PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 KzlProleccaxPrelelRC/Dateoue.indd ABSTRACT THE EFFECT OF HEAT STRESS 0N ASPECTS OF CARBOHYDRATE AND NITROGEN METABOLISM IN AGROSTIS, CYNODON AND POA SPECIES by John E. Kaufmann Supraoptimal temperatures inhibit growth of cool season turfgrasses. This investigation was designed to evaluate changes in levels of metabolites and rates of metabolism within certain intermediary pathways under high temperature growth stoppage con- ditions. Toronto creeping bentgrass (Agrostis palustris Huds.), and Merion Kentucky bluegrass (Poa pretensis L.), were used because both are widely used cultivars of cool season species that exhibit growth stoppage at supra- optimal temperatures. These species were compared to Tifgreen bermudagrass (Cynodon dactylon L.), a warm season species having a higher temperature optimum for growth. Controlled environment chambers were used to simulate optimal and supraOptimal conditions for growth in the range of 20 to 35 C and 32 to 35 C, respectively. All other environmental parameters were held constant. John E. Kaufmann Maximum dry matter production was obtained at 25 C for both cool season species and at 35 C for the warm season species. Increasing temperature decreased the nitrogen content in Kentucky bluegrass and bermudagrass leaves. No change in the level of nitrogen was found in creeping bentgrass due to temperature changes. Uptake of glucose-14C, glutamine»14 14C’ C, leucine- 14c, and acetyl-CoA- was increased in creeping bentgrass, reduced in Kentucky bluegrass, and not affected in bermudagrass by increasing the preconditioning growth temperatures from 25 to 35 C. The higher temperature caused increased incorporation of.all radioactive pre- cursors into protein in all three species. Exposure of the tissue to light for 14 hours increased the carbohydrate content of both Kentucky blue- grass and creeping bentgrass, and increased the respira— tion of glucose414 C in Kentucky bluegrass. At a 35 C preconditioning temperature the extremes in diurnal variation of the carbohydrate content were moderated, and 14C was decreased in both the respiration of glucose- Kentucky bluegrass and creeping bentgrass. Increasing incubation temperatures from 20 to 35 C, increased the respiration of glucose-14C, and decreased the percent incorporated into protein-14C. As the incubation temperatures were increased above 26 C for Kentucky bluegrass previously grown at 26 C, John E. Kaufmann the content of glutamine and asparagine increased; aspartate decreased; and glutamate and gamma aminobuty— rate were unchanged. When grown at 32 C, a significant decrease in the glutamine content occurred when the tissue was incubated at increasingly higher temperatures. As incubation temperatures were increased above 26 C for Kentucky bluegrass grown at 26 C, the percent glutamine-14C found in the tissue increased; the percent 14C decreased; and the percent aspartate-14C, 14 glutamate- asparagine- C, and 14C02 evolved was increased. As the incubation temperature was increased from 26 C to 38 C the percent glutamine-14C was halved for Kentucky blue- grass grown at 32 C while the percent evolved as 14 C02 was doubled. When Kentucky bluegrass was placed under acute heat stress, glutamine accumulation occurred with little 'conversion to glutamate. However, when Kentucky bluegrass was under chronic heat stress, rapid conversion of gluta- mine to C02 occurred, resulting in depletion of the glutamine pool. It was suggested that loss of the gluta— mine pool resulted in reduced synthesis of certain growth sustaining proteins or other macromolecules that require glutamine as a precursor. THE EFFECT OF HEAT STRESS ON ASPECTS OF CARBOHYDRATE AND NITROGEN METABOLISM IN AGROSTIS, CYNODON AND POA SPECIES BY A. a“ A )3 E John E: Kaufmann A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Sciences 1973 ‘ V5.2) qgr’1 (91 DEDICATED TO John William ii ACKNOWLEDGEMENTS The author expresses his sincere appreciation to Dr. J. B. Beard for his guidance and encouragement through- out this investigation and for his constructive criticism in reviewing the manuscript. Appreciation is also expressed for his endeavors in all aspects of