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a Dep. of Plant Agriculture, Univ. of Guelph, Guelph, ON, Canada N1G 2W1
b Centre de Recherche en Horticulture, Dép. de Phytologie, Univ. Laval, Sainte-Foy, QC, Canada G1K 7P4
c Soils and Crops Research and Development Centre, Agriculture and Agri-Food Canada, Sainte-Foy, QC, Canada G1V 2J3
* Corresponding author (jdionne{at}uoguelph.ca)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: DW, dry weight • FW, fresh weight • HPLC, high performance liquid chromatography • LT50, lethal temperature for 50% of the plants • MCW, methanol-chloroform-water • PPFD photosynthetic photon flux density
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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A major agronomic disadvantage of annual bluegrass is its susceptibility to environmental stresses (Beard, 1970; Peel, 1982). Low temperature stress restricts annual bluegrass culture on golf course greens in areas experiencing harsh winter conditions. Temperature fluctuations and extreme freezing temperatures at crown level, occurring during winter and early spring, cause recurrent losses of annual bluegrass on golf greens (Dionne et al., 1999). Susceptibility of annual bluegrass to subfreezing temperatures has been pointed out as a major factor responsible for winter damages on golf greens. In a recent study, we determined that marked differences in freezing tolerance exist among annual bluegrass ecotypes (Dionne et al., 2001). Maximum freezing tolerance was observed after exposure to nonlethal subfreezing temperatures.
Cold acclimation of a plant is a highly active process resulting from the expression of a number of physiological and metabolic adaptations to low temperature (Levitt, 1980). Major metabolic changes have been documented during the acquisition of cold tolerance including changes in carbohydrates, proteins, nucleic acids, amino acids, growth regulators, phospholipids, and fatty acids (Li, 1984). Relationships between carbohydrate levels and freezing tolerance have been studied in many plant species. We have recently demonstrated that fructans are the most abundant carbohydrates found in cold-acclimated annual bluegrass and that a marked increase in sucrose concentrations in crowns is observed after exposure to freezing temperatures, when freezing tolerance of annual bluegrass is maximum. However, variations in fructan and sucrose levels were not related to the differential freezing tolerance observed among three selected annual bluegrass ecotypes (Dionne et al., 2001).
Accumulation of free proline (Pro) in plants subjected to hyperosmotic stress induced by water deficit, elevated soil salinity or exposure to low temperature has been studied over the past 40 yr (Hare et al., 1999). Relationship between the accumulation of Pro during cold acclimation and freezing tolerance has been found in several plants (Paquin and Pelletier, 1981; Koster and Lynch, 1992; Dörffling et al., 1998). Pro overproducing mutants of wheat, Triticum aestivum L., (Dörffling et al., 1993) and transgenic plants of Arabidopsis (Nanjo et al., 1999), and tobacco, Nicotiana spp. (Kavi Kishor et al., 1995) with high Pro levels showed higher freezing or osmotic stress tolerance than wild types. Although these reports indicate a positive correlation between the accumulation of Pro and freezing tolerance, other results suggest that the increase in free Pro level is merely a result of stress exposure (Delauney and Verma, 1993; Hare and Cress, 1997; Wanner and Junttila, 1999). In addition to Pro, other amino acids have been reported to accumulate in wheat in response to low temperature including glutamic acid, glutamine, alanine, aspartic acid, and asparagine (Kaldy and Freyman, 1984; Naidu et al., 1991).
Cold acclimation is also associated with marked changes in protein composition and can affect both the amount and the type of polypeptides produced by the plant. Previous research has demonstrated that a specific subset of proteins are synthesized during cold acclimation of plants (Guy, 1990; Danyluk et al., 1991; Ryu and Li, 1994). Increases in total soluble proteins during cold hardening have been documented in many plant species (McKenzie et al., 1988). Many cold induced proteins share the unique property of heat stability (Salzman et al., 1996). Heat-stable proteins have been isolated from cold-acclimated plants and a correlation between a heat-stable protein (Wcs120) accumulation and cold-induced freezing tolerance has been observed in wheat (Houde et al., 1992). It has been suggested that cold acclimation-induced proteins may act in combination with soluble carbohydrates and other compounds in the acquisition of freezing tolerance (Gusta et al., 1996).
