JXB Advance Access originally published online on May 13, 2003
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Journal of Experimental Botany, Vol. 54, No. 388, pp. 1721-1730,
July 1, 2003
© 2003 Oxford University Press
Oxygen deficiency affects carbohydrate reserves in overwintering forage crops
Received 7 January 2003; Accepted 27 March 2003
Soils and Crops Research and Development Centre, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd., Sainte-Foy, Québec, Canada G1V 2J3
* To whom correspondence should be addressed. Fax: +1 418 642 2402. E-mail: bertranda{at}agr.gc.ca
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Abstract |
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Anaerobic conditions developing under an ice cover affect winter survival and spring regrowth of economically important perennial crops. The objective was to compare, during a prolonged period of low (<2%) O2 at low temperature, the concentration of carbohydrates of four plant species contrasting in their resistance to oxygen deficiency. Four perennial forage species, lucerne (Medicago sativa L.), red clover (Trifolium pratense L.), timothy (Phleum pratense L.), and cocksfoot (Dactylis glomerata L.) were subjected to a progressively developing oxygen deficiency stress by enclosing potted plants in gas-tight bags in late autumn for overwintering in an unheated greenhouse. Timothy was previously reported to be more resistant to oxygen deficiency than the three other species. Non-structural carbohydrates increased and remained at a higher concentration in timothy than in the other three species under low O2 concentration. Concen trations of sucrose, fructose, glucose, and fructans increased in response to oxygen deficiency in timothy, whereas the concentration of soluble sugars decreased under the same conditions in lucerne, red clover, and cocksfoot. The gene expression of glyceraldehyde-3-phosphate dehydrogenase increased in response to low oxygen concentration in oxygen deficiency-sensitive lucerne while it remained unchanged in the oxygen deficiency-resistant timothy. It is concluded that timothy maintains higher carbohydrate reserves under oxygen deficiency, a specific feature that could favour its winter survival and spring regrowth.
Key words: Carbohydrates, gene expression, glyceraldehyde-3-phosphate dehydrogenase, oxygen deficiency, perennial forages.
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Introduction |
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Perennial plants living in northern latitudes thrive under harsh winter conditions. One of the most detrimental situations is the formation of an ice layer over the plants as a result of freezing rains or freeze–thaw cycles. Under ice, plants are progressively exposed to oxygen deficiency. Rakitina (1970) reported that the gaseous composition of the atmosphere surrounding wheat seedlings encased in ice for 5 d at –5 °C was 3% O2 and up to 44% CO2 (v/v). The metabolic consequences of such O2 deficiency for the plants is a significant shift in cell energetics. The oxidative phosphorylation is inhibited under complete anoxia and is then replaced by fermentation for ATP production and regeneration of reducing power (reviewed by Dolferus et al., 1997). Fermentation is an inefficient metabolic pathway that produces only 2 mols ATP from each mol of glucose rather than 32 for aerobic respiration (Vartapetian and Jackson, 1997). Thus, one of the consequences of anoxia is the acceleration of the use of carbon (C) reserves, and a prolonged period of oxygen deficiency could lower the energy available for basic metabolism and lead to the exhaustion of substrates for respiration and regrowth (Drew, 1997). Furthermore, anaerobic respiration could cause the accumulation of potentially phytotoxic end-products (Drew, 1997). Ultimately, oxygen deficiency can affect winter survival and spring regrowth of economically important perennial crops.
Under natural conditions, complete anoxia is unlikely to occur (Saglio et al., 1999). However, oxygen deficiency (3% O2) occurs (Rakitina, 1970) and might affect the metabolism to some extent. It was previously reported that timothy (Phleum pratense L.) was unaffected after 100 d in a sealed enclosure with low O2 concentration at around 0 °C while the same conditions appreciably reduced the regrowth of lucerne (Medicago sativa L.), cocksfoot (Dactylis glomerata L.) and red clover (Trifolium pratense L.) (Bertrand et al., 2001). The slower metabolism of timothy under oxygen deficiency prevented the accumulation of potentially phytotoxic metabolites such as ethanol. Another consequence could possibly be the maintenance of a sufficient level of C reserves to sustain cold acclimation and spring regrowth of this species.