turfgrass science which helped to make this investigation possible. Grateful appreciation is extended to Drs. D. Penner, P. E. Rieke, C. J. Pollard, and R. E. Monroe for their helpful criticism and for serving on the guidance committee. Special thanks is given to my wife, Jean, who not only had the title of "wife", but also of "mother" and "secretary" during the difficult time when the manuscript was being written, organized, and typed. iii TABLE OF CONTENTS Page DEDICATION. . . . . . . . . . . . . ii ACKNOWLEDGEMENTS. . . . . . . . . . . iii TABLE OF CONTENTS . . . . . . . . . . iv LIST OF TABLES . . . . . . . . . . . - vi LIST OF FIGURES . . . . . . . . . . . viii INTRODUCTION . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . 4 Effects of Temperature on Growth . . . 4 Effects of Temperature on Carbohydrate Levels ' . . . . . . . . . 7 Effects of Nitrogen Nutrition . . . . 10 Effects of Diurnal Variation . . . . 13 Photosynthesis and Respiration . . . . 14 Nitrogen Metabolism. . . . . . . . 17 MATERIALS AND METHODS . . . . . . . . . 23 Establishment Procedures . . . . . . 23 Growth Conditions . . . . . . . . 24 Sample Preparation . . . . . . . . 25 Radioisotope Materials. . . . . . . 26 Incubation Procedures . . . . . . . 26 Total E-uptake Determination . . . . 28 Protein- 4C Analysis Procedure .‘ . . . ' 28 Thin Layer Chromatography Techniques . . 29 Dry Weight Production . . . . . . . 31 Nitrogen Analysis . . . . . . . . 31 Protein Analysis . . . . . . . . 31 Carbohydrate Analysis . . . . . . . 32 Statistical Analysis . . . . . . . 32 , iv Page RESULTS. . . . O I O O O O O O O O 33 Intermediary Metabolism Experiment. . . 33 Carbohydrate Metabolism Experiment. . . 38 Glutamine Metabolism Experiment . . . 42 DISCUSSION. 0 O O O O O O O O 0 O O 49 CONCLUSIONS 0 O O O O I O O O O 0 O 60 BIBLIOGRAPHY O O O O O O O O O O O O 6 3 APPENDIX . O O O O O O O O O O O O 69 Table LIST OF TABLES The effect of temperature on the growth and nitrogen content of three turfgrass species. . . . . . . . . . . The effegt of temperature during growth on the 1 -uptake, C02 evolved and protein- C synthesized from glucose by leaf sections of three turfgrass species. . . . . . . . . . . The effegt oftempeizture during growth on the 1 -uptake, C02 evolved and p otein- C synthesized from glutamine- C by leaf sections of three turfgrass species. . . . . . . . . . . The effegt of temperature during growth on the uptake, C02 evolved and protein-1 C synthesized from leucine-14C by leaf sections of three turfgrass species. . . . . . . . . . . The effegt of temperature during growth on the uptake, C02 evolved and pfiotein-1 C synthesized from acetyl-CoA- C by leaf sections of three turfgrass Species. . . . . . . . . . . The effect of temperature and light dura- tion on the nitrogen, protein and carbo- hydrate content of two cool season turfgrass species. . . . . . . . The effect of temperature during growth and light duration on the C-uptake, 14C02 evolved and protein-14C synthesized 1 C by leaf sections of two from glucose- cool season turfgrass species . . . vi 14C Page 33 35 35 36 38 38 40 Table 10. Page The effect of temperatuie during gigwth and incubation on the4 C uptake, C02 evolved and pigtein— C synthesized from glucose- C by leaf sections of two cool season turfgrass species . . . . 40 The effect of temperature during growth and incubation on the content of five amino acids isolated from leaf sections of Merion Kentucky bluegrass . . . . 44 The effect of temperafare during growth and incubation on the, C uptake and 14C-metabolites synthesized from gluta- mine-14C by leaf sections of Merion Kentucky bluegrass . .- . . . . . 46 vii LIST OF FIGURES Figure Page 1. Positions of amino acids isolated on a 0.5 mm layer of cellulose by thin layer chromatography . . . . . . . . . 43 2. The pathway of glutamine metabolism . . . 56 3. The effect of temperature during growsh and incubation in the percentlglutamine- , glutamate--1 , and other C metabolites in leaf sections of Merion Kentucky bluegrass . 