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a Centre de Recherche en Horticulture, Département de Phytologie, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4
b Soils and Crops Research and Development Centre, Agriculture and Agri-Food Canada, Sainte-Foy, Québec, Canada G1V 2J3
Corresponding author (julie.dionne{at}crh.ulaval.ca)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: HMW, high molecular weight • LMW, low molecular weight • LT50, lethal temperature for 50% of the plants • HPLC, high performance liquid chromatography • PPFD photosynthetic photon flux density • MCW, methanol-chloroform-water • DW, dry weight
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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 stresses restrict annual bluegrass culture on golf course greens in areas experiencing harsh winter conditions. Large temperature fluctuations and extreme freezing temperatures at crown level occurring during winter and early spring result in recurrent loss of annual bluegrass on golf greens (Dionne et al., 1999).
Cold acclimation of plants 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). Among these, the fructose polymers, fructans, were shown to accumulate during cold acclimation of grass species (Pollock and Cairns, 1991; Livingston, 1991). Although they constitute an important source of energy in overwintering plants, their depolymerization at subfreezing temperatures is thought to be an essential source of cryoprotective sugars (Olien and Clark, 1993; Livingston, 1996). Relationship between fructan accumulation and freezing tolerance have been documented in cereals (Suzuki and Nass, 1988; Pontis, 1989). Evidence suggests that soluble sugars, such as sucrose and oligosaccharides of the raffinose family, in combination with heat stable proteins could play a determinant role in stress tolerance by protecting proteins and membranes against freeze-induced denaturation (Gusta et al., 1996). No information is currently available on the expression and variability of these important traits within annual bluegrass. Our objective was to assess freezing tolerance and carbohydrate changes occurring during cold acclimation of three green-type 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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Annual Bluegrass Ecotypes
Three annual bluegrass ecotypes of contrasting cold tolerance were selected from preliminary experiments performed under environmentally controlled conditions. These annual bluegrass ecotypes originated from Western Pennsylvania (OK), Coastal Maryland (CO), and Central Québec (CR). Ecotypes from USA were obtained from Dr. David Huff's collection of annual bluegrass ecotypes at Pennsylvania State University.
Controlled Environment Experiments
Annual bluegrass tillers were transplanted individually in multi-cellular trays (size 72) filled with a 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 (400w; 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 in 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-2 s-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 under an 8-h photoperiod at low nonfreezing temperatures (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 carbohydrate analysis and freezing tolerance determination. Four pooled samples of three to five plants of each annual bluegrass ecotype were sampled at each date for carbohydrate analysis.
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°47'15''N, 71°12'00''W; elev. 
45 m) to acclimate to low temperatures under 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 was 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 carbohydrate analysis and freezing tolerance was assessed on six occasions. Five pooled samples of three to five tubes (15–25 tubes) of each annual bluegrass ecotype were sampled at each date for carbohydrate analysis (30 Oct., 19 Nov., 17 Dec. 1998, 6 Jan., 2 and 26 Feb., 11 and 26 March 1999).
Freezing Tests
Freezing tests were performed in programmed freezers following an 8-h equilibration period at -2°C according to a procedure described previously (Castonguay et al., 1993). Temperature was lowered by 3°C during a 90-min period followed by a 60-min plateau at each test temperature. Two simultaneous freezing tests were performed in separate freezers as replicates. Nonacclimated plants were tested between -17 and -5°C and cold-acclimated plants between -29 and -14°C. At the end of each temperature plateau, 12 plants (controlled environment) or eight plants (unheated greenhouse) of each ecotype were withdrawn from the freezers and thawed at 2°C for 24 h. Plants were then transferred to initial growth conditions for regrowth. After 3 wk, survival counts were taken and the 50% killing temperature (LT50) was computed by the SAS Probit procedure (SAS Institute, 1990).
Extraction of Water Soluble Carbohydrates
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 (fresh weight) of crown tissue (1 cm above and below the transition zone between shoot and root) from three to five plants was harvested and immediately used for extraction. A subsample was oven dried for 48 h at 70°C for dry matter determination. Soluble sugars were extracted as described in Castonguay et al. (1995). Tissues were ground in liquid nitrogen; 5 mL of methanol-chloroform-water (MCW, 12:5:3, v/v/v) were added and tubes were heated at 60°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 12 000 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). Fructans were extracted as follows: tissues were ground on liquid nitrogen, 5 mL water was added and tubes were heated to 100°C for 20 min and stored at -20°C until further analysis.
