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Published online 1 September 2007
Published in Crop Sci 47:2162-2169 (2007)
© 2007 Crop Science Society of America
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TURFGRASS SCIENCE

Differences in Freeze Tolerance of Zoysiagrasses: I. Role of Proteins

Aaron J. Pattona,*, Suzanne M. Cunninghamb, Jeffrey J. Volenecb and Zachary J. Reicherb

a Dep. of Horticulture, Univ. of Arkansas, 316 Plant Sciences Bldg., Fayetteville, AR 72701
b Dep. of Agronomy, Purdue Univ., 915 W. State St., West Lafayette, IN 47907-2054. Purdue Univ. Agriculture Experiment Station Journal no. 2006-18051

* Corresponding author (ajpatton{at}uark.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Genotypic variation in winter injury exists among zoysiagrasses (Zoysia spp.), but the physiological basis for these differences is not understood. Our objective was to determine the relationships between protein accumulation, polypeptide composition, and freeze tolerance of zoysiagrass. Thirteen genotypes of zoysiagrass with contrasting cold hardiness were identified. Cold acclimation was induced with 4 wk of 8/2°C day/night cycles and a 10-h photoperiod of 300 µmol m–2 s–1. Rhizomes and stolons of zoysiagrass were harvested from nonacclimated and cold-acclimated plants and used for protein analysis. Protein composition was analyzed using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with an antidehydrin polyclonal antibody. Buffer-soluble protein concentrations were higher among cold-acclimated (7.3 g kg–1 dry wt.) than nonacclimated (5.1 g kg–1 dry wt.) plants. The SDS-PAGE analysis indicated few differences in polypeptide composition among genotypes irrespective of cold acclimation. Immunoblotting indicated that dehydrin polypeptides (23 and 25 kDa) increased during cold acclimation. Abundance of the 23-kDa dehydrin polypeptide was positively associated (r2 = 0.41) with genetic variation in freezing tolerance. Our results suggest that dehydrins are associated with zoysiagrass cold acclimation, but that only the 23-kDa dehydrin plays a role in improving freeze tolerance.

Abbreviations: LT50, lethal temperature killing 50% of the plants • PAGE, polyacrylamide gel electrophoresis

Differences in Freeze Tolerance of Zoysiagrasses: I. Role of Proteins

Aaron J. Pattona,*, Suzanne M. Cunninghamb, Jeffrey J. Volenecb and Zachary J. Reicherb

a Dep. of Horticulture, Univ. of Arkansas, 316 Plant Sciences Bldg., Fayetteville, AR 72701
b Dep. of Agronomy, Purdue Univ., 915 W. State St., West Lafayette, IN 47907-2054. Purdue Univ. Agriculture Experiment Station Journal no. 2006-18051

* Corresponding author (ajpatton{at}uark.edu).

Genotypic variation in winter injury exists among zoysiagrasses (Zoysia spp.), but the physiological basis for these differences is not understood. Our objective was to determine the relationships between protein accumulation, polypeptide composition, and freeze tolerance of zoysiagrass. Thirteen genotypes of zoysiagrass with contrasting cold hardiness were identified. Cold acclimation was induced with 4 wk of 8/2°C day/night cycles and a 10-h photoperiod of 300 µmol m–2 s–1. Rhizomes and stolons of zoysiagrass were harvested from nonacclimated and cold-acclimated plants and used for protein analysis. Protein composition was analyzed using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with an antidehydrin polyclonal antibody. Buffer-soluble protein concentrations were higher among cold-acclimated (7.3 g kg–1 dry wt.) than nonacclimated (5.1 g kg–1 dry wt.) plants. The SDS-PAGE analysis indicated few differences in polypeptide composition among genotypes irrespective of cold acclimation. Immunoblotting indicated that dehydrin polypeptides (23 and 25 kDa) increased during cold acclimation. Abundance of the 23-kDa dehydrin polypeptide was positively associated (r2 = 0.41) with genetic variation in freezing tolerance. Our results suggest that dehydrins are associated with zoysiagrass cold acclimation, but that only the 23-kDa dehydrin plays a role in improving freeze tolerance.