57 viii INTRODUCTION The effect of supraoptimal temperatures in inhibiting the growth of cool season grasses has been known since the 1930's. Early investigations suggested that carbohydrate levels were lowered with increasing temperature. Thus high temperature growth stoppage was attributed to a lack of carbohydrates for growth. A revolution in turfgrass science in the 1950's resulted from: (a) development of improved cultivars of both warm and cool season species, (b) availability of fertilizers, pesticides, and fungicides formulated for turf use, and (c) development of turfgrass mainten- ance equipment. New cultivarsof cool season species provided high quality turfs in the north while warm season cultivars were well adapted in the south. How- ever, the transition zone lacked a high quality turfgrass due to summer dormancy in cool season turfgrasses. In an effort to overcome dormanCy, fertility, irrigation, disease control and maintenance practices were adjusted. However, supraoptimal temperatures, an aspect of the environment which could not be controlled, were causing growth stOppage of the cool season turfgrasses. Since that time, many investigations concerning the effect of temperature on levels of various carbo- hydrate and nitrogen fractions have been conducted. There is a widely accepted hypothesis that when turfgrasses are grown at Optimal temperatures, net photosynthesis exceeds net respiration, resulting in carbohydrate accumulation. However, when turfgrasses are grown at supraoptimal temperatures net respiration overcomes net photosynthesis resulting in loss of available carbohydrates. If tempera- tures remain above the optimum for growth, carbohydrates are exhausted and growth stops. A second hypothesis supported by more recent investigations proposes that the carbohydrate levels are inversely related to growth. Thus, as supraoptimal temperatures are approached, growth is reduced and carbo- hydrate levels increase. ‘ Exhaustion of the glutamine pool has been shown to occur at supraoptimal temperatures (4), but the exact relationship between loss of glutamine and growth stoppage has not been determined. The objectives of this investigation were to: (a) compare the effect of temperature on certain aspects of intermediary metabolism in three turfgrass species, (b) reevaluate the effect of temperature on carbohydrate levels and metabolism, and (c) determine the effect of temperature on metabolism of the glutamine pool and relate it to growth stoppage. ._..__,__. _.._,.._.. . _.._ #_- LITERATURE REVIEW Effects of Temperature on Growth The effects of temperature on growth have been investigated extensively. Brown (7) concluded that soil temperature was more important than air temperature in reporting temperature effects on growth. Maximum shoot pro- ' duction of Kentucky bluegrass was observed at an average soil temperature of 15.6 to 17.8 C. Very little shoot growth occurred at soil temperatures less than 10 C. Stuckey (37) reported that a soil temperature of 26.5 C accelerated maturation of colonial bentgrass (Agrostis tenuis Sibth.) roots. It was proposed that plant death at this temperature was due to early maturation and death of the root system. Mitchell (23) found that 20 C was the optimum temperature for dry weight production of perennial ryegrass (Lolium perenne L.), orchardgrass (Dactylis alomerata L.), colonial bentgrass, and velvetgrass (Holcus lanatus L.). In contrast, the optimum for Dalligrass (Paspalum dilatatum Pois.) was near 30 C. A rapid decline in growth occurred above the optimum temperatures with growth of the cool- season grasses ceasing above 35 C. Sullivan and Sprague (38) reported that root dry- matter production of perennial ryegrass was greatest at the 4 5 coolest temperature (15.6/10 C, day/night), and lowest at the warmest temperature regime (32.2/26.7 C). Dry- matter production of shoots was highest at day/night temper- atures of 21.1/15.6 C and lowest at 32.2-26.7 C. Harrison (12) reported that shoot growth of Kentucky bluegrass was higher at 26.7 C than at 15.6 C for the first 10-day cutting. The dry weight of the rhizomes at the end of the experiment decreased with increasing tempera- ture while root weights were maximum at 15.6 C. Schmidt and Blaser (30) noted maximum shoot growth of Cohansey creeping bentgrass after 45 days at 36 C. Reduced growth was found at 24 C and again at 12 C. Highest