Currently, information is lacking on the expression and variability of these important traits in annual bluegrass. Our objective was to relate differences in amino acid levels and changes in protein composition to freezing tolerance potential in annual bluegrass ecotypes cold hardened under both environmentally controlled and simulated-winter conditions in an unheated greenhouse.
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MATERIAL AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Controlled Environment Experiments
Annual bluegrass tillers were transplanted individually in multi-cellular trays (size 72) filled with 3:1 (v/v) sand and peat moss growing medium (Pro-mix BX, Premier Peat Moss, Rivière-du-Loup, Canada) and grown for 4 wk in a greenhouse. Environmental conditions were as follows: photoperiod, 12 h; light temperature, 22°C; dark temperature, 18°C; natural irradiance was supplemented by artificial lighting provided by high pressure sodium lamps (400 W; Philips Lighting Co., Somerset, NJ). Plants were watered daily and fertilized once a week with 20-20-20 + micronutrients (300 µg L-1 N).
Annual bluegrass was subsequently transferred to a growth chamber for low temperature acclimation at a constant temperature of 2°C, under 8-h photoperiod and a photosynthetic photon flux density (PPFD) of 150 µmol photons m-2s-1. After 2 wk of acclimation, a group of plants was transferred to a freezer at -2°C in the dark for an additional period of 2 wk to simulate natural hardening conditions in frozen soil under snow cover. Another group of plants was kept at a low nonfreezing temperature (2°C) during that additional 2-wk period. No plant mortality resulted from the incubation at -2°C. Samples were collected at different stages of cold acclimation for amino acid and protein analysis and for freezing tolerance determination. Four pools of 3 to 5 tubes (12–20 tubes total) of each annual bluegrass ecotype were sampled at each stage for biochemical analyses.
Unheated Greenhouse Experiment
Annual bluegrass tillers were transplanted in mid-September 1998 in tubes filled with a 4:1 (v/v) sand and peat moss growing medium and grown in a greenhouse as previously described. After 5 to 6 wk of growth, plants were transferred to an unheated greenhouse at a site near Québec City, Canada (46°4'15''N, 71°12'00''W; elev. 
45 m) to acclimate to low temperatures under simulated-natural environmental conditions. Air temperature inside and outside the greenhouse was monitored with copper-constantan thermocouples (Omega Engineering, Stanford, CA) connected to a data acquisition system (CR10, Campbell Scientific, Logan UT). Soil temperature in tubes was monitored with thermocouples and recorded at 40-min intervals with a temperature logger (RD-TEMP-XT; Omega Engineering, Standford, CA). When the air temperature remained permanently below freezing, plants were covered with a layer of insulating fiberglass wool to simulate snow cover. The greenhouse was constantly ventilated during daytime to maintain the inside temperature similar to that of the outside. Samples were collected every 2 to 4 wk for amino acid and protein analysis and were assessed on five occasions. Five pooled samples of 3 to 5 tubes (15–25 tubes total) of each annual bluegrass ecotype were sampled at each date for amino acid and protein analysis. (30 Oct., 19 Nov., 17 Dec. 1998, 6 Jan., 2 and 26 Feb., and 11 and 26 March 1999).