Soluble carbohydrates were analyzed by high performance liquid chromatography (HPLC) (Waters, Milford, MA) and quantitated by refractometry. The HPLC system was computer-controlled by the Millenium32 software and was composed of an automatic injector (WISP 717B), Model 510 pump, and a Model 410 Differential Refractometer. Mono and disaccharides were separated on a Sugar-Pak column (0.65 by 30 cm; Waters, Milford, MA) and eluted isocratically at 85°C with EDTA (Ca2+, Na+, 50 mg L-1) at a flow rate of 0.5 mL min-1. Peak identity and quantity were obtained by comparison to standards. High and low molecular weight (LMW) fructans were respectively separated on a KS-804 (Shodex, Tokyo, Japan) and a HPX-42A (Bio-Rad, Richmond, CA) column and eluted isocratically at 25°C with deionized water at respective flow of 0.5 and 1.0 mL min-1. Amounts of fructans were determined by reference to a fructose standard curve.
Statistical Analysis
Analysis of variance was made by 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. Fishers Least Significant Difference test (LSD) was used for comparison of LT50 and carbohydrate levels 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 Experiments
Freezing Tolerance
Changes in air and soil temperature are presented in Fig. 3
. Daily average air temperature in unheated greenhouse ranged from -20.7 to 6.8°C during the experiment. Soil temperature decreased gradually to reach 0°C in early December, remained below freezing until mid-March and progressively rose above freezing afterwards.
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-9°C at the time of their transfer to the unheated greenhouse in October to LT50 from -27.3 to -22.2°C in December and reached a maximum in early February of -30.9, -26.1, and -23.5°C for OK, CO, and CR ecotypes respectively. Although LT50 decreased from -26.6 to -20.7°C (OK), -22.3 to -19.6°C (CO), and from -17.7 to -16.8°C (CR) in March, annual bluegrass maintained significant levels of freezing tolerance up to the end of March. All ecotypes had a similar acclimation response but they differed in their capacity to tolerate subfreezing temperatures. Cold acclimated OK was significantly more cold tolerant than CO and CR at all sampling dates. Freezing tolerance of annual bluegrass ecotypes closely followed the ranking observed under environmentally controlled conditions and ecotypes retained their relative order for LT50 throughout winter: OK < CO < CR.
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10 mg g-1 dry weight up to a maximum of 78 mg g-1 dry weight. HMW and LMW fructan showed an opposite response with a decrease during fall and winter. Except for the CR ecotype that initially accumulated HMW fructans in fall, fructans progressively declined in annual bluegrass following their transfer to the unheated greenhouse. CR and OK ecotypes maintained higher fructan levels during winter than CO. The highest sucrose concentrations were measured in February, when annual bluegrass ecotypes were at their maximum freezing tolerance. At this time, the relative ranking for sucrose concentration was the reverse of LT50 ranking: CR (78 mg g-1 DW, -23.5°C), CO (63 mg g-1 DW, -26.1°C), and OK (59 mg g-1 DW, -30.9°C). As observed under environmentally controlled conditions, the cold sensitive ecotype CR accumulated significantly higher levels of sucrose, glucose, and fructose than the other two ecotypes after acclimation to simulated winter conditions in an unheated greenhouse.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cold tolerant ecotype (OK) showed a 9.5°C increase in freezing tolerance after acclimation at subfreezing temperatures as compared to a 7.1°C for CO and a 5.3°C for CR. Livingston (1996) noted that the promotive effect of subfreezing temperatures on freezing tolerance of oat was superior in the more hardy cultivars suggesting the important adaptive value of this acclimation response. These results emphasize and extend to annual bluegrass the importance of acclimation at subfreezing temperatures for the full expression of the genetic potential for freezing tolerance. Acclimation under these conditions is required to obtain an adequate assessment of annual bluegrass tolerance to subfreezing temperatures. Annual bluegrass is believed to be poorly adapted to environmental stresses and its susceptibility to subfreezing temperatures has been pointed out as a major factor responsible for winter damages. A recent study on winter protection of golf greens has indicated that winterkill of annual bluegrass is important when crown-level soil temperatures reach a threshold of -10°C (Dionne et al., 1999). Fowler et al. (1981) reported that the field survival index of 26 wheat cultivars grown under the harsh winter conditions of Western Canada was highly correlated with their ability to withstand subfreezing temperatures. Winter survival under field conditions depends on the capacity of the plant to tolerate a wide range of environmental stresses, including extreme freezing, duration of exposure, desiccation, freeze-thaw cycles, duration of snow cover, ice encasement, diseases, and other factors. Our results confirm that freezing tests are a valuable approach to assess low temperature adaptation of annual bluegrass ecotypes. However, the unexpected low LT50 of CR, an ecotype originating from central Québec suggests that additional traits must be considered to fully assess annual bluegrass adaptation to harsh winter conditions. Further studies on the relationship between field survival of annual bluegrass and freezing tolerance potential are needed to broaden our understanding on the physiological basis of adaptation to harsh winter conditions in that species.