root production was at 12 C, and was decreased with increas- ing temperature. After switching temperatures for an additional 10 day period, root and shoot growth of bentgrass increased as temperatures were decreased from 36 C. How- ever, when bentgrass maintained at 12 C was grown at higher temperatures, shoot growth increased while root growth decreased. Watschke, Schmidt, and Blaser (42) observed growth of five Kentucky bluegrass cultivars at day/night tempera- ture regimes of 18/10, 27/18, and 35/20 C. Shoot growth was greatest at the two lower temperatures and was reduced at the highest temperature. Maximum root production occurred at 27/18 C and minimum at 35/20 C. Those Kentucky bluegrass-cultivars selected from a warm climate were better adapted to heat stress. In a recent investigation by Watschke et a1. (41), ten Kentucky bluegrass cultivars were grown at a 23/15 C day/night temperature, then changed to 35/25 C. The shoot growth of all cultivars was higher at 35/25 the first week, but less than growth at 23/15 C on subsequent weekly harvests. A. Younger and Nudge (47) reported that of three Kentucky bluegrass cultivars grown at day/night temperature regimes of 27/21, 27/16, 18/12, and 16/7 C, shoot growth was greatest at the warmer day temperatures and decreased with decreasing temperature. In Fylking and NeWport, shoot growth was highest at 27/16 C, while maximum growth of Merion was found at 27/21 C. Merion produced significantly more dry matter than the other two cultivars. McKell et al.(2l) compared foliar growth of Coastal bermudagrass and NeWport Kentucky bluegrass at four day/ night temperature regimes ranging from 13/7 to 30/24 C. Shoot growth of Newport Kentucky bluegrass grown at 18/13 C increased with each sampling date. Shoot growth at the other three temperatures was similar on the first sampling date but did not increase on subsequent sampling dates. Bermudagrass shoot growth increased as temperatures increased. The shoot growth rate of Toronto creeping bentgrass was measured by Duff (10) at five day/night temperature regimes from 20/10 to 40/30 C. Shoot growth was maximum at 20/10 C and decreased with each increment of increased temperature. Martin (20) found that 25 C was the optimum for shoot growth of Merion Kentucky bluegrass. As temper- ature increased growth was reduced. Severe growth reductions were noted at a constant day/night temperature of 35 C. The shoot growth optimum for cool season grasses such as creeping bentgrass and Kentucky bluegrass is in the range of 20 to 25 C. Warm season species such as bermuda- grass and dallisgrass exhibit growth Optimums of 30 to 35 C. Root growth optimums appear to be approximately 5 C less than shoot growth optimums. Effects of Temperature on Carbohydrate Levels The effect of many environmental factors on growth and carbohydrate content of leaf tissue has been investi- A gated extensively. Factors which promote dry weight production generally cause a reduction in carbohydrate levels, or do not permit carbohydrate levels to be restored to high levels after a period of carbohydrate utilization. Seasonal fluctuations in temperature affect the carbohydrate level in grasses. Brown (7) reported produc- tion and storage of carbohydrates in Kentucky bluegrass during the cool temperatures of spring. Loss of stored carbohydrates from roots and rhizomes occurred during the summer. However, carbohydrate storage again Occurred in the fall. It was not determined whether the summer re- ductions were a result of increased growth at optimal temperature or growth stoppage at supraoptimal tempera— tures. Another investigation of seasonal effects by Zanoni et a1. (48) showed that Merion Kentucky bluegrass had increasing carbohydrate levels from late spring to mid- summer, a sharp drop in late summer, and an increase during the fall. Harrison (12) attributed reduced growth of Kentucky bluegrass at higher temperatures to carbohydrate exhaustion. The data of Sullivan and Sprague (38) indicated that maximum carbohydrate accumulation occurred in perennial ryegrass at a day/night temperature of 21.1/15.6 C, with a rapid decrease in carbohydrates at.temperatures above the optimum for growth. The highest