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First published online April 24, 2003; 10.1104/pp.102.016907 Plant Physiology 132:968-978 (2003) © 2003 American Society of Plant Biologists The Pyruvate decarboxylase1 Gene of Arabidopsis Is Required during Anoxia But Not Other Environmental Stresses[w]Institute of Plant Sciences, Altenbergrain 21, CH–3013 Berne, Switzerland
Ethanolic fermentation is classically associated with flooding tolerance when plant cells switch from respiration to anaerobic fermentation. However, recent studies have suggested that fermentation also has important functions in the presence of oxygen, mainly in germinating pollen and during abiotic stress. Pyruvate decarboxylase (PDC), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme. Here, we characterize the PDC gene family in Arabidopsis. PDC is encoded by four closely related genes. By using real-time quantitative polymerase chain reaction, we determined the expression levels of each individual gene in different tissues, under normal growth conditions, and when the plants were subjected to anoxia or other environmental stress conditions. We show that PDC1 is the only gene induced under oxygen limitation among the PDC1 gene family and that a pdc1 null mutant is comprised in anoxia tolerance but not other environmental stresses. We also characterize the expression of the aldehyde dehydrogenase (ALDH) gene family. None of the three genes is induced by anoxia but ALDH2B7 reacts strongly to ABA application and dehydration, suggesting that ALDH may play a role in aerobic detoxification of acetaldehyde. We discuss the possible role of ethanolic fermentation as a robust back-up energy production pathway under adverse conditions when mitochondrial function is disturbed.
The ethanolic fermentation pathway branches off the main glycolytic pathway at pyruvate. In the first step, pyruvate is the substrate of pyruvate decarboxylase (PDC), yielding CO2 and acetaldehyde. Subsequently, acetaldehyde is reduced to ethanol with the concomitant oxidation of NADH to NAD+ by alcohol dehydrogenase (ADH). In the present day aerobic atmosphere, ethanolic fermentation is used only by specialized organisms or under particular conditions. In plants, it has been studied because of its relevance in flooding tolerance where plant cells switch from aerobic respiration to anaerobic fermentation (for review, see Drew, 1997  ).
ADH has been the subject of numerous genetic studies, and adh mutants have been reported for a number of species, including maize (Zea mays; for review, see Freeling and Bennett, 1985
Overexpression of a bacterial PDC in transgenic tobacco resulted in constitutive high and active protein levels under both normoxic and anoxic conditions (Bucher et al., 1994
A new and exciting aspect of ethanolic fermentation is the suggested involvement in stress signaling and response to environmental stresses other than low oxygen (Tadege et al., 1999
Additional evidence indicating a role in biotic stress is provided by experiments with transgenic potato plants overexpressing bacterial PDC. These plants accumulated acetaldehyde and showed a lesion-mimic phenotype (Tadege et al., 1998
Although PDC and ADH gene induction has been demonstrated, ethanol and acetaldehyde production as a result of stress treatment has only been reported for red pine (Pinus resinosa) and birch (Betula spp.) seedlings exposed to sulfur dioxide, water deficiency, freezing, and ozone (Kimmerer and Kozlowski, 1982
One problem during aerobic stress is toxicity of reactive acetaldehyde. One of the pathways for the detoxification of aldehydes to less reactive forms is the oxidation to carboxylic acids by aldehyde dehydrogenase (ALDH). ALDH converts acetaldehyde to acetate concomitantly reducing NAD+ to NADH. In plants, ALDH is strongly expressed in germinating pollen of tobacco (op den Camp and Kuhlemeier, 1997 In this paper, we present results demonstrating the requirement for ethanolic fermentation during anoxia and its involvement during adaptation to other environmental stresses in Arabidopsis. Expression levels of the relevant mRNA from PDC, ADH, and ALDH were determined quantitatively. The identification of a pdc1 mutant by T-DNA insertional mutagenesis allowed us to study the function for PDC1 in Arabidopsis.
