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First published online November 17, 2004; 10.1105/tpc.104.023622 © 2004 American Society of Plant Biologists
The Role of
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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1-Pyrroline-5-carboxylate dehydrogenase (P5CDH), the second enzyme for Pro degradation, is encoded by a single gene expressed ubiquitously. To study the physiological function of P5CDH, T-DNA insertion mutants in AtP5CDH were isolated and characterized. Although Pro degradation was undetectable in p5cdh mutants, neither increased Pro levels nor an altered growth phenotype were observed under normal conditions. Thus AtP5CDH is essential for Pro degradation but not required for vegetative plant growth. External Pro application caused programmed cell death, with callose deposition, reactive oxygen species production, and DNA laddering, involving a salicylic acid signal transduction pathway. p5cdh mutants were hypersensitive toward Pro and other molecules producing P5C, such as Arg and Orn. Pro levels were the same in the wild type and mutants, but P5C was detectable only in p5cdh mutants, indicating that P5C accumulation may be the cause for Pro hypersensitivity. Accordingly, overexpression of AtP5CDH resulted in decreased sensitivity to externally supplied Pro. Thus, Pro and P5C/Glu semialdehyde may serve as a link between stress responses and cell death. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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1-pyrroline-5-carboxylate (P5C). Pro synthesis is catalyzed by two enzymes, P5C-synthetase (P5CS) and P5C-reductase, both using NADPH as a cofactor. Genes encoding the two enzymes for Pro anabolism have been identified in several plant species and reported to be upregulated in response to water deprivation and high salinity (Hare et al., 1999
). P5CS appears to catalyze the rate-limiting step in Pro biosynthesis because overexpression led to increased Pro levels, whereas under stress conditions P5CS antisense repressed plants were unable to accumulate Pro to levels as in the wild type (Nanjo et al., 1999). The degradation of Pro is catalyzed by sequential action of two mitochondrial enzymes, Pro-dehydrogenase (ProDH) and P5C-dehydrogenase (P5CDH). ProDH is bound to the inner mitochondrial membrane with its active site facing the matrix, whereas P5CDH is located in the mitochondrial matrix (Elthon and Stewart, 1981). Arabidopsis ProDH and P5CDH have been characterized at the molecular level (Kiyosue et al., 1996; Peng et al., 1996; Verbruggen et al., 1996; Deuschle et al., 2001). AtProDH may be upregulated by Pro, hypoosmolarity, and dark adaptation (Hayashi et al., 2000). Cofactors of ProDH are unknown, whereas P5CDH uses NAD+ or NADP+ as electron acceptors, with a preference for NAD+ (Forlani et al., 1997). P5CDH is inducible by externally supplied Pro, but induction is retarded compared to ProDH (Deuschle et al., 2001). ProDH inhibition by knockout or antisense repression resulted in no or only marginal increases in Pro levels under normal conditions. However, during stress, Pro levels were several-fold higher as in the wild type (Nanjo et al., 1999, 2003; Mani et al., 2002). Both ProDH antisense plants and prodh knockouts were sensitive to treatment with Pro during early seedling development. No data have been presented so far to explain whether this is because of a specific requirement for Pro degradation during seedling development or because of accumulation of Pro or Pro metabolism intermediates.
Accumulation of Pro is observed under water limiting conditions (e.g. drought, salinity, or cold stress) and during developmental desiccation processes, such as pollen maturation (Chiang and Dandekar, 1995; Hare and Cress, 1997). During recovery from stress, accumulated Pro is rapidly oxidized to Glu, thus serving as source of nitrogen and energy. Furthermore, Pro is an important energy source during pollen tube elongation or in insect flight (Dashek and Harwood, 1974; Zhang et al., 1982; Gade and Auerswald, 2002).