The type and quantity of sugars that accumulate in plant tissues could play an important role in the resistance to abiotic stresses. For instance, Castonguay et al. (1995) found that the raffinose family oligosaccharides (RFOs) concentration increased in crowns during cold acclimation of lucerne and reached higher concentrations in cultivars showing superior winter hardiness. When comparing sugar accumulation in related plant species either adapted to a flooded or a well-aerated environment, Albrecht et al. (1997) found that the species tolerant to an oxygen shortage accumulated reserve sugars in the form of fructans whereas sensitive species accumulated starch.
The objective was to assess the effect of oxygen deficiency during winter on C reserves that are essential for winter survival and spring regrowth of four forage species. The level of mono and disaccharides, RFOs, fructans of a low and a high degree of polymerization (DP), and starch were compared in four forage species of contrasting resistance to oxygen deficiency during a prolonged period of low O2 concentration during overwintering. The study also includes the characterization of the genetic expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme of the glycolytic pathway, which genetic expression has been shown to increase under an environmental stress in maize (Zea mays) (Russell and Sachs, 1989) and Arabidopsis thaliana (Yang et al., 1993) as well as in response to a biotic stress in potatoes (Solanum tuberosum) (Laxalt et al., 1996).
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Materials and methods |
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Plant material
Seeds of lucerne (cv. AC Caribou), red clover (cv. AC Charlie), timothy (cv. Champ) and cocksfoot (cv. Okay) were sown on 9 June 1998 in 20 cm diameter pots. The substrate was a mixture (10:3, v/v) of topsoil/general purpose growing medium (Pro-Mix BX, Premier Horticulture Ltée, Rivière-du Loup, QC, Canada). Legume seedlings were inoculated twice after emergence, at 1 week intervals. The inoculum was Sinorhizobium meliloti for lucerne and Rhizobium leguminosarum trifolii for red clover (1010 cells per plant for each inoculation). Plants were thinned to 10 seedlings per pot after emergence and grown under controlled conditions (21/17 °C day/night, 16 h photoperiod, 250 µmol photons m–2 s–1 PPFD) until mid-August. Plants were kept well-watered, fertilized once a week during the first 3 weeks with a commercial fertilizer (150 kg ha–1 of 6-11-31, N-P2O5-K2O, Plant-Prod, Brampton, ON, Canada) and cut twice (13 July and 13 August) during this period. After the first cut, 16 kg P ha–1 and 62 kg K ha–1 were applied to all species while 40 kg N ha–1 was applied to grass species only.
On 16 August, plants were placed outside for autumn acclimation. Pots were buried in the soil under field conditions and, at the end of October, transferred to an unheated greenhouse for overwintering under natural temperature variations. On 23 November, plants were cut 2.5 cm above the soil surface, leaving a minimal surface of photosynthetic tissues for all species. Half of the potted plants were sealed in gas-tight transparent plastic bags of 100 µm thickness (VAC 4, The Packaging Group Inc. NJ, USA), to induce anaerobic conditions progressively by preventing gas exchange between the plants and the atmosphere; this will be referred to as the oxygen deficiency treatment. Temperatures in enclosed pots were continuously recorded at the soil surface using stand-alone dataloggers with an external temperature probe (RD Temp XT dataloggers, Omega, Stamford, CT, USA). Pots with bare soil were included in the experimental design. The remaining potted plants were kept as controls under the same overwintering conditions.
Atmospheric conditions in the sealed bags during the winter and their impact on plant regrowth were described in Bertrand et al. (2001). Air temperature inside and outside the greenhouse and the soil temperature in the pots were continuously monitored using thermocouples and recorded with a datalogger (Doric 235, San Diego, CA, USA). When the air temperature remained continuously below the freezing point, plants were covered with a 5 cm thick layer of insulating fibreglass wool and geotextile to simulate snow cover. Potted plants were covered from 18 December until 28 February and the insulating material blocked all incoming radiation (PPFD of 0 µmol m–2 s–1 measured at the plant level). Potted plants were uncovered from the end of February until the end of the experiment (10 March 1999). The greenhouse was constantly ventilated during daytime to maintain the inside temperature similar to that of the outside (Castonguay et al., 1995). The experiment was conducted in two consecutive years (1998–1999 and 1999–2000). The results were very similar for both years. Thus, only the results of the first experimental year are presented.
