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Differences in Freeze Tolerance of Zoysiagrasses: II. Carbohydrate and Proline Accumulation

^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. 2006-18053

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

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cold hardiness among zoysiagrass (Zoysia spp.) genotypes varies, but the physiological basis for cold hardiness is not completely understood. The objective of this study was to determine the relationship of carbohydrate (starch, total soluble sugars, total reducing sugars, sucrose, glucose, and raffinose family oligosaccharides) and proline concentrations with the cold acclimation of zoysiagrass and the lethal temperature killing 50% of the plants (LT₅₀). Thirteen genotypes of zoysiagrass were selected with contrasting levels of winter hardiness. Plants were grown for 4 wk of 8/2°C day/night cycles and a 10-h photoperiod of 300 µmol m^–2 s^–1 to induce cold acclimation. Rhizomes and stolons were sampled from nonacclimated and cold-acclimated plants and used for carbohydrate and proline analysis. Concentrations of soluble sugars and proline increased during cold acclimation, while starch concentrations decreased. Starch, sugar/starch ratio, glucose, total reducing sugars, and proline in cold-acclimated plants were correlated (r = 0.61, –0.67, –0.73, –0.62, and –0.62, respectively) with LT₅₀. These correlations indicate that higher concentrations of total reducing sugars, glucose, and proline are positively associated with zoysiagrass freeze tolerance, whereas higher concentrations of starch appeared detrimental to freeze tolerance.

Abbreviations: CA, cold acclimated • LT₅₀, lethal temperature killing 50% of the plants • NA, nonacclimated • ORS, other reducing sugars • RFO, raffinose family oligosaccharides • TNC, total nonstructural carbohydrates • TRS, total reducing sugar

Differences in Freeze Tolerance of Zoysiagrasses: II. Carbohydrate and Proline Accumulation

^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. 2006-18053

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

Cold hardiness among zoysiagrass (Zoysia spp.) genotypes varies, but the physiological basis for cold hardiness is not completely understood. The objective of this study was to determine the relationship of carbohydrate (starch, total soluble sugars, total reducing sugars, sucrose, glucose, and raffinose family oligosaccharides) and proline concentrations with the cold acclimation of zoysiagrass and the lethal temperature killing 50% of the plants (LT₅₀). Thirteen genotypes of zoysiagrass were selected with contrasting levels of winter hardiness. Plants were grown for 4 wk of 8/2°C day/night cycles and a 10-h photoperiod of 300 µmol m^–2 s^–1 to induce cold acclimation. Rhizomes and stolons were sampled from nonacclimated and cold-acclimated plants and used for carbohydrate and proline analysis. Concentrations of soluble sugars and proline increased during cold acclimation, while starch concentrations decreased. Starch, sugar/starch ratio, glucose, total reducing sugars, and proline in cold-acclimated plants were correlated (r = 0.61, –0.67, –0.73, –0.62, and –0.62, respectively) with LT₅₀. These correlations indicate that higher concentrations of total reducing sugars, glucose, and proline are positively associated with zoysiagrass freeze tolerance, whereas higher concentrations of starch appeared detrimental to freeze tolerance.

Abbreviations: CA, cold acclimated • LT₅₀, lethal temperature killing 50% of the plants • NA, nonacclimated • ORS, other reducing sugars • RFO, raffinose family oligosaccharides • TNC, total nonstructural carbohydrates • TRS, total reducing sugar

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
ZOYSIAGRASS (Zoysia spp.) is grown in the transitional and warm climatic regions of the USA. Susceptibility to winter injury, however, has precluded the use of zoysiagrass cultivars that are less hardy than ‘Meyer’ zoysiagrass in the transitional climatic zone (Patton and Reicher, 2007). Concentrations of soluble proteins, sugars, and proline increase during cold acclimation (Hughes and Dunn, 1990; Harwood et al., 1994). We explored the changes in carbohydrates and proline and their association with zoysiagrass freeze tolerance to determine the physiological basis for genetic differences in freeze tolerance.

Carbohydrates play an important role in the freeze tolerance of many plant species (Sakai and Yoshida, 1968; Levitt, 1980). Protection of membranes by carbohydrates during freezing is mainly explained by the colligative action of low-molecular-weight sugars (Santarius, 1982), but also by specific sugar–membrane interactions, as is the case with sucrose (Anchordoguy et al., 1987). Sugars such as glucose, sucrose, and raffinose help maintain membrane integrity during freezing (desiccation stress) by preserving the physical characteristics of the membrane (Caffrey et al., 1988; Crowe et al., 1988). Additionally, raffinose family oligosaccharides (RFO) such as raffinose or stachyose can prevent sucrose from crystallizing at low temperatures and allow greater desiccation tolerance (Caffrey et al., 1988; Koster and Leopold, 1988). Sugars, including glucose and sucrose, can also protect protein structure during freezing (Carpenter and Crowe, 1988), and function in concert with proteins in the development of desiccation tolerance (Blackman et al., 1992).