Characterization of the PDC Gene Family in Arabidopsis
Screening several gene and protein databases for Arabidopsis Columbia (Col-0) sequences with the protein sequence of tobacco PDC2 (Bucher et al., 1995
Preliminary experiments indicated that under standard growth conditions, each of the four PDC genes was expressed at low levels, at or below the limit of detection by RNA gel-blot analysis (data not shown). To analyze the mRNA accumulation of the four different genes coding for PDC, we used real-time quantitative RT-PCR technology (see "Materials and Methods" and supplementary material, which can be viewed at http://www.plantphysiol.org). RNA was isolated from various organs, and the abundance of the four PDC mRNA and the single-ADH mRNA in these tissues was quantified based on separate RNA isolations derived from three independent experiments (Fig. 2). ADH was expressed in all organs. The highest expression was observed in imbibed seeds (50-fold higher compared with 14-d-old seedlings). The expression profile of PDC1 closely matched ADH expression. Expression of PDC2, PDC3, and PDC4 could also be seen in most organs but, in contrast to ADH and PDC1, expression was not particularly high in imbibed seeds (Fig. 2A). The real-time RT-PCR method can also be used to compare absolute transcript abundances of genes in mRNA preparations (Fig. 2B). PDC1 transcripts were the dominant PDC mRNAs in roots, flowers, siliques, and seeds. In siliques and seeds, more than 96% of all PDC transcripts belonged to the PDC1 class. PDC3 was abundant in whole seedlings, roots, and shoots, whereas PDC2 and PDC4 transcripts contributed overall only minor proportions to the total PDC mRNA population.
In maize and Arabidopsis, strong induction of fermentation genes takes place in anaerobic conditions (Dolferus et al., 1985
To settle the question of whether PDC2, PDC3, and PDC4 are induced during anoxia to levels too low to be detected by classical RNA gel-blot analysis, quantitative RT-PCR technology was used. As a positive internal control we amplified the ADH RNA, which is known to be strongly induced by anoxia (Dolferus et al., 1985
The expression analysis revealed PDC1 to be the only strongly expressed gene of the PDC gene family during anoxia. PDC2, PDC3, and PDC4 are dramatically less abundant than PDC1 transcripts and we therefore focused further on the PDC1 gene. To study the role of PDC1 in Arabidopsis, the T-DNA insertion collection of the Torrey Mesa Research Institute was screened. A multiple sequence alignment with the PDC1 sequence against the database of the T-DNA-derived flanking sequences was performed, which resulted in an alignment representing a high probability of identity with a particular line in the collection. Using a primer from the T-DNA left border plus a gene-specific primer, a single PCR product was identified in this line, and subsequent sequencing of the fragment revealed an insertion in the third exon of PDC1, at position 1,312 bp after the ATG in the coding sequence (Fig. 4A). This mutant line was designated pdc1.
At position 1,385 bp in the inserted T-DNA sequence of pdc1, a potential stop codon was identified. Furthermore, the thiamine pyrophosphate-binding site (an essential cofactor of the enzyme) is located 85 bp after the insertion site in the wild-type coding sequence. This analysis indicated a complete disruption of the PDC1 gene. To confirm this, RNA was isolated from Col-0 and Ler wild type, and pdc1 (Col-0) and the adh mutant (Ler) were subjected to a 6-h anoxic period (Fig. 4B) and analyzed by RNA gel-blot analysis. PDC1 gene expression was observed in the two different wild-type accessions and in the adh mutant, but pdc1 mutant plants showed no transcripts at all after 6 h of anaerobic conditions. To investigate whether other members of the PDC gene family might compensate for PDC1, real-time RT-PCR analysis was performed. PDC1 transcripts in pdc1 mutants were not detectable either in normoxic or in anoxic conditions, confirming the data from the RNA gel-blot analysis (Fig. 4C). None of the other three genes showed increased transcript levels in pdc1 mutant seedlings treated by anoxia compared with the wild type. Thus, the three PDC genes do not compensate for a complete loss of PDC1 transcripts in pdc1 mutant plants by induced expression. ADH expression is not influenced in the pdc1 mutant. Conversely, PDC1 expression is not influenced in the adh mutant (Fig. 4B).