Extraction and Analysis of Amino Acids
Frozen tubes were thawed overnight at 4°C, and the following day plants were washed free of soil with cold water. A pooled sample of 0.5 to 1.0 g FW of crown tissue (1 cm above and below the transition zone between shoot and roots) from 3 to 5 plants was harvested and immediately used for extraction. A subsample was oven dried for 48 h at 70°C for dry matter determination. Amino acids were extracted as follows: tissues were ground in liquid nitrogen; 5 mL of methanol-chloroform-water (MCW, 12:5:3, v/v) were added, and tubes were heated at 65°C for 20 min and stored at -20°C. A volume of 250 µL of water was added to 1 mL of extracts for phase separation and the tubes were centrifuged 4 min at 12000 g. The aqueous phase was collected, and a 750-µL subsample was evaporated to dryness on a rotary evaporator, resolubilized in water containing ethylene-diaminetetraacetic acid (EDTA; Na+, Ca2+, 50 mg L-1). The amino acid analysis system used Waters Millenium32 software and consisted of two model 510 pumps, a model 717+ autosampler and a model 774 scanning fluorescence detector. Waters Radial-Pak RESOLVE C18 (8 by 100) column (Waters Corporate Headquarters, Milford, MA) was eluted at 40°C with a gradient of buffer A [0.05 M Na2HPO4, 0.05 M NaCH3COO, 2% (v/v) methanol, 2% (v/v) tetrahydrofuran, pH 7.5] and solution B (65% methanol) with the following proportions of buffer A: 0 to 2.0 min, flow rate 0.1 mL/min, 70%; 2.0 to 2.5 min, flow rate increased from 0.1 to 2.5 mL/min, 70%; 2.5 to 6.5 min, 70%; 6.5 to 14 min, 70 to 38%, linear; 14.0 to 19.0 min, 38 to 15%, Waters curve #7; 19.0 to 20.0 min, 15 to 0% linear; 20.0 to 23.0 min, 0%; 23.0 to 24.0 min, 0 to 70% linear; 24.0 to 29.5 min, 70%; 29.5 to 30.0 min, flow rate decreased to 0 mL/min, 70%. Precolumn derivatization was made by the autosampler by mixing 10 µL of sample with 10 µL of a solution of 5 mg/mL of o-phtalaldehyde in 0.5 M K borate buffer, pH 10.0. Fluorometric detection excitation wavelength was 338 nm and the emission wavelength was 425 nm. Pro was quantitated by spectrophotometry at 515 nm by means of a colorimetric reaction with ninhydrin described by Paquin et al. (1989). Seventeen amino acids were analyzed but only aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), glutamine (Gln), arginine (Arg), tyrosine (Tyr), proline (Pro), and the sum of the 17 amino acids (TOTAL) are presented and discussed in this paper.
Extraction and Analysis of Soluble and Thermostable Proteins
Approximately 4 g FW of crown tissues (1 cm above and below the transition zone between shoot and roots) from 3 to 5 plants was ground to powder in liquid N2 with a mortar and a pestle. Samples were kept frozen at -80°C until extraction. Samples were homogenized for 2 to 3 min on ice with a Polytron (Brinkman, Rexdale, ON, Canada) homogenizer in 5 mL of a buffer containing 50 mM HEPES (N-2-hydroxyethylpiperzazine-N'-2-ethanesulfonic acid) pH 7.0, 10 mM MgCl2, 1 mM EDTA, 10% (v/v) ethylene glycol. After homogenization, samples were adjusted to 2 mM dithiothreitol and 0.02% (v/v) Triton X-100. The homogenate was centrifuged 15 min at 10000 g at 4°C and soluble proteins in the supernatant were precipitated in 65% (w/v) (NH4)2SO4. Thermostable proteins in crown tissues homogenized in the extraction buffer described above were separated by a 10-min incubation at 100°C. Samples were centrifuged at 12 000 g for 10 min and proteins in the remaining supernatant were precipitated in 65% (NH4)2SO4. Aliquots of the soluble and thermostable proteins extracts were collected for protein quantification according to the method of Lowry et al. (1951) with BSA (bovine serum albumin) as the standard protein. Protein extracts were solubilized in an inclusion buffer continuing 2.3% (w/v) SDS (sodium dodecyl sulfate), 10% (v/v) glycerol and 62.5 mM Tris, pH 6.8 and 5% (v/v) ß-mercaptoethanol. Protein samples were load-adjusted to an equivalent dry weight basis and were electrophoresed through a 12 to 18% (w/v) gradient separating polyacrylamide gel and stained with Coomassie Brilliant Blue according to the method of Laemmli (1970). Protein profiles were analyzed by densitometry using the OneDScan software (Scanalytics, Billerica, MD).
Statistical Analysis
Analysis of variance was made with the General Linear Model Procedure of SAS statistical software (SAS Institute, 1990). The controlled environment experiments were arranged as a randomized complete block with four replications and the unheated greenhouse experiments were arranged as a completely randomized design with five replications. Fisher's Least Significant Difference test (LSD) was used for comparison of amino acid level between ecotypes and sampling dates when the F-test for main effects was significant (P = 0.05).