Gas analyses
On five occasions during the winter (24 November 1998, 10 December 1998, 19 January 1999, 10 February 1999, and 10 March 1999), air in the bags was sampled using a gas-tight syringe (Luer Lok, 10 cm3, Becton Dickinson & Company, Franklin Lakes, NJ, USA) through a septum fixed on each bag with Silicone II (GE Canada INC., ON, Canada). Oxygen concentrations were analysed by gas chromatography (Varian Inc., Model 3800, Palo Alto, CA, USA) with two columns in series, HayeSep A and Molecular Sieve 5A (Chromatographic Specialties, Brockville, ON, Canada). The oven temperature was set at 60 °C and the flux of the carrier gas (helium) at 45 ml min–1. Oxygen was detected with a Thermal Conductivity Detector (TCD) at 120 °C.
Preliminary tests of the air-tightness of syringes, using O2-free gases (He and N2), showed that the samples could become contaminated with O2 due to the large difference in partial pressure between the syringe and its environment. This contamination of Luer Lok syringes by atmospheric O2 could reach 1% h–1. Even though the air in the bags was analysed within 2 h of sampling, the O2 concentration in gas samples might have been overestimated by up to 2%.
Biochemical analyses
Plant material for biochemical analyses was sampled from four pots (10 plants pot–1) of each species at six sampling dates (9 September, 14 October, 17 November, and 10 December 1998, 10 February and 10 March 1999). For each pot, approximately 1 g fresh weight (FW) of a sample of crown tissues for legumes (2 cm of the transition zone between shoots and roots) and of shoot bases for grasses (2 cm of tissues including basal leaf sheath and the upper part of the root system) were pooled from ten plants. Samples were extracted in water in a 1:8 (w/v) ratio, heated 30 min at 65 °C to stop enzymatic activity and stored at –80 °C until extraction. For each pot, a subsample was oven-dried for 48 h at 70 °C for dry matter determination and the estimation of dry weight (DW). Thawed samples were ground on ice with a polytron homogenizer (Brinkman, Rexdale, ON, Canada) with the addition of water to reach a 1:15 (w/v) ratio. Tubes were subsequently centrifuged 600 s at 1500 g and 3 ml of supernatant were collected and kept frozen at –40 °C until analysis of soluble sugars and fructans by high-performance liquid chromatography (HPLC). The non-soluble residues left after extraction were washed twice with methanol and used for starch determination. Samples were centrifuged for 180 s at 21 000 g prior to HPLC analysis. The HPLC analytical system was controlled by Waters Millennium32 software (Waters, Milford, MA, USA) and was composed of a Model 600 pump and a Model 717plus autosampler. Sugars were separated on a Waters Sugar-Pak column eluted isocratically at 85 °C at a flow rate of 0.5 ml min–1 with EDTA (Na+, Ca2+, 50 mg l–1) and detected on a differential refractometer (Waters, Model 410). Low degree of polymerization (LDP) fructans were separated on a Bio-Rad HPX-42A column (Bio-Rad, Richmond, CA, USA) while high degree of polymerization (HDP) fructans were quantified with a Shodex KS-804 column (Shodex, Tokyo, Japan). Both columns were eluted isocratically at 25 °C with deionized water at respective flow rates of 0.5 and 1.0 ml min–1. The amount of fructans was determined by reference to a fructose standard curve. Degrees of polymerization of HDP fructans were established by reference to Pullulan Standards (Shodex Standard P-82, Canada Waters Ltd. Missisauga, ON, Canada).
Starch was digested for 90 min with amyloglucosidase (Sigma Chemical Co., St Louis, MO, USA) and quantified as glucose equivalent with p-hydroxybenzoic acid hydrazide according to Blakeney and Mutton (1980). Amounts of starch were determined spectrophotometrically by reference to a glucose standard curve. The starch samples for 9 September and 14 October 1998 are missing.