percentage of carbohydrates in ryegrass was found in the subble, with reduced amounts in the leaves and roots. The reserve carbohydrate exhaustion theory was ad- vanced when Schmidt and Blaser (30) reported that acid- extractable carbohydrates in Cohansey creeping bentgrass stolons decreased from 43.4% at 12 C to 31.8% at 36 C. The carbohydrate level in leaves also decreased as temperature increased. Youngner and Nudge (47) reported that high growth temperatures resulted in lower carbohydrate reserves in Kentucky bluegrass. However, supraoptimal temperatures for growth were not included in this investigation since increasing temperature was found to increase growth. In a subsequent investigation in the same laboratory, McKell et a1. (21) measured reduced fructosan levels at higher growth temperatures in Newport Kentucky bluegrass where growth was reduced. Increased growth of Coastal bermuda— grass reflected a reduction of starch reserves at the same high temperature. Brown and Blaser (8) found similar carbohydrate levels in orchardgrass grown at 35 C and at 24 C. They attributed the lack of reduced carbohydrate levels to mois- ture stress which overcame the effects of higher tempera- ture. Watschke, Schmidt, and Blazer (42) reported that carbohydrate levels in five Kentucky bluegrass cultivars decreased as the growth temperature was increased to a maximum day/night regime of 30/20 C. It was also con- cluded that cultivars with a high carbohydrate content best supported growth at the high temperatures. Watschke, et a1. (41) found that foliar carbo- hydrate level in ten Kentucky bluegrass cultivars decreased vas the day/night growth temperature was increased from 23/15 to 35/25 C. Even though carbohydrate level was as high as 14.1% after four weeks at the 35/25 C temperature, the authors still contended that threshold carbohydrate levels were causing reduced dry matter production. 10 Supraoptimal temperatures for growth of cool season grasses have been shown to increase the carbohydrate level in both creeping bentgrass and Kentucky bluegrass. Duff (10) reported that both the 85% ethanol-soluble carbohydrate level and the water-soluble carbohydrate level increased in creeping bentgrass as.day/night temperatures were increased from 20/10 C to 40/30 C. The investigation by Martin (20) indicates that carbohydrate levels were inversely related to growth of Merion Kentucky bluegrass. Minimum carbohydrates levels were found at 25 C where growth was maximum. Maximum carbo- hydrate levels were found at 35 C where dry matter A production was severely impeded. Effects of Nitrogen Nutrition Nitrogen is an essential element for the growth of grasses. When applied to turfgrasses grown under optimum conditions it increases dry matter production. However, the reduction in growth of Italian ryegrass (Lolium multiflorum . Lam.) caused by above optimum temperatures was even greater at high nitrogen levels (36). Pellett and Roberts (27) found that Kentucky blue- grass turfs grown at low nitrogen levels were more tolerant of high temperatures than when grown at high nitrogen levels. Harrison (12) found that removing leaf tissue from Kentucky bluegrass plants supplied with a nitrogen-free ll nutrient solution, was less harmful during the hot summer months than was excessive defoliation of bluegrass plants which received a continuous supply of nitrogen. He also reported that after several defoliations, plants grown at 26.4 C and supplied with nitrogen produced no more top growth than nitrogen-free cultures. Fertilization with nitrate has been shown superior to ammonium nitrogen in supporting greater shoot growth and rhizome development in Kentucky bluegrass (12), and has been shown to be effective over a wider range of soil pH's and temperatures (9). Stoin (36) indicated that at a 21/16 C day/night temperature high rates of nitrate were more effective in promoting growth of Lolium multiflorum ,Lam. than high rates of ammonium nitrogen. At a tempera- ture regime of 32/26 C, nitrate was again more effective than ammonium nitrogen at both high and low levels of nutrition. 