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unheated Greenhouse Experiment
Variations in levels of amino acids in crowns of annual bluegrass ecotypes cold acclimated in the unheated greenhouse during winter 1998–1999 are presented in Fig. 2
. Exposure to cold temperatures induced a marked accumulation of Asp, Glu, Arg, and Pro in ecotype CR while levels of these amino acids remained relatively stable for CO and OK. Except for the CR ecotype that initially accumulated Gln in fall, Gln progressively declined in annual bluegrass following the transfer to the unheated greenhouse while Asn concentrations in crowns of annual bluegrass increased rapidly under these conditions. Levels of Tyr were similar among annual bluegrass ecotypes during cold hardening except in January, when the concentration of this amino acid was significantly higher in crowns of CR.
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Modifications in the Protein Patterns during Cold Acclimation
The electrophoretic profiles of soluble proteins in two annual bluegrass ecotypes (OK and CR) of contrasting freezing tolerance are presented in Fig. 3
. Comparison of profiles of proteins extracted on Oct. 30 indicates that polypeptides of 31, 30, 27,16, and 13 kDa were the major proteins present in non acclimated plants of both ecotypes. A distinct accumulation of polypeptides of 31, 30, 27, 16, and 13 kDa from fall until midwinter was noted in the cold tolerant ecotype OK and levels of these polypeptides subsequently decreased in spring. The highest levels of these five proteins coincided with the maximum freezing tolerance of the ecotype OK. We observed the same pattern of accumulation in ecotype CR for the 31-, 30-, and 16-kDa polypeptides while levels of the 27- and 13-kDa polypeptides remained stable throughout winter. As observed in the cold tolerant ecotype OK, the highest levels of the 31- and 30-kDa polypeptides in ecotype CR were observed at the time when it reached its maximum freezing tolerance. Densitometric analysis of protein profiles revealed that cold responsive polypeptides were present at higher levels in the cold tolerant ecotype OK than in the cold sensitive CR on 2 February, when annual bluegrass reached its maximum freezing tolerance.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to Pro accumulation, cold hardening induced other changes in amino acid composition in overwintering crowns of annual bluegrass. Transfer to low nonfreezing temperatures (H2) increased Arg concentration while Asp and Glu levels decreased under the same conditions. Meza-Basso et al. (1986) observed that Asp, Glu, and Arg levels decreased in leaves of Nothofagus dombeyi Bl., a woody species from southern Chile, after a 24-h exposure at 0°C, while Kaldy and Freyman (1984) noted an accumulation of Asp and Glu in wheat crowns after 14 d at 1°C. Naidu et al. (1991) observed that Gln, Ala, and Asp accumulated in leaves of wheat after 5 d at 4°C while Glu decreased under these conditions. These results emphasize the fact that although some amino acids have been reported to accumulate in various plant species during cold acclimation, the data are often contradictory probably as a result of difference in acclimation conditions of plant material and analytical methods. In fact, very few studies have looked at the accumulation of amino acids other than Pro in relation to cold hardening, whereas the impact of subfreezing temperature on amino acid accumulation is essentially not documented in the literature.
Maximum levels of amino acids and greater differences amongst ecotypes were observed in annual bluegrass cold acclimated under natural conditions in an unheated greenhouse. This result is in accordance with our previous report on the promotive effect of subzero temperatures on freezing tolerance and carbohydrate changes in annual bluegrass (Dionne et al., 2001). Levels of Asn and Gln were markedly higher in crowns of annual bluegrass cold acclimated in the unheated greenhouse than in crowns of plants acclimated under environmentally controlled conditions. The reasons for this large difference in amino acid levels between the experimental conditions is not clear but large variations in these amino acids have also been noted by others. For instance, Naidu et al. (1991) observed a 25-fold increase in the level of Gln from 9.6 to 242.7 µmol/g DW and a 37-fold increase in the level of Asn in leaves of wheat exposed to 4°C for 5 d. In addition, differences in irrigation prior to plant transfer in the unheated greenhouse could help to explain differences of amino acid levels since tissues of many plants are known to accumulate Asn in response to water deficit (Drossopoulos et al. 1985). The highest contributions to total amino acid accumulation after acclimation at subfreezing temperatures came from Pro, Gln, and Glu under both environmentally controlled and simulated winter conditions. These three amino acids represented about half of the total amino acids that accumulated in crowns of annual bluegrass. Our results suggest that the cold induced decline of Gln could be related to some extent to the accumulation of Pro and Glu. The exact mechanism that triggers the accumulation of specific amino acids during cold acclimation of plants is not clear; Naidu et al. (1991) suggested that cold temperature may affect the regulation of the activity of key enzymes involved in Glu and Asp synthesis and/or metabolism and may consequently induce changes in amino acid concentration.