As the Waters Sugar-Pak column could not easily separate raffinose and DP3-fructan, samples of plant material likely to contain both oligosaccharides were analysed further on a Waters Carbohydrate column eluted with 73% (v/v) acetonitrile at a flow rate of 1.5 ml min–1. This acetonitrile concentration gave a separation which was sufficient to quantify both components. Furthermore, the same samples were digested with 
-galactosidase (EC 3.2.1.22
[EC]
, Sigma-Aldrich, Oakville, ON, Canada ), 5 units l–1 of sample at 37 °C for 1 h, and the hydrolysates were analysed both on a Sugar-Pak column for the appearance of free galactose and also on Waters Carbohydrate column to verify the disappearance of the putative raffinose peak.
Extraction of RNA
At each sampling date, approximately 1 g FW of a pooled sample of crown tissues from ten plants was ground to a fine powder with a pestle in a mortar containing liquid nitrogen. Samples were kept frozen at –80 °C until their extraction. Total RNA was extracted by a method described by De Vries et al. (1988) and quantified by spectrophotometry at 260 nm. Following extraction, RNA was dissolved in TE (10 mM TRIS-HCl, 1 mM EDTA, pH 7.4) and stored at –80 °C.
Cloning of GAPDH and characterization of expression in lucerne and timothy
The glyceraldehyde-3-phosphate dehydrogenase gene from lucerne has been isolated by differential screening of a cDNA library from crowns of lucerne (cv. Apica) cold-acclimated for 2 weeks at 2 °C followed by 2 weeks at –2 °C as compared to non-acclimated material (Monroy et al., 1993). The glyceraldehyde-3-phosphate dehydrogenase gene from timothy has been isolated by screening a cDNA library from shoot bases of timothy (cv. Champ) cold-acclimated for 2 weeks at 2 °C followed by 2 weeks at –2 °C. The cDNA library from timothy has been cloned in Lambda ZAP (Stratagene, La Jolla, CA, USA). The GAPDH clone of timothy has been identified by an homology search in the Genbank/EMBL database (National Center for Biotechnology Information, Bethesda, MD, USA). Specific GAPDH probes were used for each species and the GAPDH gene expression was measured by Northern blot hybridization during the initial cold acclimation period in September and October as well as in November, December, February, and March. Ten µg of total RNA were size fractionated on a 1% formaldehyde agarose gel, transferred to a nylon membrane (Hybond-N+, Amersham Pharmacia Biotech) and hybridized overnight at 68 °C in a 2x SSC, 0.25% (w/v) low fat powder milk, with [-32P]dCTP-labelled GAPDH probes obtained as described above. Visual assessment of loaded amounts of RNA was made by adding ethidium bromide to the sample.
Experimental design and statistical analysis
The experimental design was a randomized complete block with sampling dates, treatments, and species completely randomized within each of the four blocks. The experiment included a total of 256 pots with 10 plants in each: 4 blocks x 4 sampling dates x 4 species x 2 treatments (control or oxygen deficiency) x 2 types of samples (biochemical analysis or regrowth assessment). One pot per block was sampled for each treatment, sampling date and species for biochemical analysis and gene expression. Data were subjected to an analysis of variance including sampling dates, treatments, and species as factors and using the SAS software package (SAS Institute Inc.). Statistical significance was postulated at P <0.05.
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Concentrations of O2
Daily average air temperature inside the unheated greenhouse varied between 5 °C and –18 °C throughout the experiment (Fig. 1A). Soil temperature progressively declined from 4 °C on 24 November to 0 °C by mid-December and remained below 0 °C (between –2 °C and 0 °C) until the end of the experiment. Oxygen concentrations in the enclosures decreased rapidly during the experiment (Fig. 1B). The decrease was similar for red clover, lucerne and cocksfoot for which near anaerobic conditions (
0% O2) were reached after approximately 60 d, and were sustained until the end of the experiment. The rate of O2 decrease in timothy was slower than that observed for the three other species and similar to what was observed with bare soil; the lowest O2 concentration (2%) was reached after 80 d. On the last sampling date, after the fibreglass wool insulating cover had been removed for 10 d, a 2% increase in O2 concentration was observed in timothy enclosed pots, which could indicate photosynthesis recovery of timothy upon re-exposure to light.