Abbreviations: LT50, lethal temperature killing 50% of the plants • PAGE, polyacrylamide gel electrophoresis


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
WINTER INJURY varies widely among zoysiagrass (Zoysia spp.) genotypes (Patton and Reicher, 2007). The physiological basis for these differences has only partially been explored (Rogers et al., 1975, 1976, 1977; Akiyama et al., 1994; Fuller et al., 1999; Zhang et al., 2006). Physiological changes that occur during cold acclimation in plants include increased concentrations of sugars, desaturated lipids, organic acids, proline, and soluble proteins (Sakai and Yoshida, 1968; Hughes and Dunn, 1990; Harwood et al., 1994). In this study, we explored changes in soluble proteins before and after cold acclimation among zoysiagrass genotypes.

Polyacrylamide gel electrophoresis (PAGE) has been used in the past to characterize soluble protein changes in warm-season turfgrasses during cold acclimation (Davis and Gilbert, 1970; Palmertree et al., 1973; Gatschet et al., 1994). Soluble protein from cold-acclimated centipedegrass [Eremochloa ophiuroides (Munro) Hack.] stolons and crowns increased in autumn compared with nonacclimated plants (Palmertree et al., 1973). Certain proteins in the rhizomes of hybrid bermudagrass (Cynodon dactylon x C. transvaalensis) increased during autumn, while others decreased (Davis and Gilbert, 1970; Gatschet et al., 1994). Gatschet et al. (1994) found that low-molecular-weight proteins (14–37 kDa) synthesized during cold acclimation were correlated with freeze tolerance in hybrid bermudagrass.

Extracellular voids often form from the presence of ice during low-temperature exposure. These voids form in ‘Meyer’ zoysiagrass rhizomes during freezing (Warmund et al., 1998) as a result of extracellular ice formation, which places a dehydrative stress on cells (Steponkus, 1980). Certain proteins commonly accumulate during dehydrative stress (drought, low temperature, salinity, or seed maturation), of which dehydrins (late embryogenesis abundant D-11 family) are the most common (Close, 1996). Dehydrins are hydrophilic, thermostable, and characterized by a 15 amino acid consensus sequence rich in lysine near the carboxy terminus with repeats occurring within the protein (Close et al., 1993; Close, 1996, 1997). Dehydrins appear to exist in all photosynthetic organisms (Close, 1997) including monocots such as the cereals barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), maize (Zea mays L.), and rice (Oryza sativa L.) (Close et al., 1993). Dehydrins are also present in the turfgrasses orchardgrass (Dactylis glomerata L.; Volaire, 2002) and tall fescue (Festuca arundinacea Schreb.; Jiang and Huang, 2002) when drought stressed.

Dehydrin accumulation is correlated with freeze tolerance in barley (Zhu et al., 2000), blueberry (Vaccinium corymbosum Linn.; Panta et al., 2001), citrus (Poncirus trifoliate L. and Citrus grandis L.; Cai et al., 1995), peach [Prunus persica (L.) Batsch; Artlip et al., 1997], red-osier dogwood (Cornus sericea L.; Karlson et al., 2004), Rhododendron spp. (Marian et al., 2003), and wheat (Danyluk et al., 1994). Additionally, overexpressing dehydrin genes in Arabidopsis resulted in nonacclimated plants containing similar or higher dehydrin levels than cold-acclimated wild-type plants and with greater freeze tolerance than control plants (Puhakainen et al., 2004). These findings provided evidence that dehydrins contribute to freezing tolerance (Puhakainen et al., 2004) by stabilizing membranes (Close, 1996; Puhakainen et al., 2004), cryoprotection (Wisniewski et al., 1999), antifreeze activity (Wisniewski et al., 1999), or by scavenging free radicals (Hara et al., 2003).

Although the role of dehydrins during freezing stress has been investigated in many plant species, there is no information concerning the role of dehydrins in turfgrass freezing tolerance. Additional understanding of the role of proteins in freeze tolerance of zoysiagrass will help identify physiological traits that could be exploited by breeders to increase cold hardiness of warm-season turfgrasses. The objective of this study was to determine the relationships between protein accumulation, polypeptide composition, and freeze tolerance of zoysiagrass.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Freeze Tolerance
Detailed procedures to determine the freeze tolerance of zoysiagrass genotypes and results were described previously (Patton and Reicher, 2007). Briefly, a growth chamber, cold-stress simulator, regrowth data, and nonlinear regression were used to determine the temperature resulting in no regrowth from 50% of the plants (LT50). Plants were cold acclimated for 4 wk using a controlled-environment chamber set at 8/2°C day/night cycles and a 10-h photoperiod of 300 µmol m–2 s–1 photosynthetically active radiation (Anderson et al., 1993). This experiment was replicated for a total of six experimental replications.

