Crop Science 43:266-271 (2003)
© 2003 Crop Science Society of America
TURFGRASS SCIENCE
Seasonal Changes in Carbohydrate Accumulation for Two Creeping Bentgrass Cultivars
Qingzhang Xu and
Bingru Huang*
Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901
* Corresponding author (huang{at}aesop.rutgers.edu)
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ABSTRACT
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Controlled-environment studies suggest that turf quality decline of creeping bentgrass [Agrostis stolonifera var. palustris (Huds.) Farw.] under heat stress is associated with decreases in carbohydrate availability in plants. The study was designed to examine and compare seasonal changes of carbohydrate status and C allocation pattern for two creeping bentgrass cultivars, ‘Penncross’ and ‘L-93’, that differ in heat tolerance under field conditions. The experiment was conducted from May to November in 1999 and 2000. Grasses were managed under putting green conditions with daily irrigation and mowing at a 4-mm height. Turf quality of both cultivars declined from May and reached the lowest level in August and September, and recovered in October. The content of total nonstructural carbohydrates (TNC), sucrose, and fructans in shoots and roots exhibited a similar seasonal pattern as turf quality for both cultivars in both years. Reducing sugar content decreased during the summer, but did not recover in October, except in shoots of L-93 in 2000. The decreases in carbohydrate content were more pronounced in roots than in shoots for both cultivars. Carbon allocation to roots also decreased during summer for Penncross. Cultivar differences in carbohydrate content were not consistent between fractions of carbohydrate and times of the year. The decline in carbohydrate availability, particularly in roots, and limited C allocation to roots during summer could contribute to quality decline under field conditions; however, cultivar variation in carbohydrate content was not related to the differences in turf quality decline between two cultivars.
Abbreviations: TNC, total nonstructural carbohydrate
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INTRODUCTION
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CREEPING BENTGRASS grows most vigorously in spring and fall when temperatures range from 15 to 24°C, but often suffers quality decline and root dieback during hot and humid summers (Carrow, 1996). The physiological mechanisms of summer bentgrass decline are unclear. Maintenance of high quality turf in cool-season grasses under heat stress may largely depend on the availability of carbohydrates because they provide the energy and C skeleton for plant growth and development (Hull, 1992). Nonstructural carbohydrates in plants are considered as the energy reserve to be used under stress conditions and have been widely used as a physiological measure of stress tolerance (Watschke et al., 1972, 1973; Beard, 1973, Howard and Watschke, 1985, 1991; Hull, 1992).
Various studies conducted in controlled-environment growth chambers have found that carbohydrate availability decreases with increasing temperatures (Al-Khatib and Paulsen, 1989; Moffatt et al., 1990; Liu and Huang, 2000; Huang and Gao, 2000; Xu and Huang, 2000a,b; Liu and Huang, 2001). The decline in carbohydrate availability for creeping bentgrass is associated with decreases in root growth (Carrow, 1996; Xu and Huang, 2000a,b; Sweeney et al., 2001), tiller production, and leaf growth (Xu and Huang, 2000a, 2000b, 2001). Carbon allocation to roots in creeping bentgrass is also inhibited by heat stress, which could cause root dieback (Xu and Huang, 2000b). Roots are more sensitive to high temperature than shoots and decline in root growth has been found to precede that of shoots under heat stress (Carrow, 1996; Xu and Huang, 2000a, b). This could be related to carbohydrate availability or allocation between shoots and roots.
Although carbohydrate metabolism of shoots in response to heat stress has been studied extensively for creeping bentgrass exposed to controlled environments, few studies examined seasonal changes of carbohydrate status of both shoots and roots in relation to turf performance in field conditions (Sweeney et al., 2001). Sweeney et al. (2001) reported a decline of TNC content in shoots of creeping bentgrass during summer months. The major TNC found in turfgrass consists of the monosaccharides glucose and fructose (reducing sugars), the disaccharide sucrose, and various oligosacchrides starch and fructans (Smith, 1972). Cool-season grasses accumulate mostly fructans (Hull, 1992). Little is known about seasonal changes in the status of different fractions of nonstructural carbohydrates in both shoots and roots and C allocation pattern to roots. Knowledge of seasonal changes in carbohydrate metabolism in shoots and roots is important to understanding physiological mechanism of summer bentgrass decline.