3 Sprague (31) also studied the effects of ammonium and nitrate nutrition on colonial bentgrass. Ammonium nitrogen reduced growth of shoots'and roots when compared to nitrate. Temperature effects were not considered in this investigation. I Soluble carbohydrate levels in turfgrasses are inversely related to soil nitrogen levels. Orchardgrass and perennial ryegrass had higher carbohydrate levels at low soil nitrogen levels and both decreased with increased 12 nitrogen (1, 32). More recently, Lechtenberg gt;§l. (18) reported that nitrogen fertilization reduced the fructosan and pentosan concentration in tall fescue (Festuca arundinacea Schreb.). Studies on Kentucky bluegrass indicated increased shoot growth and decreased foliar carbohydrate content with the addition of nitrogen fertilizer (27). High nitrogen reduced the levels of acid-extractable carbohydrates found in Cohansey creeping bentgrass according to Schmidt and Blaser (30). The effect of nitrogen nutrition on reserve carbo- hydrates in the leaves of creeping bentgrass and Kentucky bluegrass was investigated by Green and Beard (11). Oligosaccharides and fructosans decreased with added nitro- gen while simple sugars were not significantly affected. Total carbohydrate content was generally higher at reduced nitrogen levels. Weissman (44) reported that in the shoots of seedling wheat (Triticum aestivum), total nitrogen was higher following an ammonium treatment when compared to a nitrate treatment. This increase in total nitrogen was found to be in the nonprotein fraction. Further study indicated that amides were accumulated in the tissue when in ammonium culture. However, 12% more asparagine than glutamine was isolated after ammonium treatment and 30% more glutamine than asparagine was isolated after the nitrate treatment. 13 In a recent investigation, Watschke gt_gl. (42) reported that high nitrogen levels stimulated shoot growth of five Kentucky bluegrass cultivars at 18/10 C day/night temperatures but reduced growth at 35/20 C. Nitrate uptake was not affected by nitrogen levels of the nutrient solution. Foliar levels of carbohydrate, nitrate and ammonia also did not vary with nitrogen levels. Effects of Diurnal Variation Carbohydrate levels exhibit large fluctuations due to diurnal variation. These fluctuations are a result of net photosynthesis or carbohydrate accumulation during the light period, and respiration or carbohydrate utilization during the dark period. Diurnal variation of other metabo- lite levels, such as the nitrogenous compounds, has been' suggested by Youngberg (46) to be a result of a dilution effect from highly variable carbohydrate levels. Dirunal variation of the water-soluble carbohydrate content has been observed in forage grasses. Waite and Boyd (40) report that sucrose levels increased in ryegrass from about 5% at 9 a.m. to 7% at 3 p.m. Seventy percent of the daily increase was lost between midnight and 3 a.m. Holt and Hilst (15) state that water-soluble carbohydrates in tall fescue increased from 6% at 6 a.m. to 9% at 6 p.m., while water-soluable carbohydrates in Kentucky bluegrass increased from approximately 5% to 8% during the same period of time. 14 Lechtenberg gt_al. (18) reported that the average sugar content of tall fescue increased from about 8% at 6 a.m.-to about 10% at 6 p.m. Almost 37% of the daily in- crease in sucrose was respired or translocated between 6 p.m. and midnight, and the remaining 63% after midnight. The effect of diurnal variation on the content of both water-soluble and 85% ethanol-soluble carbohydrate of Merion Kentucky bluegrass leaves was investigated by Martin (20). The water-soluble carbohydrate content was approximately 4% at 7 a.m., 8% at noon, and 13% when the light period ended at 9 p.m. The levels dropped to about 10% at midnight and were near 4% when the dark period ended at 7 a.m. Strict controls on the time of sampling are necessary in measuring other environmental effects on carbohydrate levels. Photosynthesis and Respiration Photosynthesis rates are affected by several en- vironmental factors including temperature, light intensity, light quality, and carbon dioxide. Respiration rates are affected by temperature, light and oxygen concentration. When other factors are held constant, photosynthesis and respiration increase with