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First published online June 12, 2003; 10.1104/pp.103.022244 Plant Physiology 132:1292-1302 (2003) © 2003 American Society of Plant Biologists Enhanced Low Oxygen Survival in Arabidopsis through Increased Metabolic Flux in the Fermentative Pathway1Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 (K.P.I., M.D.P., A.G.G.); and Division of Plant Industry, Commonwealth Scientific and Industrial Research Organization, G.P.O. Box 1600, Canberra, Australian Capital Territory 2601, Australia (R.D., E.S.D.)
We manipulated the enzyme activity levels of the alcohol fermentation pathway, pyruvate decarboxylase (PDC), and alcohol dehydrogenase (ADH) in Arabidopsis using sense and antisense overexpression of the corresponding genes (PDC1, PDC2, and ADH1). Transgenic plants were analyzed for levels of fermentation and evaluated for changes in hypoxic survival. Overexpression of either Arabidopsis PDC1 or PDC2 resulted in improved plant survival. In contrast, overexpression of Arabidopsis ADH1 had no effect on flooding survival. These results support the role of PDC as the control step in ethanol fermentation. Although ADH1 null mutants had decreased hypoxic survival, attempts to reduce the level of PDC activity enough to see an effect on plant survival met with limited success. The combination of flooding survival data and metabolite analysis allows identification of critical metabolic flux points. This information can be used to design transgenic strategies to improve hypoxic tolerance in plants.
Plants are constantly challenged by environmental stresses that reduce crop yield (e.g. salinity, low temperature, drought, and flooding). Soils with excess water account for 15.7% of the arable land in the United States, and flooding accounted for 16.4% of the crop insurance claims, making it the second highest cause of crop loss in the United States (Boyer, 1982  ). Traditionally,
plant breeders have selected directly for increased stress tolerance, however,
selection for flooding tolerance has been performed only in rice (Oryza
sativa), where a genetic mapping approach was used to identify major and
minor genes involved in submergence tolerance
(Sripongpangkul et al., 2000;
Xu et al., 2000). Transgenic
plants with increased tolerance to drought, salinity, low temperatures, or a
combination of these stresses (Nelson et
al., 1998; Huang et al.,
2000) were obtained by introducing genes for the overaccumulation
of benign compounds (e.g. Gly betaine, mannitol, and Pro) that are believed to
act as osmolytes. Other groups have increased stress tolerance in Arabidopsis
by overexpressing transcription factors
(Jaglo-Ottosen et al., 1998;
Kasuga et al., 1999;
Yamaguchi-Shinozaki and Shinozaki,
2001) or signal transduction components
(Piao et al., 2001;
Tamminen et al., 2001).
There is substantial variation among terrestrial crop species in their
ability to tolerate waterlogging conditions. Even rice, which is adapted to
life in a flooded environment, incurs damage if the shoots are completely
submerged for periods of time (Drew,
1997
Because higher plants are sessile and obligate aerobes, they have evolved a
number of mechanisms to survive the hypoxic conditions that are generated by
flooding. These mechanisms include morphological adaptations, such as
formation of adventitious roots (Lorbiecke
and Sauter, 1999
This study focuses on the manipulation of the ethanol fermentation pathway.
PDC (EC 4.1.1.1) catalyzes the first step, which is responsible for the
irreversible conversion of pyruvate to acetaldehyde. ADH (EC 1.1.1.1) then
converts acetaldehyde to ethanol, with the concomitant regeneration of
NAD+ (Fig. 1). The
regeneration of NAD+ from NADH is thought to be the most important
function of the alcohol fermentation pathway under low oxygen stress
conditions, because this function is impaired by the inactivation of oxidative
phosphorylation. This function is vital, because in the absence of
NAD+, glycolysis ceases
(Kennedy et al., 1992 In this paper, we report the effect of overexpressing the enzymes of ethanol fermentation, PDC and ADH, in Arabidopsis. The combination of enzyme activity data and metabolite data from all enzymes involved in the hypoxic response allows us to critically examine carbon flow along the glycolytic and ethanol fermentation pathway. In conjunction with the survival assay, we provide a comprehensive look at overexpression of ADH and PDC and their role in hypoxic tolerance. We show that overexpression of either PDC1 or PDC2 improved plant survival under low oxygen conditions. This confirms that PDC is the metabolic control point in the alcohol fermentation pathway. ADH1 overexpression had no effect on flooding tolerance, but normal levels of ADH1 expression were shown to be critical to plant survival under low oxygen conditions.