Despite its protective role under a variety of stress conditions, external supply of Pro was found to be toxic to plant and animal cells (Hellmann et al., 2000; Maxwell and Davis, 2000; Deuschle et al., 2001; Donald et al., 2001). It has been proposed that Pro-induced damage is mediated by P5C/GSA, which, if not metabolized rapidly, might induce cell death (Hellmann et al., 2000). Consistently, yeast strains deficient in P5CDH were hypersensitive to Pro (Deuschle et al., 2001; Nomura and Takagi, 2004). In humans, defects in HsP5CDH result in type 2 hyperprolinemia, with patients experiencing seizures and variable degrees of mental retardation (Geraghty et al., 1998; Morita et al., 2002). Furthermore, both overexpression of ProDH or P5C treatment (400 µM) induced apoptosis in human tumor cell lines (Maxwell and Davis, 2000), and Donald et al. (2001) reported ProDH-dependent formation of reactive oxygen species (ROS) in human cells and induction of apoptosis. HsP5CDH seems to be a direct target of the mammalian tumor suppressor protein p53 and inhibition of expression enhanced p53-induced cell death (Yoon et al., 2004). Thus, in many organisms, synthesis, transport, and degradation of Pro and P5C/GSA must be tightly regulated to prevent cell death.
To study Pro degradation in plants, a yeast p5cdh mutant was used to clone the Arabidopsis P5CDH gene (AtP5CDH) by functional complementation (Deuschle et al., 2001). AtP5CDH is a single copy gene. Other dehydrogenases share <16% similarity on the amino acid level. Delayed induction of AtP5CDH relative to ProDH after external supply of Pro further strengthened the hypothesis that accumulation of P5C/GSA caused the symptoms of Pro-induced cell death. According to this model, P5CDH may have an important function in preventing damaging effects of P5C/GSA and subsequent cell death.
To analyze the physiological role of the ubiquitously expressed P5CDH in planta, two T-DNA insertion lines for AtP5CDH were isolated. Degradation of Pro was undetectable in p5cdh-1, indicating that P5CDH is the primary enzyme for P5C/GSA degradation. Surprisingly, the defect in P5CDH did not lead to visible alterations in phenotype under normal growth conditions, suggesting that Pro degradation is not essential for vegetative plant growth. p5cdh mutants were hypersensitive to external Pro supply and accumulated both Pro and P5C. Hypersensitivity of the p5cdh mutants to Pro, Orn, and Arg and increased tolerance of P5CDH overexpressors support the hypothesis that P5C/GSA is the causative agent of cell death induced by Pro supply.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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; Ayliffe et al., 2002). Consistently, induction of AtP5CDH in Arabidopsis was detected around lesions induced by spraying with Pro (Figures 2K and 2L).
Isolation of P5CDH Insertion Mutants
To study the role of P5CDH, insertion mutants were generated in which P5CDH expression was impaired. Screening for T-DNA insertions using the Arabidopsis Knockout Facility (University of Wisconsin) led to the identification of p5cdh-1 carrying a T-DNA in the 13th of 16 exons of AtP5CDH (Figure 3A). RNA gel blot analyses detected residual amounts of a truncated transcript, whereas protein gel blot analyses using polyclonal antibodies raised against purified potato (Solanum tuberosum) P5CDH did not show detectable amounts of P5CDH in various organs of p5cdh-1 (Figure 3B). A second insertion mutant (p5cdh-2) in the 9th exon lacking detectable transcript in the original size was identified in the Salk Insertion Sequence Database (Alonso et al., 2003) (Figures 3A and 3B). The insertion in the 9th exon is located upstream of the conserved aldehyde dehydrogenase domain, thus probably leading to a complete loss of function. In both p5cdh mutants, ProDH mRNA levels were not altered significantly as compared to the wild type (data not shown).
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; Mani et al., 2002). After salt treatment, both p5cdh-1 and the wild type accumulated Pro approximately threefold relative to pre-stress conditions. Thus, in contrast with ProDH inhibition, a block of degradation in P5CDH does not result in higher accumulation of Pro under stress conditions (Figure 5B). Consistent with the unchanged Pro levels, in a gauntlet with adverse conditions, such as salt stress, drought, temperature stress, light stress, and release of stress, no significant visible differences between the wild type and mutants were observed. These findings and the absence of growth defects of a prodh mutant (Nanjo et al., 2003) suggest that P5CDH and Pro degradation are not essential for survival under stress conditions or even during recovery after stress release. These findings prompted us to focus on the proposed function of Pro degradation in cell signaling (Deuschle et al., 2001).