Soluble sugars increase in stolons and rhizomes of warm-season turfgrasses including zoysiagrass during autumn (Dunn and Nelson, 1974; Rogers et al., 1975; White and Schmidt, 1990; Fry et al., 1991, 1993; Bush et al., 2000; Ball et al., 2002; Shahba et al., 2003; Cai et al., 2004). An increase in soluble sugar concentrations during autumn typically coincides with a decrease in tissue starch concentration (Levitt, 1980); however, reports on carbohydrate concentration in stolons and rhizomes of cold-acclimated warm-season grasses vary. Starch concentration in rhizomes and stolons of bermudagrass [Cynodon dactylon (L.) Pers.], centipedegrass [Eremochloa ophiuroides (Munro) Hack.], and St. Augustinegrass [Stenotaphrum secondatum (Walt.) Kuntze] decreases during autumn (Dunn and Nelson, 1974; White and Schmidt, 1990; Fry et al., 1991; Cai et al., 2004), whereas the starch concentration of carpetgrass (Axonopus affinis Chase) and zoysiagrass has been reported to increase during autumn (Rogers et al., 1975; Bush et al., 2000). Dunn and Nelson (1974) reported that changes in rhizome carbohydrate concentrations of three different bermudagrass cultivars were slight and not related to winter survival. Bush et al. (2000) found no relationship between seasonal carbohydrate concentrations and low-temperature tolerance for carpetgrass, and Maier et al. (1994) found no relationship between sucrose or starch concentrations and seasonal freezing resistance for three St. Augustinegrass cultivars.

Despite these reports, there is a large body of evidence showing that certain carbohydrates are correlated with freeze tolerance in cereals (Livingston, 1991), herbaceous plants (Palonen, 1999), woody plants (Cox and Stushnoff, 2001), forages (Haagenson et al., 2003), and warm-season grasses. Increased freezing tolerance in centipedegrass was correlated with seasonal increases in stolon sucrose and fructose concentrations (Fry et al., 1993; Cai et al., 2004) and total soluble carbohydrates (Cai et al., 2004). Greater buffalograss [Buchloe dactyloides (Nutt.) Engelm.] freeze tolerance was correlated with higher soluble carbohydrate concentrations such as fructose, glucose, sucrose, and raffinose in stolons (Ball et al., 2002). The freeze tolerance of saltgrass [Distichlis spicata (L.) Greene], which also has excellent freeze tolerance, is correlated with increased fructose, glucose, raffinose, and stachyose concentrations in rhizomes (Shahba et al., 2003).

In addition to sugars, plants accumulate proline during dehydrative stresses including low temperature, drought, and high salinity (Hare et al., 1999). Freeze and frost tolerance is correlated with increased proline concentrations in many crops including bermudagrass (Munshaw et al., 2006), centipedegrass (Cai et al., 2004), alfalfa (Medicago sativa L.; Paquin, 1977), potato (Solanum spp.; van Swaaij et al., 1985), and winter wheat (Triticum aestivum L.) (Dörffling et al., 1997). Some researchers have doubted, however, that proline protects plants from freezing and have proposed that proline concentrations increase as a consequence of stress rather than as a cause of stress tolerance (Wanner and Junttila, 1999). Proline accumulates slowly in plants after the onset of stress and typically after the acquisition of frost tolerance (Koster and Lynch, 1992; Wanner and Junttila, 1999). This is probably because new proteins must be synthesized to signal proline accumulation (Hare et al., 1999). Hare et al. (1999) proposed that proline synthesis, not proline levels, may be important in plant adaptation to various stresses. Mutants of Arabidopsis and winter wheat demonstrate that freezing tolerance is increased when genes are modified to prevent the degradation of proline or to overproduce proline (Dörffling et al., 1997; Xin and Browse, 1998; Nanjo et al., 1999).