Generation and Assay of Transgenic Lines for Enzyme Activity
Transgenic Arabidopsis lines containing the PDC1, PDC2, and
ADH1 sense and antisense constructs (under the control of the 35S
promoter) were screened in the T2 generation by northern-blot hybridizations
(data not shown). The line with the highest mRNA expression levels was
selected. For the antisense constructs, we selected those lines with the most
significant reduction in mRNA levels. We then used enzyme activity assays to
confirm the mRNA data and to select the best overexpressing/antisense lines.
Homozygous T3 lines were selected from these transgenic lines, and the levels
of mRNA and enzyme activity were again confirmed in this generation. The lines
overexpressing PDC1, PDC2, or ADH1 were assigned the prefix
"Ox" (Overexpressing). Lines showing a reduced enzyme activity
were generated using antisense constructs for PDC1 and PDC2
and were assigned the prefix "Ue" (Under-expressing). In the case
of ADH1, an adh1– null mutant was selected
in C24 background using allyl alcohol selection
(Jacobs et al., 1988 Enzyme activities were determined for PDC (Fig. 2, A and B), ADH (Fig. 2, C and D), LDH (Fig. 2, E and F), and AlaAT (data not shown), under aerobic and hypoxic conditions in the selected lines. In roots and shoots, the levels of PDC activity were six to ten times higher for OxPDC1 and OxPDC2, respectively, under either aerobic or hypoxic conditions. The least expressing PDC lines, UePDC1 and UePDC2, were originally selected because they had mRNA levels reduced to 30% to 50% of wild-type levels under aerobic and hypoxic conditions (data not shown). However, in terms of activity (Fig. 2, A and B), these lines had only marginally lower PDC activity compared with the wild type under both control and hypoxic conditions.
The OxADH1 line showed a 2- to 3-fold increase in ADH activity
under hypoxic conditions in both roots and shoots compared with wild-type
roots and shoots, whereas activities were increased 7- to 8-fold under aerobic
conditions (Fig. 2, C and D).
The ethyl methanesulfonate-induced adh1– mutant had
negligible ADH activity compared with the C24 wild type
(Fig. 2, C and D). In this
adh1 null mutant, Glu99 and His101 were mutated to Asp and Lys,
respectively, in a highly conserved area of the ADH enzyme
(Dolferus et al., 1990 The over- or under-expression of PDC1 or PDC2 had no effect on the levels of ADH activity in either roots or shoots (Fig. 2, C and D), nor was the total PDC activity in roots and shoots significantly affected by overexpression of ADH1 (Fig. 2, A and B). The UePDC2 and OxADH1 lines displayed increased levels of LDH activity in the roots, but in shoots, only OxADH1 showed slightly increased LDH activity levels (Fig. 2, E and F). In the adh1– line, LDH levels were decreased in roots but not in shoots (Fig. 2, E and F). No significant changes of LDH activity were observed by overexpressing the PDC1 or PDC2 genes. No differences in AlaAT activity were detected between any of the transgenic or mutant genotypes and the C24 genotype (data not shown).
To examine the effect of the introduced PDC transgenes, metabolites for the key compounds in the fermentative pathway were analyzed in roots, shoots, and the medium surrounding the roots under hypoxic conditions (Figs. 3 and 4). For acetaldehyde, only the liquid Murashige and Skoog medium was measured, because previous experience has indicated that levels in the medium accurately reflect intracellular levels, because acetaldehyde passes easily through intracellular membranes into the medium.