Development of pdc1 mutant plants and survival during anoxia were investigated. Under standard growing conditions, no morphological phenotype was apparent (data not shown). To reveal the possible consequences of a null mutation in PDC1, attention was focused on the survival of plants under anoxia. Studies on the differential adaptation of shoots and roots of Arabidopsis to low oxygen indicated that ethanol fermentation is essential in roots but not in shoots (Ellis et al., 1999
The induction of expression of the ADH gene in several species by environmental conditions such as cold, osmotic stress, or wounding suggested a role for PDC in these conditions. Therefore, we analyzed the behavior of PDC transcripts of Arabidopsis in seedlings subjected to a range of abiotic stresses. Seedlings were treated with cold, mannitol, ABA, salinity, wounding, and paraquat (a herbicide that exacerbates O2- radical production and is used to study oxidative stress; Penninckx et al., 1998
Next, we analyzed the stress-induced expression levels for all four PDC genes of Arabidopsis. PDC1 was induced by all treatments; the strongest induction was observed by ABA application (23-fold compared with the control treatment). Cold, salinity, mannitol, wounding, and paraquat induced PDC1 to comparable levels (8- to 10-fold). PDC2 was induced by several stresses compared with the control treatment (Fig. 5A). PDC3 and PDC4 mRNA levels were not markedly affected by any treatment. However, the absolute transcript abundance of PDC2 in the different stress-treated plants was always lower compared with PDC1 (Fig. 5B). PDC3 and PDC4 transcripts are in several treatments more abundant than PDC1, although these genes are not markedly induced by these conditions compared with the control treatment (Fig. 5, A and B). Because both PDC and ADH genes are inducible under stress conditions, we decided to measure the products of the pathway, acetaldehyde and ethanol, by gas chromatography. We found acetaldehyde production to be stimulated by ABA and paraquat treatment and during salt and wounding stress, but not during cold and mannitol treatments (Fig. 5C). In all cases, the amount of acetaldehyde and ethanol produced was much less than under anoxia. The expression data show that PDC1 is strongly inducible under abiotic stress conditions, that PDC2 contributed only marginally to total PDC levels, and that PDC3 and PDC4 show no induction. Detection of acetaldehyde and ethanol during stress treatments demonstrated that ethanolic fermentation was occurring during these conditions. To see whether the loss of PDC1 results in an enhanced susceptibility of the pdc1 mutant to environmental stress conditions, we determined the germination rate and the root elongation on agar plates containing different concentrations of sodium chloride and mannitol, which represent a salt stress and a dehydration stress, respectively. In both assays, no considerable differences were observed between wild-type and mutant plants in the germinating rate, and only slight effects were observed on root elongation (Fig. 6), indicating that germination under osmotic stress conditions is presumably independent of PDC1.
Acetaldehyde is a potentially toxic molecule that can be detoxified through the action of ALDH. We therefore determined the transcript profiles of each of the three Arabidopsis ALDH genes (Skibbe et al., 2002
To reveal whether ALDH genes are induced by environmental stresses other than anoxia, we performed RNA gel-blot analysis with RNAs derived from seedlings treated with different stresses. ABA and mannitol treatments strongly induced the transcription of the putative mitochondrial ALDH2B7 gene (Fig. 7B). Wounding and salinity induced the gene to a lesser extent, whereas cold and paraquat had no effect on the expression of ALDH2B7. These results indicate that there may be a function for ALDH2B7 during an abiotic stress response. The expression pattern of the putative cytosolic gene ALDH2C4 and the putative mitochondrial gene ALDH2B4 was only marginally influenced by stress treatments. The housekeeping gene ACTIN2 is barely regulated by any of the stresses, validating its use as the reference gene for the quantitative real-time PCR experiments.