The physiological basis for differences in zoysiagrass cold tolerance has only partially been explored (Rogers et al., 1975, 1976, 1977; Akiyama et al., 1994; Fuller et al., 1999). Previous work on the carbohydrate status of zoysiagrass is inconclusive (Rogers et al., 1975; Akiyama et al., 1994; Fuller et al., 1999). Starch and sucrose comprise the largest carbohydrate fractions in zoysiagrass rhizomes, with reducing sugars (glucose + fructose) comprising a small fraction of total sugars (Rogers et al., 1975; Fuller et al., 1999). Starch concentration in rhizomes and stolons of Meyer zoysiagrass has been reported to increase during cold acclimation, while total sugars increased only slightly and reducing sugars remained unchanged (Rogers et al., 1975). Fuller et al. (1999) concluded that starch, sucrose, and reducing sugars are not reliable indicators of zoysiagrass low-temperature tolerance between cultivars, whereas Akiyama et al. (1994) found that sucrose in zoysiagrass stems was related to cold tolerance. Akiyama et al. (1994) also found that proline content in leaves of three Zoysia species was related to cold tolerance based on two climatic regions in Japan where plants were selected. Additional understanding of the role of carbohydrates and proline in freeze tolerance of zoysiagrass will help identify physiological traits that could be exploited by breeders to increase the cold hardiness of zoysiagrass. The objectives of this study were to determine the relationship of nonstructural carbohydrate (starch, total soluble sugars, total reducing sugars, sucrose, glucose, and RFO) and proline concentrations in rhizomes and stolons with the freeze tolerance and acclimation status of zoysiagrass.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Freeze Tolerance
Detailed procedures to determine the freeze tolerance of zoysiagrass genotypes and results are described elsewhere (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 and after cold acclimation from additional sets of plants not used for testing freeze tolerance. Rhizomes and stolons were combined for carbohydrate and proline analysis since they have comparable anatomy, freeze tolerance, and carbohydrate concentrations in autumn and winter (Rogers et al., 1975, 1976). All rhizomes and stolons harvested were immediately frozen in liquid N₂ after washing off soil with cold water and removing roots and shoots. Samples were stored at –80°C overnight and lyophilized (Lyph Lock, Labconco, Kansas City, MO) the following day. Lyophilized tissues were ground with a mortar and pestle in liquid N₂ and stored at –80°C until quantification of carbohydrates and proline.

Total Sugar, Reducing Sugar, and Starch Analysis
Methods used to extract and quantify total sugars and starch were based on a modification from Li et al. (1996). Total sugars were extracted from 30 mg of lyophilized, ground stolon and rhizome tissue with 1 mL of 80% (v/v) ethanol in 1.5-mL microfuge tubes. Tubes were vortexed to suspend tissues, agitated for 10 min on a horizontal shaker, centrifuged at 16,000 x g for 10 min, and the supernate retained. This process was done a series of three times and the combined three supernates were diluted to 10 mL with 80% ethanol. Sugar concentrations in 200-µL aliquots from ethanol extracts were determined with anthrone (Koehler, 1952), using glucose as a standard and reading the absorbance at 625 nm with a spectrophotometer (Junior model, PerkinElmer, Wellesley, MA). The remaining ethanol extracts were frozen at –80°C for reducing sugar and individual sugar analyses.

Reducing sugars were determined using the Nelson–Somogyi method (Nelson, 1944; Somogyi, 1945), which measures total reducing sugars. Some samples were concentrated before analysis since this method does not permit accurate determination of samples containing <10 mg L^–1 of reducing sugars (Somogyi, 1945). Reducing sugars in 200-µL aliquots from ethanol extracts were added to tubes and the final volume was brought to 1.0 mL using double-deionized water. Next, 1.0 mL of the Somogyi (1945) alkaline Cu reagent was added, tubes were vortexed for 10 s, and then incubated in an oven for 1 h at 100°C. The reaction was stopped after incubation by placing tubes in an ice bath. Tubes were removed from the bath and 1 mL of Nelson's (1944) arsenomolybdate reagent was added to each tube. Tubes were vortexed and absorbance was read at 520 nm in a spectrophotometer (Spectronic GENESYS 10, Thermo Electron Corp., Waltham, MA). Reducing sugar concentrations were determined using glucose as a standard. The concentration of other reducing sugars (ORS) was estimated as the difference between total reducing sugars and glucose concentrations.

Pellets remaining after the supernates were removed for sugar analysis were dried in an oven (50°C) overnight and used for starch analysis. Pellets were resuspended in 500 µL of double-deionized water and heated for 10 min in a heat block (105°C) to gelatinize the starch. Tubes were then cooled in an ice bucket and 400 µL of 0.2 mol L^–1 NaOAc buffer (pH 5.1) was added. Starch was digested by adding 0.2 units of amyloglucosidase (Product A1602, from Aspergillus niger, Sigma-Aldrich Co., St. Louis, MO) and 40 units of -amylase (Product A2643, from porcine pancreas, Sigma-Aldrich Co.) in 100 µL of 0.2 mol L^–1 NaOAc buffer (pH 5.1) to each tube. Tubes were capped to prevent evaporation and incubated at 50°C for 16 h. Tubes were centrifuged at 16,000 x g for 10 min at 4°C, and a 25-µL aliquot removed. The final volume was adjusted to 1 mL with double-deionized water and then 1.0 mL of Trinder reagent (Product 220-32, glucose oxidase–peroxidase, Glucose Trinder Assay, Diagnostic Chemicals Ltd., Oxford, CT) was added. Glucose concentrations were determined using glucose as a standard and absorbance was read at 505 nm in a spectrophotometer (Spectronic GENESYS 10). Starch concentration was then estimated as 0.9 x glucose concentration.