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Carbohydrates
Total non-structural carbohydrates (TNC) increased in legume species during autumn acclimation, reached a maximum in November and then decreased during the overwintering period (Fig. 2). This decrease in TNC concentration was faster in the oxygen deficiency treatment than in the controls and the concentration of TNC was lower than 150 mg g–1 DW by the end of the experiment. The TNC concentration in cocksfoot remained low at near 150 mg g–1 DW during autumn and winter and was not affected by O2 deficiency. The TNC concentration increased progressively in timothy from September to February. Contrary to the other species, the oxygen defi ciency increased the TNC concentration up to 200 mg g–1 DW at the end of the experiment.
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In legumes, the main storage carbohydrate was starch, which concentration decreased from November to February (Fig. 3A). Starch concentration did not differ between the treatments in lucerne and red clover, except at the end of the oxygen-deficiency treatment when the starch concentration of lucerne was greater in the control than in the oxygen-deficiency treatment. In grasses, carbohydrates were stored under the form of HDP fructans which were more abundant in cocksfoot (P <0.0001) (60–120 mg g–1 DW) than in timothy (30–75 mg g–1 DW) (Fig. 3B). Concentrations of fructans did not decrease during the winter in contrast to starch. The concentration of HDP fructans decreased slightly in response to oxygen deficiency in timothy whereas the same treatment had no effect on their concentration in cocksfoot. The average degree of polymerization of fructans significantly decreased from 225 to 50 during winter for timothy while it remained relatively stable near 80 for cocksfoot (Fig. 3C). The degree of polymerization of fructans was slightly higher under the oxygen-deficiency treatment than in control plants of timothy.
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During cold acclimation, from September to November, a significant increase was observed in sucrose concentration in lucerne, red clover, and timothy. The sucrose concentration increased significantly under oxygen deficiency in timothy while it decreased in the three other species (Fig. 4). In cocksfoot, the concentration of sucrose remained low throughout the experiment as compared to the other species. The concentration of fructose (Fig. 5A) also increased in timothy under the oxygen deficiency treatment (P <0.0001) while fructose concentration either decreased or was not affected by the treatments in the three other species. Glucose concentration was significantly higher in oxygen-deficiency-treated plants of timothy and red clover while it became significantly lower under oxygen deficiency in cocksfoot and remained unaffected in lucerne (Fig. 5B).
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The oligosaccharides of the raffinose family, raffinose (Fig. 6A) and stachyose (Fig. 6B) increased significantly during cold acclimation in lucerne and red clover. Furthermore, the trisaccharide raffinose was significantly higher under the oxygen-deficiency treatment in both legume species while stachyose showed superior accumulation in oxygen-deficiency-treated lucerne only. Although raffinose was not observed in cold-acclimated cocksfoot, its accumulation was induced in oxygen-deficiency-treated plants. In oxygen-deficiency-treated timothy, raffinose was undetectable, but the concentration of a DP3-fructan was increased (Fig. 6C). In cocksfoot, the concentration of DP3-fructans increased during cold acclimation, but did not differ between oxygen-deficiency-treated plants and control plants.
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GAPDH gene expression
The level of gene expression of GAPDH differed between lucerne and timothy during both cold acclimation and oxygen deficiency treatment (Fig. 7). Transcripts of GAPDH increased in lucerne during cold acclimation and reached a maximum in November. An increase in the gene expression of GAPDH in lucerne was also observed in response to the oxygen-deficiency treatment in December, February and March. In timothy, the GAPDH gene expression decreased from September to December when a minimum was reached. Although the level of expression subsequently increased during winter, there was no specific induction of expression by oxygen deficiency as observed in lucerne.