OxPDC2, OxPDC1, and UePDC2 lines had decreased levels of pyruvate in roots and shoots under hypoxic conditions (Fig. 3, A and B). The decrease in pyruvate levels was associated with a 2- or 4-fold increase of acetaldehyde in the medium for OxPDC1 and OxPDC2 (Fig. 3C). Both lines also showed a corresponding increase in ethanol concentration in the medium (1.6 times; Fig. 3D). OxPDC1 was the only line that showed significantly higher ethanol levels in roots (1.8 times) and shoots (Fig. 3, D and E). The effect of PDC overexpression on the metabolites of the other fermentation pathways that are derived from pyruvate was also measured (Fig. 4). Decreased levels of both lactate and Ala were found in the roots, shoots, and Murashige and Skoog medium for OxPDC1. These metabolites remained unchanged in OxPDC2 (Fig. 4, A–F). No significant differences were observed in Glu or 2-oxoglutarate levels for OxPDC2, but OxPDC1 had significantly lower levels of Glu in the medium (data not shown). These data show that overexpression of the Arabidopsis PDC1 and PDC2 enzymes, although overexpressed to similar total activity levels (Fig. 2), had different effects on metabolite accumulation, suggesting that they may function differently. If overexpression of PDC leads to increased carbon flow through the ethanol pathway, then repression of PDC could act to decrease carbon flow and reduce ethanol levels. But the decrease in PDC activity that we observed in the UePDC lines was not nearly as dramatic as the increase in activity of the overexpressing lines (Fig. 2). Residual PDC activity in the UePDC lines could still account for a significant flow of carbon through the alcohol fermentation pathway. The levels of pyruvate, acetaldehyde, and ethanol production of UePDC1 and UePDC2 were not significantly different from wild type (Fig. 3, A–F), but UePDC1 and UePDC2 both showed increased levels of lactate production in roots but not in leaves (Fig. 4, A and B). This suggests that under hypoxic conditions even a minor reduction in PDC activity may be sufficient to increase the amount of carbon flow from pyruvate to lactate. Only small differences were observed in lactate and Ala (Fig. 4) and in Glu and 2-oxoglutarate levels (data not shown).
Pyruvate levels were not affected in roots, but they decreased in shoots of the OxADH1 and adh1– lines (Fig. 3, A and B). Acetaldehyde levels were reduced to 0.1% of wild-type levels in roots in OxADH1, whereas the adh1– null line had a 9-fold increase in acetaldehyde production in the medium (Fig. 3C). Although there were no significant changes in tissue ethanol levels in roots or shoots of the OxADH1 line (Fig. 3, E and F), a strong reduction in ethanol production (80%) was observed in the roots of the adh1– mutant; no change was found in the shoots (Fig. 3, D and E). No significant changes were found in lactate or Ala levels in roots, shoots, or the medium for OxADH1 transgenic lines, whereas the adh1– mutant showed a 3- to 4-fold increase in lactate levels in the medium and in roots (Fig. 4, A–C).
Survival assays were conducted on each genotype to assess the contribution
of the transgene to whole-plant survival under low oxygen conditions. We used
two experimental conditions that were previously shown to discriminate between
wild-type and adh1– null mutants
(Ellis et al., 1999
Both the OxPDC1 and OxPDC2 lines performed better than
the control line in the 24-h NHPT and 48-h HPT treatments. These data suggest
that PDC enzyme activity levels are a limiting factor for survival of
moderately severe oxygen conditions. We did not see any significant
differences between C24 and the two UePDC lines that we tested. The
Ox-ADH1 line did not perform better than the C24 wild-type line, but
as was previously shown (Ellis et al.,
1999 The data for the root and shoot weight measurements were less consistent than those for root tip growth (Table I), which could partially be due to the variation in stringency of the stress treatment between the two independent experiments. OxPDC1 showed increased root formation in the 24-h NHPT and the 48-h HPT treatments. In contrast, root development of the OxPDC2 line was higher in the 48-h HPT treatment, but in one of the 24-h NHPT treatments, results were significantly worse than for C24. No significant changes were found for the UePDC1, UePDC2, and OxADH1 lines, whereas the adh1– showed decreased root formation in the 48-h HPT treatment. For the shoot weight measurements, only the OxPDC2 line showed significantly higher shoot weights in the 24-h NHPT treatment. The data for the 24-h NHPT (OxPDC1) and 48-h HPT (OxPDC1 and OxPDC2) treatments showed increased shoot weights but were not significant due to variation within each experiment.