Individual Sugar Analysis
Methods used to quantify glucose, sucrose, and RFO were based on modifications of a diagnostic kit (Product K-RAFGL, Megazyme Int. Ireland Ltd., Bray, Ireland). Aliquots (200 µL) of 80% (v/v) ethanol sugar extracts were added to three small test tubes (13 by 100 mm) labeled A, B, and C. Tube A was directly assayed for glucose. Sucrose was hydrolyzed to glucose plus fructose by digesting Tube B with 138 units of invertase (Product E-INVRT, from Saccharomyces cerevisiae, ß-fructofuranosidase, EC 3.2.1.26, Megazyme Int. Ireland Ltd.) in 200 µL of 50 mmol L^–1 NaOAc buffer (pH 4.8). Raffinose family oligosaccharides were determined by digesting Tube C with 8 units of -galactosidase (Product E-AGLAN, from Aspergillus niger, EC 3.2.1.22, Megazyme Int. Ireland Ltd.) and 138 units of invertase in 200 µL of 50 mmol L^–1 NaOAc buffer (pH 4.8). The RFOs in Tube C were hydrolyzed into galactose, glucose, and fructose. All tubes were brought to a final volume of 1 mL using 50 mmol L^–1 NaOAc buffer, lightly vortexed to mix the solutions, and digested at 50°C for 1 h (Somiari and Balogh, 1995). One milliliter of Trinder reagent was added to each tube, and tubes were vortexed for 5 s and then incubated at room temperature for 20 min to allow color development. Glucose concentrations were determined using glucose as a standard and absorbance was read at 505 nm in a spectrophotometer (Spectronic GENESYS 10).

The absorbance value for Tube A was used to calculate glucose concentration. The difference in absorbance between Tubes A and B was used to calculate sucrose concentration (B – A), since sucrose in the samples was hydrolyzed to glucose + fructose by invertase. The difference in absorbance between Tubes B and C was used to calculate RFO concentration (C – B), since sucrose and RFO in the samples were converted to equivalent moles of glucose by invertase and -galactosidase. This method does not distinguish between raffinose and stachyose, but concentrations of RFO can be expressed on a molar basis using the average molecular weight of raffinose and stachyose (585.5 g mol^–1), since each mole of RFO contains 1 mol of glucose.

Proline Analysis
A modification of the Bates et al. (1973) procedure was used to extract and quantify proline. Proline was extracted from 50 mg of lyophilized, ground stolon and rhizome tissue with 1 mL of 3% (w/v) 5-sulfosalicylic acid in 1.5-mL microfuge tubes. Tubes were vortexed for 15 s to suspend tissues a total of three times, shaken for 15 min, and centrifuged at 16,000 x g for 10 min; 100-µL aliquots were removed for proline quantification, and test tubes were adjusted to 1 mL using double-deionized water. Next, 1.0 mL acid ninhydrin (1.25 g ninhydrin in 30 mL glacial acetic acid, 13.8 mL 85% H₃PO₄, and 6.2 mL double-deionized water) and 1.0 mL glacial acetic acid were added. Tubes were vortexed for 15 s and then incubated in an oven for 1 h at 100°C. The reaction was stopped after incubation by placing the tubes in an ice bath. The tubes were removed from the bath and 4 mL of toluene was added to each tube. The tubes were then vortexed for 20 s, and 5 min was allowed for phase separation. Three milliliters of the upper chromophore containing toluene was pipetted into 13-mm test tubes and absorbance was read at 520 nm in a spectrophotometer (Spectronic GENESYS 10), using toluene as a blank. Proline concentrations were determined using L-proline (Sigma P-1428) as a standard and calculated on a dry-weight basis.

Statistical Analysis
Differences among carbohydrate and proline concentrations in genotypes, species, and acclimation status were analyzed using PROC ANOVA and PROC TTEST (SAS Institute, Cary, NC). The change in tissue concentration (carbohydrates and proline) caused by cold acclimation was calculated for each sample (concentration = {100[(1 + cold-acclimated [CA] concentration) – (1 + nonacclimated [NA] concentration)]/(1 + NA concentration)}) with the integer 1 added to each value to prevent division by concentrations <1. Means were separated using Fisher's protected least significant difference when F tests were significant at 0.05. Correlation coefficients between LT₅₀ and tissue concentration in cold-acclimated tissue, nonacclimated tissue, as well as the change in tissue concentration caused by cold acclimation were determined using PROC CORR. A negative correlation coefficient indicates that freeze tolerance improves (LT₅₀ decreases) as concentrations of carbohydrates and proline increase.