Induction of Cell Death by Pro Supply
Spraying Arabidopsis leaves with Pro or its degradation product P5C led to cell death (i.e., foci of dead cells), a phenotype comparable to leaf spot disease or ozone induced lesions (Figure 6A) (Rao and Davis, 1999; Pilloff et al., 2002). The damaged tissue accumulated phenolics, callose, and H2O2 as indicated by autofluorescence, aniline blue, and 3,3-diaminobenzidine (DAB) staining, respectively. Trypan blue staining confirmed cell death in the center of the lesions (Figures 6B to 6D). Pro-dependent induction of cell death seems to be signal mediated because transgenic NahG plants, in which the stress hormone salicylic acid is degraded, were less sensitive to external Pro in the dark (Figure 6E) (Bowling et al., 1994). Mutants with altered induction of hypersensitive cell death in response to pathogens (e.g., eds1 and eds8 [enhanced disease susceptibility]) were hypersensitive to Pro (Figure 7). Eds1 was found to encode a protein with similarity to eukaryotic lipases, whereas eds8 has not been characterized at the molecular level yet (Falk et al., 1999). Also, ndr1 (non-race specific disease resistance) and ecotype Cvi-0 (Cape Verde Islands) displayed hypersensitivity to Pro. Cvi-0 plants were reported to accumulate higher levels of salicylic acid as compared to other ecotypes in response to ozone treatment, whereas Ndr1 encodes a presumptive membrane-associated protein required for salicylic acid–dependent, resistance gene–mediated pathogen defense (Century et al., 1997; Rao et al., 2000).
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). Addition of Pro in the presence of salt ameliorated toxicity shown by improved growth (Figure 8A).
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p5cdh Mutants Are Pro Hypersensitive
The p5cdh mutant lines were used to assess the involvement of P5CDH in Pro-induced cell death. Previous studies suggested that accumulation of the Pro degradation intermediate P5C/GSA may be the causing agent of the observed stress responses. Thus, p5cdh mutants are expected to accumulate P5C and therefore be hypersensitive toward externally supplied Pro.
A method for P5C determination was developed for plant extracts. P5C was below the detection limit (<0.05 µmol/g fresh weight) in untreated Columbia-0 (Col-0), Wassilewskija (Ws), and p5cdh-1 and -2 mutant plants, as well as in Pro-treated Col-0 and Ws wild-type plants (Table 1). P5C was detected only in the p5cdh mutants when exposed to Pro. A 24-h exposure of p5cdh-2 to 10 mM Pro was sufficient to obtain P5C levels that were more than threefold above the detection limit. When exposed to 60 mM Pro, P5C was more than threefold higher as compared to treatments with 10 mM Pro. Longer treatments did not further affect P5C levels (data not shown).
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Treatment of 3-week-old p5cdh-1 plants with 100 mM Pro significantly enhanced expression of 1320 genes, many of which have a proposed function in signal transduction, secondary metabolism, or stress defense. A total of 317 genes were significantly downregulated, 64 of them are predicted photosynthesis-related genes, which is in agreement with the observed bleaching of the p5cdh mutant lines grown on Pro (see Supplemental Table 1D online).
Alternative Sources of P5C Cause Symptoms Similar to Pro
The effect of Orn and Arg on p5cdh-1 and -2 was tested to analyze whether other compounds producing P5C have similar effects as Pro. Orn also induced toxicity in Arabidopsis wild-type plants, and p5cdh mutants were hypersensitive to Orn. Symptoms resembled those of Pro treatment, but were less severe (Figure 9C). Supply of Arg as the sole nitrogen source in the medium led to a dwarf phenotype, shortened roots, and premature bolting and flowering in axenic culture (Figure 9D). Inhibition of root elongation by Arg was more severe in p5cdh-1 than in the wild type. Hypersensitivity of p5cdh mutants to Orn and Arg further supports the hypothesis that cell death induced by Pro supply is linked to P5C accumulation.