In the absence of oxidative phosphorylation, the glycolytic conversion of Glc to pyruvate is the only means to generate limited amounts of ATP under anaerobic conditions. Plants then switch to fermentation pathways to regenerate NAD+ (Roberts et al., 1984a ,
1984b). Of the three main
fermentation pathways in plants, only two—alcohol and lactic acid
fermentation—regenerate NAD+
(Fig. 1). Ala fermentation does
not produce NAD+, but Ala accumulation under low oxygen conditions
is thought to play a role in nitrogen assimilation
(Good and Muench, 1993).
The three fermentation pathways are relatively simple, enabling us to start
a metabolic engineering approach in Arabidopsis. The three final metabolites
of these pathways are also easily measured, and because they have a common
starting point, pyruvate, we can monitor changes in metabolites and study the
impact on carbon flow in the branches of the fermentative pathway. We
established a survival assay to study the influence of metabolic changes on
low oxygen stress survival (Ellis et al.,
1999
We have successfully developed plants with modified levels of PDC and ADH
activities. Six different lines were analyzed for over- and under-expression
of these enzymes using enzymatic, metabolic, and survival data. This allows us
to study their role in ethanol fermentation and their impact on the other
branches in the fermentation pathway. Overexpression of any of these genes
using the cauliflower mosaic virus 35S promoter resulted in high levels of
constitutive enzyme activity that was insensitive to hypoxia
(Fig. 2). Overexpression of
either PDC1 or PDC2 enhanced plant tolerance to low oxygen
conditions (Fig. 5;
Table I). Similarly, Quimio et
al. (2000
The PDC overexpressors produce much higher levels of acetaldehyde
(Fig. 3), as has been observed
in PDC-overexpressing tobacco plants
(Bucher et al., 1994
The UePDC1 and UePDC2 lines displayed no visible changes
in low oxygen tolerance relative to the wild type. These transformants also
did not show significant differences in pyruvate, acetaldehyde, and ethanol
production, although there are small differences at the level of lactate and
Ala (Table I; Figs.
3 and
4). Given our current knowledge
about gene silencing (Waterhouse et al.,
2001b
The adh1– mutant had minimal ADH activity,
resulting in a dramatic increase in acetaldehyde in the medium surrounding the
roots (Fig. 3). The mutant also
had poor tolerance to low oxygen conditions
(Fig. 5). These results confirm
earlier studies showing that at least low to normal levels of ADH activity are
necessary for short-term, waterlogging tolerance
(Johnson et al., 1994
PDC activity occurs at a critical branch point between aerobic and
anaerobic metabolism (Roberts et al.,
1989
Although our lines overexpressing the anaerobically inducible
(PDC1) and constitutively expressed (PDC2) gene had similar
PDC activity levels and low oxygen survival properties, our results indicate
that PDC1 and PDC2 may have different enzymatic properties. Overexpression of
the PDC1 gene, but not PDC2, leads to a reduction in
metabolites of the other fermentation pathways (lactate and Ala;
Fig. 4). Biochemical
characterizations have demonstrated that, like yeast and some bacterial PDC
enzymes, plant PDC has nonlinear kinetics and is activated by its substrate
pyruvate (Mücke et al.,
1996
The experiments described in this paper were carried out with plants grown
in Murashige and Skoog medium containing 3% (w/v) Suc. Therefore our data
showing that PDC overexpression increases metabolic flux through the alcohol
fermentation pathway and improves low oxygen stress survival may only be true
when sugar supply is adequate. Under sugar-limiting conditions (e.g. in the
field), a faster depletion of carbohydrate stores may lead to decreased
survival. An exogenous supply of Glc has been shown to ameliorate the effects
of anoxia (Xia and Saglio,
1990 In conclusion, our data support the idea that PDC is a critical flux control point in hypoxic metabolism and that PDC activity levels can determine plant survival under low oxygen conditions. PDC activity controls carbon flow through the ethanol fermentation pathway and affects flow through the lactate and Ala pathways. On the basis of this study, improved flooding survival may be achieved by overexpressing a plant PDC1 or PDC2 gene. Whether overexpression of both PDC and ADH activity would further improve low oxygen stress survival remains to be established. Additional work is also required to determine first, whether continual overexpression has significant negative effects on productivity and second, whether the results of the survival assay can vary, depending on the conditions of evaluation.