	RESULTS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Carbohydrates
Total soluble sugars averaged 32 g kg^–1 dry wt. for NA zoysiagrass plants and 81 g kg^–1 dry wt. for CA plants (Table 1 , Fig. 1A ). Soluble sugar concentrations for CA plants were higher (P = 0.01) for Z. japonica (84 g kg^–1 dry wt.) genotypes than Z. matrella (75 g kg^–1 dry wt.) genotypes. Total soluble sugars of CA plants were not correlated with LT₅₀ (r = –0.23, P = 0.45). There was a correlation, however, between LT₅₀ and the change in total soluble sugars (sugars) (r = 0.55, P = 0.05) (Fig. 2A ) during cold acclimation as well as between total soluble sugars in NA plants and LT₅₀ (r = –0.61, P = 0.03).

View larger version (44K): [in this window] [in a new window]   Figure 1. Concentrations of (A) total soluble sugars, (B) starch, (C) sugar/starch ratio, (D) total reducing sugars, (E) glucose, (F) other reducing sugars, (G) sucrose, and (H) total nonstructural carbohydrates in zoysiagrass stems (rhizomes and stolons) before (nonacclimated) and after cold acclimation (cold acclimated by 4 wk at 8/2°C day/night cycles with a 10-h day photoperiod of 300 µmol m–2 s–1) for 13 genotypes. Error bars represent one standard error of the mean (n = 6). Species are indicated by (m) or (j) for Zoysia matrella or Z. japonica, respectively.

View larger version (15K): [in this window] [in a new window]   Figure 2. Relationships between zoysiagrass freeze tolerance (LT50, the lethal temperature killing 50% of the plants) and (A) the change in total soluble sugars during acclimation (sugars) (r2 = 0.31, P = 0.05), (B) starch concentration in cold-acclimated plants (r2 = 0.37, P = 0.03), (C) the sugar/starch ratio in cold-acclimated plants (r2 = 0.45, P = 0.01), and (D) the change in the sugar/starch ratio (sugar/starch) during acclimation (r2 = 0.44, P = 0.01). Each point represents the mean of six replications. *Significant at the 0.05 probability level.

The ratio of total sugar to starch (sugar/starch = total soluble sugars/starch) was calculated to determine its impact on freezing tolerance. The sugar/starch ratios averaged 5.2 among CA zoysiagrass genotypes and ranged from 1.9 to 9.2 in CA plants (Fig. 1C). The sugar/starch ratio was higher (P < 0.0001) for CA Z. japonica (6.2) genotypes than for CA Z. matrella (3.6) genotypes. Sugar/starch ratios in CA plants were highest in Meyer, Companion, J-36, Zenith, and DALZ0102 zoysiagrass and correlated with LT₅₀ for CA plants (r = –0.67, P = 0.01; Fig. 2C). Additionally, the change in the sugar/starch ratio (sugar/starch) during acclimation was also correlated with LT₅₀ (r = –0.66, P = 0.01; Fig. 2D).

Total reducing sugar (TRS) concentration was 3.4 g kg^–1 dry wt. in NA zoysiagrass plants and 19 g kg^–1 dry wt. in CA plants (Fig. 1D). The TRS concentrations were higher (P = 0.004) for CA Z. japonica (21 g kg^–1 dry wt.) genotypes than for CA Z. matrella (14 g kg^–1 dry wt.) genotypes. The TRS concentration of CA plants was correlated with LT₅₀ (r = –0.62, P = 0.02) (Fig. 3A ) and TRS concentration of NA plants was also correlated with LT₅₀ (r = –0.57, P = 0.04).

View larger version (14K): [in this window] [in a new window]   Figure 3. Relationships between zoysiagrass freeze tolerance (LT50, the lethal temperature killing 50% of the plants) and (A) total reducing sugars in cold-acclimated plants (r2 = 0.41, P = 0.02), (B) glucose in cold-acclimated plants (r2 = 0.55, P = 0.004), (C) other reducing sugars in cold-acclimated plants (r2 = 0.29, P = 0.06), and (D) sucrose in cold-acclimated plants (r2 = 0.08, P = 0.34). Each point represents the mean of six replications. *, ** Significant at the 0.05 and 0.01 probability levels, respectively; NS, not significant.

Other reducing sugar concentrations averaged 2.3 g kg^–1 dry wt. for NA zoysiagrass plants and 10.6 g kg^–1 dry wt. for CA plants (Fig. 1F). Other reducing sugars, as with glucose, comprised little of the total sugar concentration in either NA or CA plants (Table 1). The relative proportion of ORS to total sugars in rhizomes and stolons increased from 7 to 13% during acclimation, similar to glucose. Other reducing sugar concentrations in CA zoysiagrass plants averaged 10.6 g kg^–1 dry wt., which was similar to the average glucose concentration (8.0 g kg^–1 dry wt.) in CA plants. Additionally, there was a close correlation (r = 0.95, P = 0.0001) between ORS and glucose concentrations in CA zoysiagrass. Other reducing sugar concentrations were higher (P = 0.02) for CA Z. japonica (12.0 g kg^–1 dry wt.) genotypes than CA Z. matrella (8.4 g kg^–1 dry wt.) genotypes. Trends in ORS concentration were similar to glucose and TRS, but ORS concentrations did not correlate with LT₅₀ of CA plants (r = –0.54, P = 0.06; Fig. 3C).