P5CDH Overexpression Leads to Reduced Pro Sensitivity
Hypersensitivity of p5cdh mutants to Pro and other agents degraded via P5C as well as the direct demonstration of P5C accumulation in p5cdh mutants after Pro supply indicated that P5C/GSA accumulation may induce cell death. Thus, increased P5CDH activity should lead to amelioration of Pro-induced cell death. P5CDH was overexpressed under control of the 35S promoter of Cauliflower mosaic virus. Under normal growth conditions, plants overexpressing P5CDH were indistinguishable from the wild type, but consistent with a function of P5CDH in protection from P5C effects, the overexpressing lines tolerated higher concentrations of external Pro than the wild type (Figure 10).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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). Surprisingly, external supply of Pro, or the degradation intermediate P5C/GSA, were found to be toxic (Bonner et al., 1996; Hellmann et al., 2000; Deuschle et al., 2001; Hare et al., 2002). Although synthesis has been studied extensively, little is known about the events after stress relief, when Pro is degraded. Here, we show that the second enzyme of Pro catabolism, P5CDH, is essential for Pro degradation. Furthermore, we provide evidence that the induction of cell death by Pro is caused by P5C/GSA, which triggers a salicylic acid–mediated signaling cascade leading to cell death. An increase in the capacity to metabolize P5C/GSA consistently leads to a protection from cell death induced by Pro supply.
Role of P5CDH in Metabolism under Normal Growth Conditions
Promoter analysis revealed expression of P5CDH in all tissues, especially in pollen, where ProDH is also expressed to high levels (Nakashima et al., 1998). Pollen grains are known to have a high content of free Pro, supposedly functioning as an osmolyte during the desiccation process and as an energy source during pollen germination (Schwacke et al., 1999). To obtain more information about Pro degradation and P5CDH function, two insertion mutants for P5CDH were isolated. Both mutant lines were visibly indistinguishable from the wild type under normal growth conditions, and also the level of free Pro was not significantly elevated in the p5cdh-1 mutant. However, no degradation of externally supplied Pro was detected, indicating that alternative Pro degradation pathways not using P5CDH do not exist. Pollen germination and pollen tube growth in vitro were unaffected in p5cdh-1, suggesting that Pro is not the only/major energy source of pollen tube growth (data not shown). Another situation where Pro might play an important role is during the early phases of development. Seed imbibition was recently reported to induce a fourfold increase in free Pro levels, and seedling development was inhibited by externally supplied Pro in ProDH-antisense lines (Mani et al., 2002; Hare et al., 2003). Because the germination rate of p5cdh mutants was reduced, Pro degradation seems to be important for seed maturation or germination.
P5C/GSA as a Feedback Regulator for Pro Biosynthesis and Degradation
Because Pro levels in p5cdh-1 were not significantly higher as compared with the wild type both under stress or nonstress conditions, Pro synthesis must be subject to allosteric regulation. Feedback inhibition of Pro synthesis by Pro had previously been described (Hu et al., 1992; Zhang et al., 1995; Hong et al., 2000). Inhibition of Pro degradation by knockout or antisense repression of ProDH resulted in no or only marginal increases in Pro levels under normal conditions. However, during stress, Pro levels increased severalfold higher than in the wild type (Nanjo et al., 1999, 2003; Mani et al., 2002). Enhanced accumulation of Pro in prodh but not p5cdh mutants was unexpected and indicates the existence of a feedback mechanism that determines the upper limit of Pro accumulation and is dependent on ProDH but not P5CDH activity. It is conceivable that the degradation and synthesis intermediate P5C/GSA may be the signal limiting Pro biosynthesis under stress. Because synthesis and degradation occur in the cytosol and mitochondrial matrix, respectively, the P5C pools must communicate, potentially involving a P5C transporter in the inner mitochondrial membrane (proposed in Phang, 1985), or the P5C-generated signal must be perceived and converted within the mitochondria.