Tissue Preparation
Whole rhizomes and stolons of zoysiagrass containing crown tissue were harvested both before acclimation (nonacclimated) and after cold acclimation from additional sets of plants not used for freeze tolerance testing. Rhizomes and stolons were combined for protein analysis since they have comparable anatomy, freeze tolerance, and solute levels in autumn and winter (Rogers et al., 1975, 1976). All rhizomes and stolons harvested were frozen in liquid N2 after washing off soil and removing leaves, roots, and stems. Samples were then stored at –80°C overnight and lyophilized (Lyph Lock, Labconco, Kansas City, MO) the following day. Lyophilized tissues were ground in liquid N2 using a mortar and pestle and stored at –80°C until protein analysis.

Protein Analysis
Methods used to extract and quantify proteins were based on a modification from Li et al. (1996) and performed at 4°C or on ice unless stated otherwise. Buffer-soluble proteins were extracted from ~50 mg of lyophilized, ground tissue with 1 mL of 0.1 mol L–1 sodium phosphate buffer (pH 6.8) containing 1 mmol L–1 phenylmethyl sulfonyl fluoride and 10 mmol L–1 2-mercaptoethanol in 1.5-mL microfuge tubes. Tubes were vortexed 15 s then set in ice a series of four times, centrifuged at 16,000 x g for 10 min at 4°C, and three 25-µL aliquots of supernate removed for protein quantification. The remaining supernate was retained for sodium dodecylsulfate (SDS) PAGE and immunoblotting. Buffer-soluble protein was estimated using the Bradford procedure (Bradford, 1976). Buffer-soluble protein concentrations were determined using bovine serum albumin as a standard and absorbance was read at 595 nm in a spectrophotometer (Stasar II, Gilford Instrument Laboratories, Oberlin, OH).

Buffer-soluble protein concentrations in the supernate ranged from 0.15 to 0.61 g L–1, so proteins were concentrated with trichloroacetic acid (TCA) before gel electrophoresis (Peterson, 1983). After TCA precipitation, the pellet was washed three times with 1 mL cold (–20°C) acetone. The acetone was evaporated off and 70 µL of 2x SDS buffer (0.0625 mol L–1 Tris/HCl pH 6.8, 20% (v/v) glycerol, 0.004% (v/v) bromphenol blue, 1.28 mol L–1 2-mercaptoethanol, and 0.08 mol L–1 SDS) was added to resuspend the pellet for SDS-PAGE analysis (Laemmli, 1970). Lanes were loaded with 25 µg protein, and proteins were separated in 1.5-mm-thick gels containing 12% (w/v) acrylamide each. Coomassie Brilliant Blue R-250 was used to stain proteins overnight (Merril, 1990). After scanning images of Coomassie gels, gels were rinsed in 50% methanol (v/v) and Ag stained with a modified Wray et al. (1981) method. The SDS-PAGE analysis was conducted for three separate harvests.