The objective of this study was to examine seasonal changes in the status of different fractions of nonstructural carbohydrates in both shoots and roots and C allocation pattern in relation to turf performance for two creeping bentgrass cultivars, L-93 and Penncross. L-93 was previously reported to have better heat tolerance than Penncross (Liu and Huang, 2000, 2001).
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MATERIALS AND METHODS
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The experiment was conducted on an United States Golf Associaton-specification putting green at the Turfgrass Research Center, Manhattan, KS, in 1999 and 2000. L-93 and Penncross were seeded in 205 cm x 318 cm plots in September 1996. Grass was mowed daily except Sunday at a 4-mm height from early May to early November. During this period, the green was irrigated daily to replace 100% daily water loss estimated by measuring evapotranspiration rate using mini-lysimeters. The green received four applications (May, June, September, and October) of N fertilizers for a total of 98 kg N ha-1 in 1999 and 65 kg N ha-1 in 2000. The two cultivars were arranged in a randomized complete block design with three replications. All measurements were taken monthly on two samples in each plot. Effects of cultivars and dates of measurement were determined by analysis of variance according to the general linear model procedure of Statistical Analysis System (SAS Institute, Cary, NC). Differences between cultivars and dates of measurement were determined by the LSD test at the 0.05 probability level.
Turf quality was visually rated based on color, uniformity, and density on a 1 to 9 scale, with 9 being the best and 1 being the worst. Plants with soil cores of 5 cm in diameter and 20 cm deep (from soil surface to 20 cm depth) were collected using a core collector. Roots and shoots were washed free of soil and separated to measure dry weight and carbohydrate content after oven drying (killed at 105°C for 30 min and dried at 70°C until no further weight loss).
For carbohydrate analysis, 150 mg dry tissues were influxed in a water bath with 20 mL of 80% ethanol for 30 min at 78°C. The tissues were filtered and ethanol was removed from the filtrate by heating at 75°C. One mL of 5% ZnSO4 was added to the cooled aqueous solution to precipitate the proteins. The solution was neutralized with NaOH to phenol red, diluted to 50 mL and allowed to stand overnight, and filtered through no. 40 Whatman filter paper. One milliliter of the filtered solution was transferred to a 20-mL volumetric tube and 1 mL of ferricyanide reagent was added. The solution was boiled in a water bath for 10 min and then cooled in running water. A 2-mL aliquot of 2 M sulfuric acid was added to partially neutralize the solution. After neutralization, the solution in the tube was shaken until gas evolution ceased, and 0.8 mL of arsenomolybdate solution was added. The solution was again shaken and diluted to volume (20 mL). The absorbance of the solution was measured at 490 nm using a spectrophotometer (Spectronic Genesys 2, Spectronic Instruments Inc., Rochester, NY). The reducing sugar content was calculated as mg glucose g-1 dry weight.
A 2-mL solution was transferred to a test tube with 2 mL of 4% H2SO4 (w/v) to determine the content of all soluble sugars and sucrose. The mixer was boiled for 15 min and then neutralized with 1 mL of 1N NaOH. Sucrose content was calculated by subtraction of reducing sugar content from total sugar content.
For measurement of fructans, the residue from the ethanol extraction was washed from the filter paper with 80% ethanol into a flask and evaporated at 75°C. Twenty milliliters of water were added into the flask. The flask was shaken for 30 min at room temperature. The sample was filtered and checked for starch with iodine (no water-soluble starch). 0.7 mL of 25% HCl was added in the solution for 30 min at 75°C to degrade fructans to reducing sugars. Proteins were removed and the aliquot was neutralized as described above. After diluted to 50 mL, the reducing sugars were determined to obtain fructan content. The residue from the cold-water extraction was incubated at 38°C for 44 h with 
-amylase (EC 3.2.1.1, Sigma Chemical Co., St. Louis, MO) to extract starch. The TNC content was the sum of all soluble sugars, fructans, and starch.
Carbon allocation pattern was determined using the pulse 14CO2 labeling technique (Huang et al., 1993). Plant canopy was enclosed in a clear plexiglass chamber (5 cm tall and 10 cm in diameter) and exposed to 37 x 107 Bq 14CO2 for 20 min. Three days after 14CO2 labeling, shoots and roots were washed free of soil, killed at 105°C for 30 min, and dried at 75°C for 48 h. A 20-mg sample was digested with 4 mL solubilizing liquid (solvable; Dupond, Wilminton, DE) at 50°C for 24 h and bleached with 0.1 mL of 30% H2O2 for 1 h at 25°C (Huang et al., 1993). Fifteen milliliters of scintillation cocktail for 14C counting were added to the solutions and were measured in a scintillation counter (Packard, Deers Grove, IL). The proportion of C allocation to roots was calculated using 14C in roots and shoots.