As in yeast, elevated amounts of P5C were observed only in p5cdh mutants after supply with exogenous Pro (Nomura and Takagi, 2004). In Arabidopsis, Pro levels were the same as in the wild type, and P5C levels were 30 times lower, indicating that ProDH activity was also impaired. It remains to be determined if ProDH activity is inhibited already by low concentrations of P5C or if accumulating P5C is exported into the cytosol and converted to Pro by P5C-reductase.
Role of P5CDH in Metabolism under Stress Conditions and in Pathogen Infection
During salt stress, Pro levels increased threefold both in p5cdh-1 and in the wild type. In all experiments performed, stress tolerance of p5cdh mutants was similar as in the wild type, supporting the hypothesis that accumulation of Pro is important for stress resistance, whereas redox shuttling between the mitochondria and the cytosol by means of Pro synthesis and degradation probably does not contribute to stress tolerance, as postulated for animal cells (Phang, 1985). The presence of high Pro levels in p5cdh-1 for extended periods after stress release did not seem to have obvious consequences.
The spectrum of stress symptoms induced by Pro application is very similar to the well-characterized hypersensitive response (HR) of plants to incompatible pathogens. Strikingly, Fabro et al. (2004) recently reported that plants undergoing HR accumulated Pro, probably because of upregulation of the key enzyme in Pro biosynthesis, P5CS, at and around the sites of HR. Further evidence for a function of Pro degradation or more specifically P5CDH in PCD derived from the regulation of the flax ortholog of AtP5CDH, LuFIS1 (Roberts and Pryor, 1995). LuFIS1 was upregulated around rust infection sites only during susceptible infection with flax rust but not when the plants were resistant because of a hypersensitive reaction (Ayliffe et al., 2002). The pathogen may induce P5CDH to prevent accumulation of P5C and induction of PCD by the plant. Expression of P5CDH around HR-like lesions might contribute to restrict HR to the area of infection and prevent uncontrolled spreading of cell death. Reduced sensitivity of salicylic acid–deficient NahG plants to exogenous Pro application in the dark but not in plants exposed to the normal dark–light cycles further supports the concept of a P5C-mediated signal because darkness was reported to induce ProDH expression (Hayashi et al., 2000). Damages occurring in the light seem to depend on salicylic acid–independent mechanisms, potentially mediated by ROS formation. Consistently, Cvi-0, an Arabidopsis ecotype overproducing salicylic acid in response to ozone stress, is hypersensitive to Pro (Rao et al., 2000). The link between P5C and pathogen-induced cell death is further substantiated by the finding that cpr5, eds1, eds8, and ndr1 are hypersensitive to Pro. Thus, what had been termed Pro toxicity is most probably not a direct toxicity of P5C/GSA but cell death induced via a signaling cascade.
P5C/GSA as a Downstream Effector after Pro Treatment
Symptoms of cell death observed after external Pro application included local browning, accumulation of fluorescent compounds, callose deposition, DNA laddering, and local cell death, and Arabidopsis plants died after long-term incubation, suggesting a link between P5C accumulation and PCD. Hare et al. (2002) showed disruption of chloroplast and mitochondria ultrastructure among the early symptoms of Pro application, which might be connected to the mitochondrial permeability transition frequently observed during the onset of apoptosis (Tiwari et al., 2002).
Interestingly, PCD was also observed when plants were exposed to salt stress (Katsuhara, 1997; Huh et al., 2002). It is therefore conceivable that Pro accumulation under salt stress or during HR to pathogens provides the link to cell death. This hypothesis may be tested by analyzing the effect of salt stress on PCD in P5CS mutants. By contrast, growth amelioration of NaCl-treated BY-2 suspension cultures by addition of Pro shows that Pro supply can have positive effects on plant growth, at least when supplied under stress conditions.
P5C application in animals and plants led to cell death–like symptoms similar to those observed as a consequence of external Pro supply, the simplest interpretation being that the effects of Pro treatment are mediated by P5C (Hellmann et al., 2000; Maxwell and Davis, 2000). P5C accumulation may be because of delayed induction of P5CDH relative to ProDH during external Pro supply (Deuschle et al., 2001). Consistently, P5C was detectable in p5cdh mutants after external Pro supply but not in the wild type. Hypersensitivity of p5cdh-1 mutants to other P5C-producing substances, such as Arg and Orn, may support the hypothesis that the cellular damage caused by Pro application is induced by P5C or P5C-derived signals.