Transfer to low nonfreezing temperatures induced major changes in carbohydrate content in overwintering crowns of annual bluegrass. Although LMW fructans initially increased at low temperature, a gradual increase of HMW fructans took place as hardening progressed over a 4-wk period at 2°C. Large accumulations of HMW fructans were also observed during cold hardening of winter cultivars of rye (Secale cereale L.), barley (Hordeum vulgare L.), and wheat (Livingston, 1991). Fructans are primarily considered as a storage carbohydrate and their function as a cryoprotectant remains controversial (Livingston and Henson, 1998). Recent studies on the relationship between fructans and stress tolerance support their adaptive value in helping plants cope with adverse environmental conditions. Bromus pictus Hook. f., a grass species native to a cold arid region with constitutive fructan synthesis showed greater root survival to desiccation than the closely related Bromus auleticus Trin. ex Nees, which accumulates fructan only when exposed to low temperature (Puebla et al., 1997). Although a direct protective role of fructans remains conjectural, the recent demonstration by Demel et al. (1998) of a strong and specific interaction between HMW fructans with model membranes suggests that fructans might help prevent lipid condensation and phase transitions to take place in water stress or freeze-desiccated cells. The accumulation of comparable levels of HMW fructans in two annual bluegrass ecotypes of contrasting freezing tolerance (OK and CR) does not indicate a close link between their accumulation and differential freezing tolerance among annual bluegrass ecotypes. This result differs from previous reports of a close relationship between fructan accumulation and freezing tolerance in other Poaceae species (Pontis, 1989; Santoiani et al., 1993). Suzuki and Nass (1987) noted that hardier winter cereal cultivars generally accumulated higher levels of HMW fructans when compared to less hardy cultivars. In the current study, a closer relationship seemed to exist between the variations in LMW fructans and levels of freezing tolerance of annual bluegrass ecotypes acclimated under environmentally controlled conditions. The fact that no such link was apparent when ecotypes were acclimated to simulated winter conditions suggests the complexity of the interactions between the environment and the biochemical bases of adaptation to low temperature.
Sucrose started to accumulate in crowns of annual bluegrass at low temperature above freezing, and its levels markedly increased at temperatures below freezing under both controlled and natural acclimation conditions. Under both hardening conditions, peak sucrose concentration in crowns coincided with maximum freezing tolerance of annual bluegrass. Our results agree with previous observations of marked hydrolysis of both HMW and LMW fructans into mono and disaccharides at subzero temperatures in temperate grass species (Chatterton et al., 1988; Tronsmo et al., 1993) and winter cereals (Livingston, 1996; Olien and Clark, 1993). It has been suggested that sucrose plays an important cryoprotective role by stabilizing membranes and proteins (Hoekstra et al., 1989), preventing adhesion of ice to critical cellular tissue during freezing (Olien, 1984) or by modifying ice crystal formation (Olien and Lester, 1985). Sucrose levels were not related to the differential freezing tolerance among the three annual bluegrass ecotypes tested in the current study: the cold sensitive ecotype CR accumulated significantly higher levels of sucrose, glucose, and fructose than the cold tolerant ecotype OK. This differs from Livingston (1996) observation of a strong correlation between soluble sugar levels and freezing survival of 23 oat cultivars. On the other hand, Castonguay et al. (1995) reported that levels of sucrose in fully acclimated alfalfa (Medicago sativa L.) were poorly related to the differences in freezing tolerance between cultivars. Conflicting literature reports on the association between freezing tolerance and soluble sugars might be resolved by integrating additional factors like potential interactions between heat stable proteins and soluble sugars for maximum protection of membranes and proteins against stress-induced denaturation (Gusta et al., 1996). Seed desiccation tolerance was found to be more closely related to the ratio of raffinose to sucrose than to the level of sucrose alone (Black et al., 1996). Such ratio between soluble sugars might also be required in annual bluegrass to achieve superior levels of freezing tolerance.
The annual bluegrass ecotype CR showed lower levels of freezing tolerance and higher carbohydrate content than the other two ecotypes (OK and CO). CR originates from central Québec, where the depth and duration of the snow cover are important factors. It is noteworthy that superior accumulation of soluble carbohydrates in wheat has been linked to both reduced freezing tolerance and increased tolerance to snow molds (Yoshida et al., 1998; Gaudet et al., 1999). Gaudet et al. (1999) noted that wheat cultivars tolerant to snow mold accumulate higher levels of carbohydrate and metabolize them at slower rates than cultivars sensitive to snow molds. A wheat cultivar resistant to snow mold exhibited reduced freezing tolerance and accumulated more carbohydrates than freezing tolerant cultivars (Yoshida et al., 1998). An interesting parallel between these results and our observations on annual bluegrass in the current study can be drawn.
We observed differences in freezing tolerance and carbohydrate levels among the three annual bluegrass ecotypes tested in the current study, which strongly suggests that differences in genetic potential for freezing tolerance exists within that species. Screening of a larger number of ecotypes will provide an assessment of the extend of variation of freezing tolerance and associated biochemical changes in green-type annual bluegrass.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication March 17, 2000.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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