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RESULTS
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Daily maximum air temperature increased to >24°C at the end of May in both years and reached the highest level of 40°C at the end of July in 1999 and of 43°C at the end of August in 2000 (Fig. 1). In 2000, there was a prolonged period of heat in August where air temperature exceeded 40°C for 
14 d. Air temperature decreased to <24°C by the middle of October 1999 and 2000.
Turf quality for both L-93 and Penncross was the highest (8.6 for L-93 and 8.2 for Penncross) in June in both years, declined in July, and reached the lowest level in August 1999 (6.2 for L-93 and 5.0 for Penncross) and in September 2000 (6.8 for L-93 and 5.0 for Penncross) (Fig. 2). Turf stands returned to spring quality in October and November (>8 for L-93 and >7 for Penncross). L-93 exhibited better turf quality than Penncross during most of the experimental period, and this difference was more pronounced in 2000 than in 1999. The decline of quality for L-93 during summer months was less dramatic than that for Penncross compared with their respective levels in May and June.
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Fig. 2. Seasonal changes of turf quality for two creeping bentgrass cultivars in 1999 and 2000. Vertical bars on the top indicate LSDs (P = 0.05) for turf quality comparison between cultivars at a given date of year. Vertical bars on the right indicate LSDs (P = 0.05) for turf quality comparison over time for a given cultivar.
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Total nonstructural carbohydrate content in shoots (Tables 1 and 2) and roots (Tables 3 and 4) decreased from May to August for both cultivars in both years. Total nonstructural carbohydrate content in both shoots and roots increased to above the initial level by October 1999 for both cultivars and to the initial level by November 2000 for L-93. The lowest levels of TNC in both shoots and roots occurred in August in both years. Shoot TNC content in L-93 was higher than Penncross from May to November 1999 and in September and October 2000 (Table 1 and 2). Root TNC content in L-93 was higher than Penncross in August 1999 and in October 2000 (Tables 3 and 4).
Reducing sugar content in shoots decreased from May to August for Penncross in 1999 and for both cultivars in 2000 (Tables 1 and 2). Reducing sugar content in roots was significantly lower in August than in May in both years for Penncross (Tables 3 and 4). The decline in reducing sugar content for L-93 was not detected from May to August in 1999, but occurred from May to September in 2000. L-93 had higher reducing sugar content in shoots than Penncross only in November 1999 and October 2000 (Tables 1 and 2). No cultivar differences in reducing sugar content in roots were found during most months, except in May 1999 and 2000 when reducing sugar content was higher in Penncross than in L-93 (Tables 3 and 4).
Sucrose content in shoots decreased to the lowest level in August for both cultivars and increased to above the level of May by November 1999 for both cultivars (Table 1) and by October 2000 for Penncross (Table 2). L-93 had a higher sucrose content in shoots than Penncross in November 1999 and from July to September 2000. Root sucrose content decreased from May to August 1999 for Penncross, but not for L-93 (Table 3). Significant summer decline in sucrose content occurred in both cultivars in 2000 (Table 4). No cultivar differences in sucrose content in roots were found during most months of both years, except in November 1999 and October 2000 when sucrose content was higher in L-93 and Penncorss, respectively.
Fructans content in shoots (Table 1) and roots (Table 3) in both cultivars decreased to the lowest level in August 1999. In 2000, the lowest fructans content in shoots occurred in July for both cultivars (Table 2). Significant decline in fructans content in roots from May to August was detected only in Penncross (Table 4). L-93 contained higher fructans content in shoots in May and August 1999 (Table 1) and June, September, and October 2000 (Table 2). Roots had higher fructans content in L-93 than Penncross in May and November 1999 and June, August, and October 2000.
Carbon allocation to roots decreased from May to August and increased in October 2000 for Penncross (Fig. 3). No difference in C allocation to roots between cultivars was found in 1999, except in May. Carbon allocation to roots of L-93 was higher than that of Penncross in July and August 2000. Carbon allocation to roots was higher in Penncross than L-93 in October 2000.