A similar conclusion may be drawn from studies in human cell lines, where induction of ProDH expression by p53 or ProDH overexpression induced apoptosis (Polyak et al., 1997; Maxwell and Davis, 2000). Accordingly, HsP5CDH was also found to be a direct target of p53, and overexpression was able to reduce ROS accumulation caused by H2O2 or UV treatment (Yoon et al., 2004). Alternative to the hypothesis that an increase in P5C as a consequence of Pro treatment leads to cell death via a signaling cascade, it has been suggested that ROS triggering cell death are produced by a hypothetical Pro cycle (Yoon et al., 2004). In this model, both Pro and P5C cycle across the mitochondrial membrane. The oxidation of Pro to P5C by ProDH can contribute to the energy supply of the cell and enhance generation of ROS. P5CDH would normally remove P5C irreversibly from this cycle by converting it to Glu, reducing the production of ROS. The existence of a mitochondrial P5C/Pro shuttle remains to be demonstrated.
Unexpectedly, plants impaired in ProDH expression were also developmentally arrested by the supply of exogenous Pro (Mani et al., 2002; Nanjo et al., 2003). To differentiate between the possibilities whether P5C also accumulates in these mutants or whether other factors play a role in Pro-induced developmental arrest, it will be important to determine P5C and ROS levels in prodh mutants.
However, strong support for the role of P5C/GSA in Pro-induced cell death derives from constitutive overexpression of AtP5CDH in Arabidopsis, which resulted in decreased sensitivity to externally supplied Pro.
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METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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BY-2 tobacco (Nicotiana tabacum) suspension culture was grown in constant darkness in liquid culture. Fresh weight is an average of five independent treatments and measurements.
DNA Extraction from Tobacco BY-2 Cells
Tobacco BY-2 cells, a kind gift from T. Merkle (University of Freiburg, Germany), were maintained on an orbital shaker at 25°C in the dark in 125-mL Erlenmeyer flasks containing MS medium. Cells were subcultured once a week. At 48 h after subculturing, different concentrations of Pro and NaCl were added to the culture medium. At 48 h after treatment, tobacco BY-2 cells were collected by filtration and weighed, and genomic DNA was isolated according to Young and Gallie (2000). For DNA fragmentation analysis, 20 µg of each sample was resolved on a 1.8% TBE agarose gel.
Stress Treatment of Arabidopsis Plants
Cold stress was imposed by growth at 4°C or incubation at –20°C for different periods of time. Heat treatment was performed at 37°C. Salt stress was applied by either growth on MS medium supplemented with different concentrations of NaCl, by watering plants with water containing different concentrations of NaCl, or by transfer of plants grown on solid 2MS medium to liquid medium containing 200 mM NaCl. Light stress was applied by shifts from low light (50 µmol/s/m2) to high light (400 µmol/s/m2) and back. Drought stress was achieved by lack of watering of plants in soil for different periods of time.
Mutant Identification
The Wisconsin mutant population (Krysan et al., 1999) (www.biotech.wisc.edu/Arabidopsis/default.htm) was screened using the gene-specific primer 5'-TTATTAGTGGACACACCACTTCTAAGTAG-3'. Out of 94 plants from pool 4005, four plants carrying the p5cdh-1 insertion were recovered. For further analyses, the mutant was backcrossed twice to the Ws wild type.
Insertion mutant information about p5cdh-2 (Salk_018453) was obtained from the SIGnAL Web site at http://signal.salk.edu. The insertion was verified by PCR with primers designed by the SIGnAL iSect Web tool and sequencing of the obtained PCR product from the T-DNA left boarder.