Plant Material, Growth Conditions, and Stress Treatment
Arabidopsis ecotype C24 was used in all our experiments. Seeds were
germinated, and seedlings were grown as outlined by Ellis et al.
(1999
Standard cloning and recombinant DNA methods were used throughout
(Maniatis et al., 1982
RNA extractions, blot hybridizations using RNA probes, and filter washings
were performed as described previously
(Dolferus et al., 1994
Each overexpressing line was assessed alongside its matching
under-expressing line and the wild-type C24 line, in both aerobic and hypoxic
environments (Howard et al.,
1987
Gas chromatography (GC) was used to determine acetaldehyde and ethanol
concentrations in the liquid medium. Roots of 3-week-old plants were
harvested, transferred into 10-mL syringes containing 10 mL of growing medium
gassed with 100% argon to exclude N2. The syringes were outfitted
with 18-gauge needles containing glass wool at the air lock to prevent roots
from clogging the needle. After the plunger had been inserted into the
syringe, a 1-mL argon gas bubble was pulled into the syringe's chamber, and
the needle tip was fitted with a rubber stopper to prevent gas exchange or
leakage. The syringes were left on a rotary shaker at room temperature. Within
a 24-h period, 1-mL samples were taken at regular intervals and stored at
4°C. Fresh weights of the roots were taken at the experiment's conclusion.
Measurement of acetaldehyde and ethanol in the medium was performed using a
Varian 3700 GC, fitted with a Varian 800 series auto-sampler and flame
ionization detector. The stainless steel column (2-m x 2.16-mm i.d.) was
packed with Poropak QS. The GC set-up is as follows: injector, 170°C;
detector, 250°C; column programmed between 110°C to 130°C; and
N2 carrier gas flow, 30 mL min–1.
Calibration was done using samples spiked with known amounts of acetaldehyde
and ethanol. All other metabolites were assayed as described previously. In
brief, roots were ground in sand and perchloric acid in a 3:1 (w/v) ratio as
described by Good and Muench
(1993
The survival assay protocol of Ellis et al.
(1999
Two separate survival assay experiments were completed for each transgenic
or mutant line. The data for each measurement for the two experiments were
analyzed using a two-tailed Student's t test. For each experiment the
measured means between the transgenic/mutant and wild-type plants were used
for the t test. Differences were considered significant providing
that differences from both experiments had P < 0.05. Presented
values in the table indicate significant differences where 0.01
We thank Dr. Marc Ellis for valuable assistance in all aspects, particularly the survival assay and experimental set-up for GC analysis. Special thanks go to Drs. Lorraine Tonnet (GC analysis), Nic Savidov, and Peter Hunt for their discussions, comments, and assistance. We also appreciate the excellent technical work performed by Sandra Stops. Received February 25, 2003; returned for revision March 6, 2003; accepted March 21, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.022244.
1 This work was supported in part by a Natural Sciences and Engineering
Research Council of Canada (NSERC) grant to A.G.G.
2 R.D. is financially supported by the Cooperative Research Centre for
Sustainable Rice Production, c/o New South Wales Institute of Agriculture,
Private Mail Bag, Yanco, NSW 2703 Australia. * Corresponding author; e-mail allen.good{at}ualberta.ca; fax 780–492–9234.
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ABSTRACT
RESULTS
, P < 0.05; 
, P < 0.01.
P < 0.05 and where P < 0.01. Data for all enzyme and
metabolite experiments are based on a minimum of two separate experiments.
Each experiment consisted of three replicate plates per treatment with a C24
control included in each hypoxic chamber as a control. Student's t
test (two-tailed) was used to assess whether there was a significant
difference between the means of the wild-type and transgenic/mutant
plants. 