Dominance of PDC1 Transcript Abundance during Anoxia The four PDC genes annotated in the Arabidopsis Genome Sequence Bank are highly homologous to each other and to other plant PDC genes (Fig. 1; Table I). All functional motifs are conserved, and thus they are likely to encode proteins with bona fide PDC enzymatic activity. This is supported by the reduced accumulation of the product of the enzyme, acetaldehyde, in the pdc1 null mutant (Fig. 4E). No conserved stress-related cis-elements were present in any of the other PDC genes.
The transcript abundance of all PDC genes under normal conditions was at the detection limit of classical RNA gel-blot analysis preventing accurate quantitation. Quantitative real-time RT-PCR enabled us to determine transcript levels of each of the PDC genes independently. The careful analysis of the mRNA populations with three independent experiments and RNA extraction for the real-time RT-PCR analysis made it possible to obtain reliable expression profiles. We could demonstrate that among the four PDC genes only PDC1 showed appreciable variation in expression between different tissues and high levels of induction by anoxia. Previous sequence inspection reported two anaerobic response elements in the 5' upstream region of PDC1 from Arabidopsis (Hoeren et al., 1998
Under normal conditions PDC1 transcripts make up about 10% to 40% of the total PDC mRNA in seedlings (Figs. 2B and 5B). Under oxygen limitation, PDC1 transcripts are strongly induced during anoxia and represent in these cells about 99% of the total PDC mRNA (Fig. 3C). This dominant status of one PDC gene in Arabidopsis is different from tobacco. In tobacco, at least two copies exist for PDC (Bucher et al., 1995
Root tips of pdc1 mutant seedlings are less tolerant to oxygen deprivation than the wild type (Fig. 4D), indicating that the reduced PDC levels compromise survival. It should be noted that for up to 12 h, there is no significant difference, but prolonged anoxia results in the accelerated death of the root tip in mutant plants. The real-time PCR analysis showed that under anoxia, the total PDC mRNA concentration in pdc1 mutants was reduced to approximately 1% of wild-type levels. No compensation of the loss of pdc1 expression through induction of the other genes was observed (Fig. 4C). The absence of the PDC1 transcript in the pdc1 mutant had no effect on the level of expression of ADH, and respectively, the absence of ADH transcripts in the adh null-mutant did not influence the expression of PDC1 (Fig. 4, B and C). This indicates, that the regulation of the expression of these two genes is independent of the presence of the other. The reduction of PDC mRNA levels is accompanied by a reduced production of acetaldehyde and ethanol. However, there is a quantitative difference between the reduction in mRNA and enzyme product levels, with acetaldehyde accumulating to 26% of wild-type levels (Fig. 4E). We envisage three possibilities for the comparatively high acetaldehyde production in pdc1 knock-out roots. First, acetaldehyde production may be the result of the activity from the enzymes encoded by PDC2, PDC3, and PDC4. The latter mRNAs may be more efficiently translated, or the proteins be more stable or have a higher specific activity. Second, in the anoxically induced wild type, PDC enzymes may be in large excess, and the flux through the pathway may be limited by other factors than enzyme abundance. Third, acetaldehyde in pdc1 roots may be the product of a yet unknown PDC-independent pathway. In any case, knock-out of the major gene, PDC1, compromises survival under oxygen deprivation.
Increase of PDC expression has been reported by cold and mannitol treatments in Arabidopsis (Dolferus et al., 1997
Taken together, these results suggest the involvement of ethanolic fermentation in response to abiotic stress, as indicated both at the transcriptional level and by the accumulation of ethanolic fermentation products. Abiotic stress can disrupt respiratory activities and it has been proposed that compensation for impaired mitochondrial function will occur by shifting the metabolism form aerobic to anaerobic fermentation (Levitt, 1980 However, germination assays with the pdc1 knock-out plants on osmotic pressure producing medium, indicated, that PDC1 seems not to be essential for survival in these conditions (Fig. 6). Thus, ethanolic fermentation might be a part of abiotic stress adaptation but how much it contributes to stress-tolerance has to be investigated.