Construction of Overexpressers, Antisense Lines, and GUS Plants
For stable transformation, Agrobacterium tumefaciens strain pGV2260 (Deblaere et al., 1985) was transformed with the mini binary vector pCB302.3 containing an AtP5CDH cDNA in sense or antisense orientation and used for transformation of Arabidopsis by floral dip (Clough and Bent, 1998; Xiang et al., 1999). The AtP5CDH coding sequence was amplified by PCR from a vector containing the cDNA from ecotype Landsberg erecta and inserted into pCB302.3 using the unique BamHI site. T0 seeds were sown in the greenhouse, and transformants were selected by spraying the plants with 0.1% BASTA (Aventis CropScience, Gent, Belgium) at the four leaf stage. AtP5CDH-GUS plants are described by Deuschle et al. (2001).
Array Analysis and RNA Gel Blot Analyses
Plants were cultured on solid MS medium containing 60 mM sucrose. Three-week-old plants were transferred to chambers (Weck round-rim jar 100) in which roots had contact with 10 mL of liquid MS medium supplemented with salt, sugars, or different amino acids. After 48 h of incubation, the roots were washed in distilled water, and the plants were rapidly frozen in liquid N2. Analysis of the expression profile at earlier time points (0.5 to 6 h) showed that transfer of plants led to high variability in both Pro-treated and untreated samples, thus, data were not considered. RNA extraction, gel electrophoresis, and blotting were performed according to Lehrach et al. (1977) and Logemann et al. (1987). Hybridizations were conducted according to Martin et al. (1992) using the cDNA of AtP5CDH labeled with [
32P]dCTP by random priming. For the array analysis, RNA from 3-week-old plants grown on solid 2MS and transferred to liquid MS medium supplemented with either 100 mM Pro or 100 mM glucose for 48 h was used. The array containing 3292 Arabidopsis plastidic protein GSTs, probing, and data analysis are described by Pesaresi et al. (2003) and Richly et al. (2003). The custom-made Arabidopsis transporter array carries gene-specific cDNA fragments (cGSTs) for 1274 (putative) membrane proteins with four or more transmembrane spans and 222 soluble proteins spotted on Hybond N+ filters. The filters were hybridized overnight with 33P-labeled cDNA in DIG EasyHyb solution (Roche, Indianapolis, IN) and washed two times for 5 min at 65°C in 2x SSC/0.1% SDS (1x SSC is 0.15 M NaCl and 0.015 M sodium citrate) and once for 20 min at 65°C in 0.2x SSC/0.1% SDS before exposure and evaluation.
Expression levels of AtP5CDH were determined by RT-PCR using primers specific for the coding sequence of AtP5CDH (forward, 5'-GCTTTTTGTTCATGAGAACTGGTC-3'; reverse 5'-CAGCCCGGGAGTAGATGGAGGAAGTTCCC-3'; 23 cycles), specific for endogenous AtP5DCH mRNA (forward, 5'-GCTTTTTGTTCATGAGAACTGGTC-3'; reverse, 5'-TTTATTTGATGCAACAGCACACTAAG-3'; 25 cycles), and for AtActin2 (forward, 5'-TCCAAGCTGTTCTCTCCTTG-3'; reverse, 5'-GAGGGCTGGAACAAGACTTC-3'; 20 cycles).
Staining Procedures and Detection
Histochemical GUS assays were performed according to Martin et al. (1992). Seedlings were stained for 24 h, flowers for 12 to 14 h.
For detection of dead cells, leaves were boiled for 3 min in trypan blue staining solution (10 mL of lactic acid, 10 g of phenol, 10 mL of glycerol, 10 mL of deionized water, 10 mg of trypan blue; freshly diluted 1:1 in 100% ethanol). Destaining was conducted overnight in chloral hydrate (2.5 g/mL).
H2O2 was visually detected in the leaves of plants using DAB. Briefly, leaves or whole plants were excised with a razor blade and supplied through the cut petioles or stems with a 1-mg/mL solution of DAB, pH4, for 10 h in the dark at 21°C. As positive control, leaves were wounded with a needle in the upper half of the leaf right before the DAB treatment. The experiments were terminated by immersion of the plants or leaves in warm ethanol (80%) until they were decolorized except for the deep brown polymerization product produced from DAB in the presence of H2O2.