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Discussion |
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The link between the accumulation of organic reserves and winter hardiness or spring regrowth of perennial species is firmly established (McKenzie et al., 1988). Thus, any stress that restricts the accumulation or accelerates the utilization of carbohydrate reserves in plants could impair winter hardiness and regrowth. Anaerobic metabolism has a low energetic efficiency. For this reason, plants overwintering under near anaerobic conditions could see the level of their carbohydrates reach a critical low concentration, below which spring regrowth could be impaired. It was recently reported that the regrowth of lucerne, red clover and cocksfoot was markedly reduced under conditions of low O2 (<2%) and high CO2 (around 25%) concentrations occurring at low temperature, whereas timothy regrowth remained unaffected (Bertrand et al., 2001). Timothy had a greater TNC concentration under low O2 condition than the other three species. This higher concentration of TNC in oxygen-deficiency-treated plants of timothy could be related to its lower metabolic rate under oxygen deficiency compared with the other three species. It was previously observed that the metabolic rate of timothy was much reduced under oxygen deficiency compared with the sensitive species, lucerne, red clover and cocksfoot (Bertrand et al., 2001). The increase in TNC concentration under oxygen deficiency could also be due to an import of carbohydrates from the roots to the shoot bases of timothy. Schlüter and Crawford (2001) observed that the mechanisms of carbohydrate transport remained active under long-term anoxia in Acorus calamus and Iris pseudacorus, two species considered as anoxia-resistant. These specific features could allow timothy to maintain adequate levels of carbohydrate reserves for spring regrowth.
A 2% O2 increase was observed in sealed enclosures with plants of timothy after the fibreglass wool covers were removed for a period of 10 d, during which air temperature inside the unheated greenhouse oscillated around the freezing point. This increase, only observed with timothy, could be due to photosynthesis occurring in the remaining leaf tissues. Öquist (1983) observed that many cold-acclimated species are able to photosynthesize at temperatures below zero as long as unfrozen water remains in the tissues. Other studies indicated that photosynthesis might occur under ice at low temperature (Andrews, 1988). Whether this occurs in timothy under field condition and its importance for spring regrowth might require further studies.
In legumes, the main C reserve is starch and its concentration in overwintering organs increases during autumn. The typical disappearance of starch reserves was observed in the crowns of lucerne and red clover during the winter (McKenzie et al., 1988). Anoxia-induced reduction of amylolytic activity could limit the availability of substrate and could lead to carbohydrate starvation (Arpagaus and Braendle, 2000). In this study, however, starch hydrolysis was not impaired by oxygen deficiency in contrast to Perata et al. (1992), who observed that starch breakdown stopped under oxygen deficiency in flood-intolerant plant tissues.
The major soluble sugars whose concentrations increased during cold acclimation were sucrose for legumes and fructose for grasses. The protective effect of soluble sugars through membrane stabilization in response to freeze-induced desiccation in plants is well documented (Sun et al., 1994). Under the oxygen deficiency treatment, an increase in concentrations of sucrose, fructose, and glucose was observed in the oxygen deficiency-resistant timothy, whereas the concentration of soluble sugars decreased under the same conditions in the oxygen deficiency-sensitive species. This confirms the conclusion of other studies which reported that energy and C skeletons are important determinants of the duration of survival under oxygen deficiency (Vartapetian and Jackson, 1997). Furthermore, the accumulation of soluble sugars observed in timothy is consistent with previous results (Bertrand et al., 2001) that indicated that the greater resistance of this species to oxygen deficiency at low temperatures could be associated with a slower glycolytic metabolism; a strategy that allows timothy to maintain oxidizable substrates to support spring regrowth. Because glycolysis is being favoured in response to oxygen-deficiency-treatment in the three sensitive species, it is not surprising to observe a depressed sucrose formation as this process requires a large quantity of fructose-6-P.
Temperate grasses store carbohydrates in the form of fructans of a high degree of polymerization rather than starch (Chatterton et al., 1989). Fructans were shown to accumulate during cold acclimation of grass species (Pollock and Cairns, 1991) and the relationship between fructan accumulation and freezing tolerance has been documented in cereals (Suzuki and Nass, 1988). The observation of a lower concentration of HDP fructans throughout the winter in cold-tolerant timothy (lethal temperature for 50% of the plants (LT50) of –14 °C) (Gudleifsson et al., 1986) as compared to cocksfoot (LT50 of –9 °C) does not support a link between fructan accumulation and cold-tolerance in these grass species. However, the progressive decrease of the degree of polymerization of fructans toward spring observed in timothy gives to this species a readily mobilizable source of carbohydrate for regrowth (Suzuki, 1993). The increase of 10 mg g–1 DW of fructose concentration induced by oxygen deficiency in timothy corresponds quantitatively to the decrease in HDP fructans, which hydrolysis was increased by oxygen deficiency only in this species. Fructan depolymerization at subfreezing temperature is thought to be an essential source of cryoprotective sugars (Livingston, 1996) and the increase in free fructose has been associated with depression of the freezing point of cold-hardened crowns of rye (Olien and Lester, 1985).