Cold acclimation-induced accumulation of amino acids might not be related exclusively to freezing tolerance but could also play a role in coping with other stresses associated with overwintering under a deep and long-lasting snow cover. It has been suggested that Pro degradation upon relief from stress might provide carbon, nitrogen, and energy for regrowth (Hare and Cress, 1997). Similarly, mobilization of high levels of Pro and other amino acids that accumulated in the CR ecotype, originating from central Quebec, may be required to provide the energy and nutrients required for the long overwintering period and spring regrowth. Volenec et al. (1996) noted that although carbohydrate accumulation in relation to cold stress has been studied intensively, nitrogen reserves might be equally important for winter hardiness and spring regrowth.
We observed quantitative and qualitative changes in protein composition in crowns of annual bluegrass in response to cold acclimation. A distinct accumulation of polypeptides from fall until midwinter was noted and levels of these polypeptides subsequently decreased in the following spring. Protein accumulation follows a similar pattern to the acquisition of freezing tolerance, and peak levels of soluble protein in crowns coincided with maximum freezing tolerance of annual bluegrass. Guy et al. (1985) established that changes in gene expression occur during cold acclimation and since then many cold responsive proteins have been isolated in many plant species (Thomashow, 1999). The cold tolerant ecotype OK accumulated higher levels of proteins than the cold sensitive ecotype CR during cold acclimation. Differential accumulation of cold-regulated proteins has been reported for members of the Poacea differing in freezing tolerance (Houde et al., 1992; Ouellet et al., 1993).
Stress-induced proteins from numerous plant species share the unique property of remaining soluble upon boiling. Dehydrins belong to this class of proteins and were shown to be cold inducible in many plant species. Houde et al. (1992) isolated a heat-stable protein (Wsc120), related to dehydrins, from cold-acclimated wheat and found a correlation between the accumulation of this protein and freezing tolerance of wheat. Considering that the cold-tolerant ecotype OK accumulated higher levels of proteins and that peak levels of proteins coincided with the maximum freezing tolerance of annual bluegrass, our results strongly suggest that cold acclimation-induced protein accumulation plays a determinant role in freezing tolerance of annual bluegrass. One of the important conclusions to emerge from recent studies is that cold acclimation includes the expression of certain cold-induced genes encoding proteins involved in membrane stabilization during freezing stress (Thomashow, 1999).
The fact that the ecotype CR, originating from northern climate, accumulated higher levels of amino acids and carbohydrates than the other two ecotypes (Dionne et al., 2001), and its low freezing tolerance suggest that many factors need to be considered to assess annual bluegrass adaptation to harsh winter conditions fully. Large differences in freezing tolerance and in biochemical changes observed between CR and the other two ecotypes may be the result of genetic adaptation to their specific growing conditions. Genetic requirements to survive under deep and long lasting snow cover could be independent of adaptation to withstand low, subfreezing temperature. Although ecotypes OK and CO exhibited high freezing tolerance in both experiments, persistence of these ecotypes, originating from Maryland and Pennsylvania respectively, under Quebec climatic conditions can only be confirmed by field evaluation. Additionally, further studies on the relationship between field survival of annual bluegrass and freezing tolerance potential and associated biochemical changes are needed in a larger annual bluegrass ecotype collection to broaden our understanding of the physiological basis of adaptation to harsh winter conditions in this species.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication February 9, 2001.
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TOPNOTESABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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