temperature to a maximum rate then decrease. It has been widely accepted that the maximum rate of photosynthesis occurs at a lower tempera- ture than fOr respiration. Therefore, at some point 15 along the temperature curve respiration rates overcome photosynthetic rates and a net loss of carbon skeletons occur. Temperature optimum of photosynthesis between temperate and tropical origin forage grasses was reported by Murata and Iyama (25). Apparent photosynthesis in Italian and perennial ryegrass was greatest at 5 to 15 C, and at 35 C for bermudagrass and bahiagrass (Paspalum notatum Flugge). Respiration increased with increasing temperature through 40 C for all species. Moss gt_al. (24) showed respiration increased in corn (Egg_may§ L.) more rapidly than photosynthesis as. temperature was increased to 43.9 C. However, the magnitude of photosynthesis was so much greater that net assimilation was greater at the higher temperature. 3 According to Miller (22) maximum net photo- synthesis of Seaside creeping bentgrass occurred at 25 C and was from 35 to 40 C for common bermudagrass. At a temperature of 40 C, photosynthesis was 62.2% of maximum in the cool season species, and 97.7% of maximum in the warm season species. Schmidt and Blaser (30) reported 80% more C02 fixed in Cohansey creeping bent- grass grown at 24 C when compared to 12 C or 36 C. 16 Duff (10) found that photosynthesis of leaf sections of Toronto creeping bentgrass grown at both 20/10 and 40/30 was greater at a test temperature of 30 C than 20 C or 40 C. When grown at 40/30 C., the reduction of photosynthesis caused by increasing test temperature from 30 C to 40 C was much less compared to 20/10 C. Maximum respiration was found at 40 C for both temperatures. However, the rates of photo- synthesis at 40 C far exceeded respiration rates. Data from ten bluegrass cultivars investi- gated by Watschke gg_gl. (41) indicated that day/night temperatures of 35/25 C did not cause the sum of the rates of dark resPiration and photorespiration to exceed photosynthetic rates measured in'a low oxygen atmosphere. Martin (20) found that increasing test tempera— tures at 5 C increments from 20 C to 40 C increased both respiration and photosynthesis of Merion Kentucky bluegrass regardless of the temperature of the pre- conditioning period. When preconditioned for one week at 35 C, however, both photosynthesis and respiration were reduced compared to plants grown for one week at 20 C. Respiration did not exceed photosynthesis at any temperature. 17 Nitrogen Metabolism Nitrogen exists in leaf tissue as proteins, amino acids, amides, free ammonia, free nitrate, and other nitrogenous compounds. While temperature changes do not greatly alter total nitrogen, significant changes in the levels of one or more of the nitrogen containing compounds could be responsible for growth stoppage of cool season turfgrasses. Steward (33) has suggested that environmental factors may interact with metabolic processes at the point of contact between carbohydrate and nitrogen metabolism involving keto acids, amino acids, and amides. His studies with the mint plant (Mentha pipereta L.) indicated daylight, long days, and night temperatures promoted protein synthesis and glutamine accumulation in the leaves. In contrast, darkness, short days, and high night temperatures favored asparagine accumulation. Beard and Daniel (4) studied the seasonal varia- tion in the total, nonprotein, glutamine, asparagine and total amide nitrogen fractions of creeping bentgrass leaf tissue. Temperature was the major environmental factor affecting seasonal variations in these fractions. Total nitrogen increased with average daily soil tempera- tures through 24 C and then decreased at higher temper- atures. Glutamine decreased sharply with increasing 18 temperatures, dropping to very low values at soil temperatures above 24 C. Asparagine was also reduced, but to a lesser extent. In another investigation by Beard (3), total nitrogen increased in both creeping bentgrass and bermudagrass with increasing temperature. Nonprotein nitrogen increased with increasing temperature in bent- grass but decreased with increasing temperature in bermudagrass. The free ammonia level increased from glow to optimum temperatures and decreased at higher temperatures. The amide level in bermudagrass decreased with increasing temperature, but not of the magnitude of decrease found for bentgrass. Stoin (36) indicated that the protein nitrogen content of tall fescue and perennial ryegrass was rela- tively unaffected by growth temperature, but the soluble amino nitrogen content was increased with temperature.