H2O2 was also detected using dihydrorhodamine 123 (Sigma, St. Louis, MO), which was added from a 2.5-mg/mL stock solution in ethanol to give a final concentration of 5 µg/mL in MS medium. Seedlings were incubated for 30 min at room temperature and viewed under a Zeiss Axioplan epifluorescence microscope (Carl Zeiss, Oberkochen, Germany) through a rhodamine optical filter. For the detection of autofluorescence, plants were viewed directly under an epifluorescence stereomicroscope (Leica, Wetzlar, Germany) with a green fluorescent protein filter set. For callose visualization, plants were infiltrated with 4% paraformaldehyde in PBS and fixed overnight at 4°C. Plants were rinsed once in PBS before staining in 0.5% aniline blue in PBS, pH 8.5. Stained plants were mounted onto glass slides and viewed with a 4',6-diamidino-2-phenylindole filter set.
For viability staining, 0.5 µL 0.5% fluorescein diacetate solution was added to each 10-µL aliquot of BY-2 culture. Living cells accumulate green fluorescent fluorescein, and dead cells appear light blue because of autofluorescence.
Pro and P5C Determination
Pro content of 18-d-old Arabidopsis seedlings was determined according to Bates et al. (1973). The standard isolation protocol for amino acids proved to be unsuitable for P5C detection, thus a special assay had to be developed and was applied. Plant material was resuspended in 10 mL g-1 50 mM HCl and extracted in a Teflon-in-glass Potter homogenizer by 2 x 12 strokes. After centrifugation for 10 min at 18,000g, 10-mL aliquots of the supernatant were immediately loaded onto Dowex AG50 (200 to 400 mesh) columns (5 mL bed-volume) equilibrated with water. After extensive washing with 50 mM HCl, P5C was eluted with 1 M HCl, collecting 1.5-mL fractions. P5C and Pro content in the eluate were quantified with the o-aminobenzaldehyde and the ninhydrin assay method, respectively, as previously described (Williams, 1975).
14C Pro Feeding Studies
Rosette leaves from 4-week-old Arabidopsis plants were cut under 2.5 mM EDTA, pH 6, and fed through the petiole with 160 µM Pro solution (86 µM labeled Pro [U-14C Pro; Amersham Biosciences, Piscataway, NJ], 74 µM unlabeled Pro, and 60 mM sucrose in deionized water) for different periods of time. After incubation, the leaves were rinsed with distilled water, and the amino acids were analyzed using HPLC.
Determination of Amino Acids
One hundred milligrams of ground Arabidopsis material was extracted once in 0.5 mL of methanol/water (80% methanol [v/v], 3 min at 95°C) and the sediment once again in 0.5 mL of methanol/water (20% methanol [v/v], 3 min at 95°C) after centrifugation (3 min at 20000g). The supernatants were combined, and the liquid was evaporated to dryness in a speed vac. The sediment was redissolved in 150 µL of lithium diluent Li220 (Pickering Lab, Mountain View, CA), and 10 µL were separated by HPLC on a cation-exchange column on a Kontron HPLC system (Neufahrn, Germany). Single amino acids were detected and quantified photometrically at 440 and 570 nm after post-column derivatization with ninhydrin in a derivatization oven (Pickering).
Analysis of Organic Acids and Sugars
After sample preparation as for amino acid analysis, 10 µL of the sample were separated by HPLC on an Aminex HPX 87-H column (Bio-Rad, Hercules, CA) using 2 mM H2SO4 as eluant. The retention times of 14C-labeled compounds were monitored with an LB 507B radiodetector (Berthold, Wildbad, Germany).
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2 Current address: Department of Botany, Stockholm University, Lilla Frescativägen 5, 10691 Stockholm, Sweden.
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: Wolf B. Frommer (wfrommer{at}stanford.edu).
Online version contains Web-only data.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.104.023622.
Received April 22, 2004; accepted September 6, 2004.
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1-Pyrroline-5-Carboxylate Dehydrogenase in Proline Degradation
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