The concentration of the trisaccharide raffinose increased in legumes as well as in cocksfoot in response to oxygen deficiency. Raffinose concentration increased exclusively in the three sensitive species under oxygen deficiency conditions whereas another trisaccharide, a DP3 fructan, increased in the oxygen deficiency-resistant timothy. The increase in raffinose could be linked to plant dormancy induced by low oxygen concentration as observed in canarygrass (Phalaris minor Retz.) and wild oat (Avena fatua L.) (Parasher and Singh, 1985; Foley, 1996). Castonguay et al. (1995) measured a superior accumulation of raffinose and stachyose in winter hardy cultivars of lucerne that differ in their freezing tolerance as well as in their autumn dormancy (Brummer et al., 2000). In timothy, the accumulation of readily usable DP3-fructan could play a role in the resistance of this species to oxygen deficiency at low temperature. In related species either tolerant or intolerant to flooding, it has been shown that the species accumulating storage carbohydrates in the form of fructans rather than starch survived longer periods of oxygen deficiency (Hanhijarvi and Fagerstedt, 1995). Albrecht et al. (1997) speculated on the advantages for plants to accumulate fructans in response to oxygen deficiency. One of those advantages could be the storage of sugars in the form of fructose polymers without the intermediate stages and energy-using processes which are required for starch synthesis. For both species of grasses, the accumulation of fructans of low DP during the winter could be an adaptive advantage: the monosaccharides produced by fructan breakdown are transported to the growing regions and could play a role through turgor regulation for cell expansion during spring regrowth (Pollock, 1986).
The enzyme GAPDH, a key glycolytic enzyme, is known as one of the "anaerobic polypeptides" induced by anaerobic conditions in maize (Sachs et al., 1996). An increase of the genetic expression of GAPDH under oxygen deficiency at low temperature was observed in lucerne. Umeda and Uchimiya (1994) observed a significant increase in the amount of GAPDH transcripts in submergence-tolerant rice compared to an intolerant cultivar. Ricard et al. (1989) concluded that the expression of GAPDH was related to the capacity of rice seedlings to maintain a high metabolic rate under anaerobiosis. The increase in GAPDH transcripts induced by oxygen deficiency in lucerne could be an adaptive advantage to improve lucerne survival under short-term anaerobic conditions by enabling the plants to produce ATP through increased glycolysis. Contrastingly, GAPDH gene expression in timothy was not induced by oxygen deficiency although transcript levels were relatively high in overwintering plants. Lack of GAPDH induction under oxygen deficiency agrees with a previous observation that metabolic activity is not enhanced by oxygen deficiency in timothy (Bertrand et al., 2001).
In addition to their differential response to oxygen deficiency, the two species showed contrasting modulations of GAPDH gene expression during autumn acclimation. The transcript level of GAPDH increased from September to November in lucerne while it decreased in timothy. The general observation of increased transcript levels of GAPDH in response to environmental stresses such as heat (Yang et al., 1993), anoxia (Manjunath and Sachs, 1997), dehydration (Velasco et al., 1994), and biotic stress (Laxalt et al., 1996) suggested that a higher rate of glycolysis allowed plants to cope with stress (Velasco et al., 1994; Laxalt et al., 1996). It was observed that it was not the case for timothy in response to an anaerobic stress nor during cold acclimation.
In a previous paper, it was reported that timothy is more resistant to long-term oxygen deficiency at low temperature than cocksfoot, lucerne and red clover. In this paper, it is confirmed that slower metabolic rates of timothy allowed it to maintain higher carbohydrate reserve under oxygen deficiency. This feature, specific to timothy, could favour its winter survival and capacity for spring regrowth
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Acknowledgements |
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We thank Mrs Ginette Devarennes, Annie Létourneau, Lucette Chouinard, and Mr Pierre Lechasseur for their technical contribution to this study.
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References |
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