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Fig. 3. Seasonal changes of 14C allocation to roots for two creeping bentgrass cultivars in 1999 and 2000. Vertical bars on the top indicate LSDs (P = 0.05) for 14C allocation comparison between cultivars at a given date of year. Vertical bars on the right indicate LSDs (P = 0.05) for 14C allocation comparison over time for a given cultivar.
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DISCUSSION
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Seasonal changes in turf quality and carbohydrate content in shoots and roots for both cultivars of creeping bentgrass were closely related to temperature changes. Decreases in the content of TNC, sucrose, and fructans in shoots and roots, as well as C allocation to roots were accompanied by turf quality decline as temperature increased to the supraoptimal levels in summer months. The decreases of carbohydrate content were more pronounced in roots than in shoots for both cultivars, particularly in 2000. This could be related to the reduction in C allocation to roots during the summer stress period. These results suggest that the decline both in carbohydrate availability of shoots and roots and in C allocation to roots could contribute to poor turf performance during summer months. Aldous and Kaufmann (1979) suggested that decline in carbohydrate content in roots appeared to be related to cessation of shoot growth at supraoptimal temperatures for Kentucky bluegrass (Poa pratensis L).
Others have also reported decreases in TNC content during summer for creeping bentgrass (Sweeney et al., 2001) and other cool-season turfgrasses (Youngner et al., 1978). Our study demonstrated that the decline of TNC in shoots and roots in midsummer was related to the reduction of reducing sugars, sucrose, and fructans, as they generally exhibited pattern of changes similar to TNC. Chatterton et al. (1987) reported that concentrations of starch, sucrose, and fructans decreased while glucose concentration remained constant with increasing temperature for crested wheatgrass [Agropyron cristatum (L.) Gaertn.] and redtop [Agrostis alba var. gigantea (Roth) G. Mey., nom. illeg. (= A. gigantea Roth)]. Howard and Watschke (1991) reported that fructan content was two-fold lower at 30°C than at 10°C although the sum of glucose, fructose, and sucrose did not change in Kentucky bluegrass during higher temperature stress. Fructans and sucrose may serve as buffer fractions of carbohydrates in plants exposed to heat stress (Howard and Watschke, 1991).
The decline of carbohydrate content in both shoots and roots during summer may result from an imbalance between C production in photosynthesis and consumption in respiration (Carrow, 1996; Huang and Gao, 2000; Xu and Huang, 2000b; Liu and Huang, 2001). Liu and Huang (2001) reported that photosynthetic rate decreased while respiration rate increased and photosynthesis exceeded respiration rate during summer months for both L-93 and Penncross under putting green conditions similar to the present study. Carbohydrate consumption to production ratio was several times greater under heat stress than under optimum temperature conditions (Huang and Gao, 2000; Xu and Huang, 2000b). Watschke et al. (1972) also reported that photosynthesis was reduced by 47%, dark respiration was increased by 42%, and TNC was reduced by 36% when cool-season grasses (P. pratensis, P. trivialis L., P. compressa L., and Lolium perenne L.) were transferred from 23/15 to 35/25°C (day/night). Volenec et al. (1984) reported that tall fescue (Festuca arundinacea Schreb.) genotypes with high dark respiration rate had lower yield per tiller and water soluble sugar content per stem base when compared with genotypes having low respiration rate, suggesting that reduction of respiration rate of mature tissues could increase leaf biomass production, especially during summer.
Comparing between cultivars, L-93 performed better than Penncross during most of the time from May to November. However, L-93 had a higher content of TNC, sucrose, fructans, and reducing sugars only occasionally during this period, and the cultivar differences were not consistent between carbohydrate fractions and at different times of the year. Sweeney et al. (2001) also reported inconsistent differences in TNC level among 15 creeping bentgrass cultivars at different times of the year. This suggests that cultivar variation in carbohydrate content could not explain the differences in turf quality and other factors might account for consistent cultivar differences in turf quality during the growing season. L-93 has higher tiller density, more and narrower leaves, and larger root systems (Xu and Huang, 2001), which may contribute to its better performance than Penncross.
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ACKNOWLEDGMENTS
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Funds for this study was provided by the United State Golf Association. We thank Dr. Zhaolong Wang, Michelle DoCasta, and John Pote for helpful comments on the manuscript.
Received for publication December 1, 2001.
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ABSTRACT
INTRODUCTION