Immunoblotting
Methods used for immunoblotting were based on a modification from Cunningham and Volenec (1996). ‘White Icicle’ radish (Raphanus sativus L.) was used as a positive dehydrin control (Close et al., 1993). Proteins from SDS-PAGE gels (15% w/v) were transferred overnight to a nitrocellulose membrane (Protran BA83, Schleicher & Schuell, Keene, NH) using 60 V, 12 W, and 200 mA (Towbin et al., 1979). Membranes were blocked with tris-buffer saline plus polyethylene glycol sorbitan monolaurate (Tween 20 or TBST) for 30 min. Membranes were incubated with a 1:250 or 1:333 dilution of rabbit antidehydrin polyclonal antibody (PLA-100, Stressgen Bioreagents Corp., Victoria, BC, Canada) in TBST for 1.5 h. After primary antibody incubation, membranes were washed in TBST for 30 min and then immersed in TBST containing the secondary goat antirabbit IgG antibody (dilution 1:3000) conjugated to alkaline phosphatase (170-6518, Bio-Rad Laboratories, Hercules, CA) for 1.5 h. Secondary antibodies were detected using 5-bromo-4-chromo-3-indolyl phosphate and nitro blue tetrazolium substrate solution (Blake et al., 1984). Samples were kept at room temperature throughout all electrophoresis and immunoblotting procedures. Optical density of bands was determined using analysis software (Quantity One, Bio-Rad Laboratories, Hercules, CA). Immunoblotting was conducted for three separate harvests.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Mean buffer-soluble protein concentrations were 5.1 g kg–1 dry wt. for nonacclimated (NA) zoysiagrass plants and 7.3 g kg–1 dry wt. among cold-acclimated (CA) plants with concentrations ranging from 3.5 to 7.6 g kg–1 dry wt. in NA plants and 6.3 to 9.0 g kg–1 dry wt. in CA plants (Fig. 1 ). An increase in zoysiagrass buffer-soluble protein concentration during acclimation was expected since proteins are synthesized during cold acclimation in hybrid bermudagrass (Davis and Gilbert, 1970), centipedegrass (Palmertree et al., 1973), and other crops (Sakai and Yoshida, 1968). Buffer-soluble protein concentrations across acclimation status were similar among Z. japonica (6.9 g kg–1 dry wt.) and Z. matrella (6.2 g kg–1 dry wt.) genotypes. Among all the genotypes, ‘Zenith’, ‘J-36’, and ‘Companion’ were among the genotypes with the highest concentration of buffer-soluble proteins in both NA and CA plant tissues. Zenith, J-36, and Companion all have low winter injury (Patton and Reicher, 2007) and were the only cultivars in this study commercially available by seed. The LT50 was not correlated (r = 0.35, P = 0.25) with buffer-soluble protein in CA tissues, however, despite the fact that soluble protein content is known to parallel freezing tolerance across seasons (Levitt, 1980).


Figure 1
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  		Figure 1. Buffer-soluble protein concentrations as influenced by cold acclimation and zoysiagrass cultivar. Error bars represent one standard error of the mean (n = 6). Species is indicated by (m) or (j) for Zoysia matrella or Z. japonica, respectively.

 
Since there was no relationship between buffer-soluble protein concentration and zoysiagrass LT50, we explored protein composition and its relationship with freezing tolerance using SDS-PAGE and immunoblotting. The SDS-PAGE analysis revealed several prominent polypeptides using both Coomassie blue (Fig. 2A ) and Ag-staining techniques (Fig. 2B); however, levels of only five polypeptides changed in response to cold acclimation. The amounts of 41- and 75-kDa polypeptides decreased during cold acclimation (Fig. 2B), while the amount of 18- and 21-kDa polypeptides increased during cold acclimation and were more prominent in Ag-stained gels (Fig. 2B). Additionally, the amount of a 23-kDa polypeptide increased during cold acclimation and was more prominent in Coomassie gels (Fig. 2A). The 23-kDa polypeptide was readily visible in ‘Companion’, ‘J-36’, and ‘Zenith’, all of which have low winter injury (Patton and Reicher, 2007). The relatively few changes in polypeptide composition in zoysiagrass during cold acclimation were unexpected since several changes in polypeptide composition were observed in hybrid bermudagrass and centipedegrass in response to cold acclimation (Davis and Gilbert, 1970; Palmertree et al., 1973; Gatschet et al., 1994).


Figure 2
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  		Figure 2. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles and immunoblots of buffer-soluble protein from zoysiagrass genotypes with different freeze tolerances. Gels show buffer-soluble protein profiles from nonacclimated (Lanes 2–14) and cold-acclimated (Lanes 15–27) zoysiagrass plants. Gels were loaded with equal amounts of buffer-soluble protein (25 µg) per lane. Gels were first stained with (A) Coomassie brilliant blue R-250 and then (B) Ag stained. Solid lines indicate predominant proteins present in nonacclimated and cold-acclimated plants and dashed lines indicated protein changes in response to cold acclimation with their predicted molecular masses (A and B, far right). (C) Immunoblots show 23- and 25-kDa dehydrin-like polypeptides probed with a 1:250 dilution of rabbit antidehydrin polyclonal primary antibody. ‘White icicle’ radish (Raphanus sativus) seed was used as a positive control with 3 µg of protein loaded in Lane 1. Solid lines indicate dehydrin-like polypeptides and their predicted molecular masses (C, far right). Molecular weight (MW) markers represent proteins sized 104, 81, 48, 36, 27, and 19 kDa (A, B, and C, far left).