Sucrose increased on average 113% during cold acclimation (Fig. 1G). Although sucrose comprised the majority of the total sugar concentration in both NA and CA zoysiagrass plants, the proportion of sucrose to total soluble sugars decreased during cold acclimation (Table 1). Sucrose concentrations in CA plants were similar for Z. japonica (51 g kg^–1 dry wt.) genotypes and Z. matrella (50 g kg^–1 dry wt.) genotypes. Sucrose concentrations in CA plants were not correlated with LT₅₀ (r = 0.29, P = 0.34; Fig. 3D), but the change in sucrose concentrations (sucrose) during cold acclimation correlated with LT₅₀ (r = 0.71, P = 0.006; Fig. 4A ). Additionally, the ratio of sucrose/TRS correlated with LT₅₀ (r = 0.61, P = 0.03; Fig. 4B).

View larger version (9K): [in this window] [in a new window]   Figure 4. Relationships between zoysiagrass freeze tolerance (LT50, the lethal temperature killing 50% of the plants) and (A) the change in sucrose during acclimation (r2 = 0.51, P = 0.006), and (B) sucrose/total reducing sugars in cold-acclimated plants (r2 = 0.43, P = 0.02). Each point represents the mean of six replications. *, ** Significant at the 0.05 and 0.01 probability levels, respectively.

Proline
Proline concentration increased dramatically during cold acclimation. Concentrations ranged from 5.4 to 14.1 mmol kg^–1 dry wt. in NA zoysiagrass plants and from 74 to 178 mmol kg^–1 dry wt. in CA plants (Fig. 5 ). Proline concentrations for CA plants were higher (P = 0.0003) among Z. japonica (132 mmol kg^–1 dry wt.) genotypes than Z. matrella (90 mmol kg^–1 dry wt.) genotypes. Companion, J-36, and Zenith had the highest concentrations of proline in CA plant tissues, whereas ‘Victoria’, ‘Cavalier’, ‘Zorro’, and ‘Diamond’ contained the lowest concentrations. Zoysiagrass LT₅₀ was correlated with stolon and rhizome proline concentrations in CA tissues both before (r = –0.62, P = 0.02) and after logarithmic transformation (r = –0.67, P = 0.01; Fig. 6 ).

View larger version (29K): [in this window] [in a new window]   Figure 5. Proline concentration in zoysiagrass rhizomes and stolons before (nonacclimated) and after cold acclimation (cold acclimated by 4 wk at 8/2°C day/night cycles with a 10-h day photoperiod of 300 µmol m–2 s–1) for 13 genotypes. Error bars represent one standard error of the mean (n = 6). Species are indicated by (m) or (j) for Zoysia matrella or Z. japonica, respectively.

View larger version (11K): [in this window] [in a new window]   Figure 6. Freeze tolerance (LT50, the lethal temperature killing 50% of the plants) is increased as proline concentrations in cold-acclimated rhizomes and stolons increase. Correlation coefficient (r2 = 0.44, P = 0.013) between LT50 and logarithmically transformed proline concentration. Each point represents the mean of six replications.

	DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Total reducing sugars comprised a small fraction of the total carbohydrates in zoysiagrass rhizomes and stolons, which is consistent with earlier reports (Rogers et al., 1975; Fuller et al., 1999). Genotypes with good freezing tolerance contained higher concentrations of reducing sugars in cold-acclimated rhizomes and stolons in our study. Total reducing sugars were previously reported to be unrelated to freezing tolerance in bermudagrass, carpetgrass, and zoysiagrass (Dunn and Nelson, 1974; Rogers et al., 1975; Fuller et al., 1999; Bush et al., 2000). This is contrary to our findings, possibly because of differences among zoysiagrass, bermudagrass, and carpetgrass. Furthermore Rogers et al. (1975) tested only one zoysiagrass cultivar for one season and Fuller et al. (1999) considered TRS to be a minor factor in freezing tolerance because of their low concentrations.

Glucose and fructose are known to comprise virtually all the reducing sugars in grass tissues and their concentrations typically occur in about a 1:1 ratio (Smith, 1981). Glucose concentrations were slightly lower than ORS, but levels were similar and highly correlated, suggesting that most ORS were probably fructose. These sugars are important for freezing tolerance in buffalograss and saltgrass (Ball et al., 2002; Shahba et al., 2003). Glucose is able to maintain membrane integrity and protect protein structure during freezing (Carpenter and Crowe, 1988; Crowe et al., 1988), which could be the potential mechanism by which it was able to improve the freezing tolerance of zoysiagrass in our study.