None of the three ALDH genes from Arabidopsis were induced during anoxia (Fig. 7A). This is in agreement with the situation in tobacco leaves (op den Camp and Kuhlemeier, 1997
ALDH2B7 expression is strongly induced by cold and mannitol treatments (Fig. 7B). This increase in transcripts suggests a specific function or regulation in stress response. Op den Camp and Kuhlemeier (1997
Plant Growth Conditions
Seeds of Arabidopsis, accessions Col-0 or Ler were surface sterilized and plated on 0.5x Murashige and Skoog medium containing 10% (w/v) Suc, vitamins (1 x 10-4% myo-inositol, 1 x 10-6% nicotinic acid, 1 x 10-6% pyrodoxin-HCl, 1 x 10-5% thiamine-HCl, and 2 x 10-6 % Gly) and 0.85% (w/v) agar. Plates were incubated at 4°C for 3 d to break seed dormancy and then transferred to the growth room (23°C ±2°C, 16-h-light/8-h-dark cycles) in vertical position for 2 to 3 weeks as described by Chung and Ferl (1999
The pdc1 mutant was identified in the T-DNA population (accession Col-0) originating at the Torrey Mesa Research Institute (San Diego) by a random sequencing based screen of the T-DNA left border (line no. 688.D02). Genetic analysis by PCR using primers from the left border (5'-TAG CAT CTG AAT TTC ATA ACC AAT CTC GAT ACA C-3') and a PDC1-specific primer (5'-ACA TTC AGA AGA TGC TCT CTA GTG AAA C-3') identified homozygous mutant lines and enabled the isolation of a fragment covering the insertion site for sequencing. Homozygous pdc1 mutant plants were further examined. The adh mutant in the background Ler was obtained directly from the Nottingham Arabidopsis Stock Center online catalog (35 N8095; http://nasc.nott.ac.uk/home.html).
For anoxic treatments, 2- to 3-week-old plantlets on plates were placed in an anoxia work bench (Forma Scientific, Mariatta, OH) in the dark. Cold and mannitol treatments were performed as previously described by Dolferus et al. (1994
Roots of hydroponically grown plants or leaves from plants grown on soil were prepared as follows. Material was washed three times with distilled water. Roots or leaves were then infiltrated with a 50 mM Glc and 0.1 mM CaSO4 solution three times for 2.5 min under vacuum. Infiltrated samples were briefly blotted on tissue paper, placed in 10-mL gas-tight glass-bottles, and sealed after transfer either to the anaerobic working bench and kept in the dark according to Bucher and Kuhlemeier (1993
Survival assay for anoxia was done according to Ellis et al. (1999 Survival assay for dehydration and salt stress was performed on Murashige and Skoog plates containing the indicated concentrations of either mannitol or NaCl. Seeds of wild type and pdc1 were surface sterilized and placed on the indicated plates. These were then incubated at 4°C for 2 d and then transferred to the growth room (23°C ±2°C, 16-h-light/8-h-dark cycles) in vertical position for 1 week. Germination rate and root length were scored after this period.