 
An antidehydrin antibody based on the lysine-rich, 15 amino acid consensus sequence EFFGIMDKIKEKLPG of dehydrins detected six polypeptides (12, 14, 16, 18, 23, and 25 kDa) in CA zoysiagrass, suggesting that these are dehydrins or dehydrin-like polypeptides (Fig. 3 ). Immunoblotting indicated these dehydrin-like polypeptides were prevalent in CA plants, but were often lacking in NA plants (Fig. 2C and 3). Although dehydrin-like polypeptides were generally lacking in NA plants, ‘Zorro,’ Companion, and J-36 contained low concentrations in NA rhizomes and stolons (Fig. 2C). Two dehydrin-like polypeptides (23 and 25 kDa) were most prominent in CA plants (Fig. 3) and thus are displayed for all genotypes across treatments (Fig. 2C).


Figure 3
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  		Figure 3. Immunoblots of buffer-soluble protein from zoysiagrass genotypes with different freeze tolerances. Immunoblots show dehydrin-like polypeptides probed with a 1:333 dilution of antidehydrin polyclonal primary antibody for nonacclimated (NA: Lanes 3, 5, 7, and 9) and cold-acclimated (CA: Lanes 4, 6, 8, and 10) zoysiagrass plants. Equal amounts of protein (25 µg) were loaded into each lane. Radish (Raphanus sativus) seed was used as a positive control with 10 µg of protein loaded in Lane 2. Dehydrin-like polypeptides and their predicted molecular masses are indicated (far right). Molecular weight (MW) markers represent proteins sized 104, 81, 48, 36, 27, and 19 kDa (Lane 1).

 
All five Z. matrella and some Z. japonica (‘Victoria’, DALZ0102, ‘El Toro’, and ‘Palisades’) genotypes contain high levels of a 25-kDa dehydrin-like polypeptide, while other Z. japonica (Companion, J-36, Zenith, and Meyer) genotypes do not. The differences in abundance of the 25-kDa dehydrin-like polypeptide may be due to genotypes classified as Z. japonica that are actually hybrids of Z. japonica x Z. matrella. Engelke and Anderson (2003) proposed that many commercially available Z. japonica cultivars are not a single species as classified by their morphological characteristics, but instead are interspecific hybrids. This is supported by the work of Anderson (2000), who found probable interspecific hybrids based on morphological and restriction fragment length polymorphism fingerprint characterization, and by the work of Yaneshita et al. (1997), who found interspecific hybridization of Zoysia spp. among natural populations. Cultivars such as Victoria, El Toro, and Palisades are probably crosses of Z. japonica with Z. matrella (Anderson, 2000; Engelke et al., 2002; Gibeault, 2003), but are classified as Z. japonica because of their predominant morphological characteristics. Therefore, the genetic makeup of Victoria, DALZ0102, El Toro, and Palisades may help explain why they contain high levels of the 25-kDa dehydrin-like polypeptide, while other Z. japonica genotypes do not. The 25-kDa dehydrin-like polypeptide is apparently specific to Z. matrella or hybrids of Z. japonica x Z. matrella. This is similar to findings among the genera Cornus, Vaccinium, and Rhododendron, where dehydrin molecular masses varied depending on species (Karlson et al., 2004; Panta et al., 2001; Marian et al., 2003).

Immunoblots indicated that the 23-kDa dehydrin-like polypeptide was present in low concentrations in some genotypes (‘Cavalier’, ‘Diamond,’ ‘Royal’, and Victoria) and in larger concentrations in other genotypes (Companion, J-36, Meyer, Zenith, and Zorro) (Fig. 2C and 3). The 23-kDa dehydrin-like polypeptide concentration mirrored the 23-kDa polypeptide visible in some genotypes with Coomassie blue stain (Fig. 2a). Higher optical density values in immunoblots for the 23-kDa dehydrin-like polypeptide were associated (r2 = 0.41, P = 0.018) with lower LT50 values (Fig. 4 ), whereas optical density values for the 25-kDa dehydrin-like polypeptide were not correlated (r = 0.47, P = 0.11) with LT50 values (Fig. 5 ). These results indicate that the 23-kDa dehydrin-like polypeptide plays a role in improving freezing tolerance, whereas the 25-kDa dehydrin-like polypeptide does not.