We attempted to detect total RFO in our samples using an enzymatic method for quantifying RFO in seeds. Raffinose family oligosaccharides are known to be important in freeze tolerance of other warm-season grasses (Ball et al., 2002; Shahba et al., 2003), although their concentrations are low (total RFO <2 mmol kg^–1 dry wt.). Due to high concentrations of sucrose and low concentrations of RFO in our samples, RFO concentrations were below our detection limit. We were only able to ascertain that the concentration of RFO in our samples was probably < 2 mmol kg^–1 dry wt.

Starch concentration decreased in rhizomes and stolons of all 13 of our genotypes of zoysiagrass during cold acclimation, which is consistent with findings in bermudagrass, centipedegrass, and St. Augustinegrass (Dunn and Nelson, 1974; White and Schmidt, 1990; Fry et al., 1991; Cai et al., 2004), but contrary to previous findings in Meyer zoysiagrass (Rogers et al., 1975). Our study found a correlation between LT₅₀ and starch concentration in cold-acclimated rhizomes and stolons, suggesting that genotypes that contain less starch after cold acclimation are more freeze tolerant; however, this also is contrary to a previous report on Meyer zoysiagrass that hypothesized that high concentrations of starch would be an advantage in winter survival (Rogers et al., 1975). Rogers et al. (1975) found that starch in rhizomes and stolons of Meyer zoysiagrass increased during cold acclimation to a maximum in December and decreased in March following winter. In our study, starch concentration in Meyer rhizomes and stolons decreased from 38 to 10 g kg^–1 dry wt. during cold acclimation, which was similar to other genotypes. Additionally, Rogers et al. (1975) reported that stolons and rhizomes of Meyer had starch concentrations sixfold higher than total sugars in December, which is opposite of our data showing nearly ninefold more soluble sugar than starch in CA Meyer plants.

Although starch was probably hydrolyzed during cold acclimation, the decrease in starch (26 g kg^–1 dry wt. [NA – CA]) does not fully explain the increase in total soluble sugars (49 g kg^–1 dry wt. [CA – NA]) during cold acclimation. Amylotic enzymes are known to remain high in zoysiagrass leaves during cold hardening (Rogers et al., 1977), which allows the continued breakdown of starch during cold acclimation. Although photosynthesis declines in zoysiagrass during cold acclimation (Rogers et al., 1977), photosynthesis is still higher than respiration at lower temperatures (Fry and Huang, 2004). Sugar accumulation in leaves and stolons during cold acclimation may be a direct result of higher photosynthetic rates than respiration rates (Levitt, 1980). Translocation of sugars from leaves to rhizomes and stolons during cold acclimation could account for the increase in sugars not explained by starch hydrolysis.

Consistent with a previous report in centipedegrass (Cai et al., 2004), the change in the sugar/starch ratio during acclimation was correlated with LT₅₀ (Fig. 2D). This suggests that genotypes are more freeze tolerant when soluble sugars increase or starch decreases during cold acclimation. Since the relationship between sugars, sucrose, and LT₅₀ indicated that genotypes with poor freeze tolerance accumulate more sugars during acclimation, the relationship between sugar/starch and LT₅₀ probably indicates that the decrease in starch during cold acclimation is more important to improving zoysiagrass freezing tolerance than total soluble sugar concentration.

An increase in TNC during cold acclimation in our study is similar to findings with bermudagrass and carpetgrass, which increase TNC during autumn (Bush et al., 2000; Dunn and Nelson, 1974; White and Schmidt, 1990). Total nonstructural carbohydrates are made up of total soluble sugars and starch. Starch is proposed by some researchers (Rogers et al., 1975; Cai et al., 2004) to be a determining factor in freezing tolerance since it can serve as an energy reserve during winter. Because of the large size of individual starch molecules, however, starch is unlikely to significantly contribute to freeze point depression inside plant cells. White and Schmidt (1990) proposed that the accumulation of TNC in stolons and rhizomes of warm-season grasses may not be as important for cold hardiness as the hydrolysis of starch (insoluble polysaccharides) to water-soluble sugars. This hypothesis is in agreement with our data.

Total nonstructural carbohydrates were initially believed to be a good predictor of cold tolerance in bermudagrass and other warm-season grasses since TNC concentrations increased in rhizomes and stolons in autumn (Dunn and Nelson, 1974), and because carbohydrates were known to be important factors in cold hardiness (Levitt, 1980). Total nonstructural carbohydrates were found by many, however, to be a poor predictor of cold hardiness in warm-season grasses (Dunn and Nelson, 1974; White and Schmidt, 1990; Fry et al., 1991; Maier et al., 1994; Bush et al., 2000). Recent evidence, including ours, has shown stronger relationships between LT₅₀ and fructose, glucose, raffinose, and stachyose in warm-season grasses despite their presence in much lower concentrations than sucrose (Ball et al., 2002; Shahba et al., 2003; Cai et al., 2004). Although there are obvious differences in sugar and starch concentrations between species, future work should focus on fructose, glucose, raffinose, and stachyose as predictors of cold hardiness in warm-season grasses in addition to sucrose, total soluble sugars, and TNC.

