grant 3510014857 to E.V.
1 Current address: Warwick Medical School, University of Warwick, Gibbet Hill Campus, Coventry CV4 7AL, UK.  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.plantcell.org) is: Elizabeth Vierling (vierling{at}email.arizona.edu).
[W] Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.107.052647 Received May 2, 2007; Revision received February 4, 2008. accepted February 15, 2008.
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INTRODUCTION
-aminocaproic acid, pH 8.0), centrifuged to remove insoluble material, and then clarified with a desalting column (Zeba desalting column; Pierce). Enzyme activity was determined at 25°C by incubating the desalted fraction (10 µL) in 180 µL of 0.1 M phosphate buffer containing 10 µL of 6 mM NADH as cofactor and 10 µL of 6 mM GSNO as substrate. GSNOR activity was monitored for 1 min after the addition of NADH using an Agilent 8453 UV spectrophotometer. The rates were corrected for background NADH decomposition of each extract containing no GSNO. Rates were averaged over selected intervals during which the absorbance decline was linear. Final NADH decomposition values were normalized against total protein amount. Data are means of three independent experiments. 