Figure 4
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  		Figure 4. (A) Immunoblot of a 23-kDa dehydrin-like polypeptide from zoysiagrass genotypes with different freeze tolerances, showing a 23-kDa dehydrin probed with a 1:250 dilution of antidehydrin polyclonal primary antibody for cold-acclimated plants. Equal amounts of protein (25 µg) were loaded in each lane. (B) There was a relationship (r2 = 0.41, P = 0.018) between freeze tolerance (LT50, the lethal temperature killing 50% of the plants) and optical density (O.D.) of the 23-kDa dehydrin-like polypeptide band.

 

Figure 5
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  		Figure 5. (A) Immunoblot of a 25-kDa dehydrin-like polypeptide from zoysiagrass genotypes with different freeze tolerances, showing a 25-kDa dehydrin probed with a 1:250 dilution of antidehydrin polyclonal primary antibody for cold-acclimated plants. Equal amounts of protein (25 µg) were loaded in each lane. (B) There was no correlation (r = 0.47, P = 0.11) between freeze tolerance (LT50, the lethal temperature killing 50% of the plants) and optical density (O.D.) of the 25-kDa dehydrin band.

 
Our results suggest that dehydrin-like polypeptides are associated with zoysiagrass cold acclimation, and that the 23-kDa dehydrin-like polypeptide plays a role in improving freezing tolerance. This is similar to findings in Arabidopsis, barley, blueberry, citrus, peach, red-osier dogwood, Rhododendron spp., and wheat (Danyluk et al., 1994; Cai et al., 1995; Artlip et al., 1997; Zhu et al., 2000; Panta et al., 2001; Karlson et al., 2004; Marian et al., 2003; Puhakainen et al., 2004). The contribution of dehydrins to freezing tolerance is achieved by stabilizing membranes (Close, 1996; Puhakainen et al., 2004), cryoprotection (Wisniewski et al., 1999), antifreeze activity (Wisniewski et al., 1999), and by scavenging free radicals (Hara et al., 2003). Additionally, dehydrins are known to form amphipathic {alpha} helices (Ismail et al., 1999), which give them the ability to minimize protein denaturation (Artlip et al., 1997) and preserve enzymatic activity (Rinne et al., 1999).

Jessup et al. (2006) recently identified quantitative trait loci in Z. matrella for salinity tolerance. Future marker-assisted breeding could allow improvement of zoysiagrass tolerance to abiotic stress. As suggested by Houde et al. (1992), dehydrins could be a useful tool for breeders to select freeze-tolerant phenotypes. The 23-kDa dehydrin-like polypeptide could be a potential genetic marker for cold hardiness in zoysiagrass. Additionally, identification and overexpression of transcription activators (C-repeat/dehydration-responsive element binding factor homologs) or dehydrin genes in zoysiagrass could also be used to enhance freezing tolerance, as has been done with Arabidopsis (Jaglo-Ottosen et al., 1998; Puhakainen et al., 2004).

Cold-regulated proteins, carbohydrates, lipids, and proline are known to play important roles in the cold hardiness of hybrid bermudagrass (Dunn and Nelson, 1974; de los Reyes et al., 2001; Munshaw et al., 2006), but the mechanisms involved in the cold hardiness of zoysiagrass are not as well understood. Zhang et al. (2006) recently discovered that lipid changes in zoysiagrass membranes were correlated with freeze tolerance. This research shows that a 23-kDa dehydrin-like polypeptide improves freezing tolerance of zoysiagrass. We are currently investigating the relationship of carbohydrate and proline levels with genetic variation in the freeze tolerance of zoysiagrass. Additional research is needed to determine how management practices such as mowing and fertilization impact zoysiagrass freeze tolerance, and whether the 23-kDa dehydrin-like polypeptide could be used as a diagnostic indicator of the impact of cultural practices on freeze tolerance or as a potential genetic marker for zoysiagrass cold hardiness.


   ACKNOWLEDGMENTS
 
This research was supported by the Midwest Regional Turfgrass Foundation. We thank Jon Trappe, Kyle Bongen, and Daniel Weisenberger for helping with planting and harvesting.


   NOTES
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ABSTRACT
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 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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Received for publication November 27, 2006.


   REFERENCES
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