Overall, zoysiagrass freeze tolerance is enhanced when the total soluble sugar/starch ratio, glucose concentrations, and total reducing sugar concentrations are high in CA plants. Interestingly, the change in total soluble sugars and sucrose during cold acclimation suggests that concentrations of total soluble sugars and sucrose increased more during cold acclimation in genotypes that were less freeze tolerant. Starch decreased in all genotypes during cold acclimation in our study and appeared to be detrimental to freeze tolerance, which is contrary to Rogers et al. (1975). Additionally, the relationship between the sugar/starch ratio and LT₅₀ indicates that hydrolyzing starch during cold acclimation is more important to zoysiagrass freeze tolerance than total soluble sugar concentration.

Proline
An increase in proline concentrations in zoysiagrass rhizomes and stolons during cold acclimation was expected since proline is synthesized in bermudagrass, centipedegrass, alfalfa, potato, and winter wheat during cold acclimation (Paquin, 1977; van Swaaij et al., 1985; Dörffling et al., 1997; Cai et al., 2004; Munshaw et al., 2006). Consistent with reports on zoysiagrass (Akiyama et al., 1994), other turfgrasses (Cai et al., 2004; Munshaw et al., 2006), and other crops (Paquin, 1977; van Swaaij et al., 1985; Dörffling et al., 1997), zoysiagrass LT₅₀ was correlated with stolon and rhizome proline concentrations in CA tissues (Fig. 6). Both linear and logarithmic regressions indicate that freeze tolerance improves with increasing proline concentration, but the relationship between freezing tolerance and proline concentration is better explained when proline concentration is transformed logarithmically. Correlation coefficients between frost tolerance and proline concentration in potato were also higher after logarithmic transformation of the proline concentration. These results also indicated that proline is less beneficial at lower temperatures and that as proline increases to high concentrations, the benefit to freeze tolerance is reduced (van Swaaij et al., 1985). Santarius (1992) evaluated the cryoprotectiveness of proline on chloroplast membranes of spinach (Spinacia oleracea L.) and found that proline helps protect membranes through hydrophobic interactions, but that proline is less efficient as temperatures decrease. This may explain our results where proline concentrations in zoysiagrass rhizomes and stolons were less beneficial at lower temperatures. In addition to stabilizing membranes (Santarius, 1992), proline aids with osmotic adjustment and decreases water potential, allowing plants to tolerate dehydrative stresses (Delauney and Verma, 1993), and proline is known to quench singlet O₂ and other reactive O₂ species (Matysik et al., 2002), which are known to increase during freezing (Kendall and McKersie, 1989).

Before examining the physiological basis for differences in cold hardiness, we first determined the freeze tolerance of several zoysiagrass genotypes. There was a positive relationship (r² = 0.48, P = 0.009) between freeze tolerance (LT₅₀) determined with growth or freeze chambers and winter injury in the field, indicating that growth-chamber-based procedures produced results that can predict winter injury in the field (Patton and Reicher, 2007).

Although we measured the concentrations of carbohydrates and proline from plants cold acclimated in a growth chamber, it is probable that plants sampled in the field contain similar concentrations. Researchers should expect similar results from either sampling zoysiagrass plant tissues from the field during winter or using the growth chamber cold-acclimation procedure used in this study; however, future analysis of the seasonal changes in carbohydrate concentrations of multiple zoysiagrass cultivars will be needed to test this hypothesis.

The mechanisms involved in the cold hardiness of zoysiagrass are beginning to be understood. Zhang et al. (2006) recently discovered that lipid changes in zoysiagrass membranes were correlated to freeze tolerance. A specific 23-kDa dehydrin-like polypeptide has also recently been discovered, whose abundance is positively associated with improved zoysiagrass freezing tolerance (Patton et al., 2007). Before the initiation of this study, previous knowledge about the role of carbohydrates in zoysiagrass cold hardiness promoted a hypothesis that high concentrations of starch were advantageous to zoysiagrass cold hardiness and that reducing sugars were probably not important. This study establishes that high concentrations of glucose, total reducing sugars, and proline, as well as total soluble sugar/starch ratios play a role in improving freezing tolerance, whereas high concentrations of starch are detrimental to freeze tolerance. Understanding the roles of carbohydrates and proline in zoysiagrass freeze tolerance will help breeders select freeze-tolerant phenotypes and improve the winter performance of future releases. Furthermore, understanding management factors that may influence carbohydrate and proline concentrations may help golf course superintendents minimize winter injury of their current zoysiagrass swards.

	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

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Received for publication December 11, 2006.

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