Total RNA was isolated from seedlings of control or stress treatments or mutants and transferred to nylon membranes as described by Caderas et al. (2000
Total RNA (2 µg per reaction) was DNase I treated. First-strand synthesis of cDNA was performed by using oligo(dT) primer and avian myeloblastosis virus RT. The following primers were used for RT-PCR experiments: ADH forward primer, 5'-AGT TGT GGT TTG TCT ACT GGG TTA G-3', and reverse primer, 5'-AGA GTC CTC TCA TTC AAG AAA TTC A-3'; PDC1 forward primer, 5'-CTC GTT GAC GCC ATT CAT AAC-3', and reverse primer, 5'-CCA TGA TAA AGC GTA CAT GGA A-3'; PDC2 forward primer, 5'-TTT GGT AGT GTC TTC ACC GTT C-3', and reverse primer, 5'-TTC TTG GGA TGG GAT CTC AAC-3'; PDC3 and PDC4 forward primer, 5'-CTG GTC TTG TCG ATG CTA TTC A-3', and reverse primer, 5'-AAA CTT TGT CAA CAA GGG GTT C-3'; PDC4 reverse primer, 5'-CAC CAT CAA TGG TAA TGG TAC A-3'; RD29a forward primer, 5'-GTG GAG AAG ATC TCT ACC GAG AAG G-3', and reverse primer, 5'-CAT CAA AGA CGT CAA ACA AAA CAC A-3'; and ACT2 forward primer, 5'-ATT CAG ATG CCC AGA AGT CTT GTT-3', and reverse primer, 5'-GAA ACA TTT TCT GTG AAC GAT TCC T-3'. Primers were optimized for amplification in a gradient cycler with various annealing temperature from 47.5°C to 63.1°C.
For quantitative real-time PCR experiments, the LightCycler system (Roche Diagnostics, Mannheim, Germany) was used. For PCR-reactions, a mastermix of the following reaction components was prepared (the end-concentration is indicated in parentheses): 12 µL of water, 2 µL of MgCl2 (2.5 mM), 1 µL of forward primer (0.5 µM), 1 µL of reverse primer (0.5 µM), and 2 µL of LightCycler (Fast Start DNA Master SYBR Green I, Roche Diagnostics). LightCycler mastermix was filled in the LightCycler glass capillaries, and 2 µL of cDNA was added as PCR template. Capillaries were closed, centrifuged, and placed into the LightCycler rotor. The following LightCycler experimental run protocol was used: denaturation program (95°C for 10 min), amplification and quantification program repeated 45 to 55 times (95°C for 15 s, annealing temperature: 63°C for 15 s for all primer combinations except for PDC3 amplification:58°C, 72°C for 20 s with a single fluorescence measurement), melting curve program (65°C–95°C with heating rate of 0.1°C s-1 and a continuous fluorescence measurement), and finally a cooling step at 40°C.
For relative quantification, PCR efficiencies for each gene were determined as follows: Standard curves for each gene was performed using the cDNA with the highest abundance of the gene to cover the range of all template concentrations. For PDC2, an external standard curve was performed on a purified PDC2-PCR fragment. Real-time PCR efficiencies (E) were calculated from the given slopes in the LightCycler software of the standard curves according to the equation: E = 10 [-1/slope]. PCR efficiencies for the used target and reference genes: ADH, 1.95; ACT2, 1.99; RD29a, 1.90; PDC1, 1.88; PDC2, 1.70; PDC3, 1.57; and PDC4, 1.92 (see also supplementary materials; they can be viewed at www.plantphysiol.org). Crossing points, defined as the point at which the fluorescence rises above the background fluorescence was determined using the "Fit Point Method" in the LightCycler software 3.5.3 (Roche Diagnostics). cDNA abundance of the PDC genes was calculated by crossing point differences between amplification of the different genes after a baseline adjustment. Gene-specific PCR efficiency was used to calculate the induction level. A mathematical model, which was shown recently to hold in experimental environments, was used to quantify the expression of target genes relative to the expression of a reference gene (Pfaffl, 2001
We thank A. Sessions and P. Ho (Torrey Mesa Research Institute, San Diego) for providing the seeds of the pdc1 mutant. We also thank the Nottingham Arabidopsis Stock Center (Nottingham, UK) for providing the seeds of the adh mutant. We furthermore thank R. Brändle and S. Zeeman for critical reading of the manuscript and our colleagues in the laboratory for stimulating discussions and general support. Received November 5, 2002; returned for revision November 29, 2002; accepted January 30, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.102.016907.
[w] The online version of this article contains Web-only data. The supplemental material is available at http://www.plantphysiol.org. * Corresponding author; e-mail cris.kuhlemeier{at}ips.unibe.ch; fax 41–31–332–2059.
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