- In an earlier investigation, Stoin (3S) concluded that the soluble nitrogen content was higher when Kentucky bluegrass was grown at 35 C compared to 21 C. ASpara- tate and glutamate levels decreased, while asparagine increased. Glycine, valine, alanine, serine, threonine, isoleucine, and lysine increased with increasing temper- ature. Glutamine was reported to be highly variable with no definite trend. l9 Watschke 3E_§l. (42) reported that nitrate uptake by five Kentucky bluegrass cultivars was stimu- lated by increasing temperatures. However, even though growth was reduced at this temperature, the increased nitrate uptake did not result in increased levels of foliar nitrate or ammonium nitrogen. Kaufmann EE_E£' (16) reported that nitrate reductase activity of creeping bentgrass leaf tissue grown at 35 C was reduced when compared to 25 C. Nitrate reductase isolated from bermudagrass exhibited activity through 40 C. Data from Schmidt and Blaser (30) indicated that total nitrogen in the stolons of creeping bent- grass increased with increasing temperature. In the leaf tissue the minimum total nitrOgen was found at 24/18 C compared to 12/10 C and 36/30 C day/night temperatures. Sullivan and Sprague (30) found that the 80% ethanol-soluble nitrogen fraction increased relative to total nitrogen in perennial ryegrass leaf tissue fol- lowing clipping. The largest increase of this fraction occurred at the highest temperature treatment. Total nitrogen declined with time after clipping but the 20 decline was much less at the highest temperature treat- ment. The authors suggested the possibility of ammonium toxicity due to a rapid digestion of proteins at high temperatures. In a review of the biochemical aspects of tempera- ture effects Langridge (17) listed five possible causes for high temperature effects. Several of the possible causes involved reduced Synthesis or accelerated breakdown of enzymes and/or amino acids and therefore, would involve protein or nitrogen metabolism. Petinov and Molokovskii (28, 29) reported an aCcumulation of ammonia in several plant Species subjected to temperatures above 45 C. High temperature treatment caused intensified proteolysis, ammonia accumulation, impaired amino acid synthesis, and abnormal amino acid metabolism. The abnormal amino acid metabolism included accumulation of large amounts of gamma aminobutyrate. They were able to partially overcome heat injury by treating the plans with organic acids. These, they suggested, neutralized the ammonia and provided energy sources for the neutralizing reaction. Ultimately the excess ammonia was stored in amide pools and was avail- able for resynthesis of proteins. Steward and Margolis (34) noted that a manganese deficiency produced an accumulation of amides in tomato 21 plants. This deficiency was found not to interfere with nitrate reduction or the immediate conversion of nitrogen to an organic form, but rather to control the supply of carbon accepters for nitrogen via Krebs cycle. Thus existing amino acids were converted to amides. The effects of light on the metabolism of glucose and glutamine in wheat leaves were investigated by Bidwell et al. (6). In the light, 68% of radioactive glucose was converted to sucrose, while in the dark only 33.2% was isolated as sucrose. Greater quantities of aSpartate, glutamate, asparagine, glutamine,.alanine, and carbon dioxide were formed in the dark. When radioactive glutamine was fed in the light, 30.5% of the radioactivity remained as glutamine and 28.9% was isolated as glutamate. In the dark 48.4% remained as glutamine and 16.0% was isolated as glutamate. Greater quantaties of asparagine, aspartate and carbon dioxide were isolated after the dark treatment, but greater quantities of sugars and other amino acids were iSOIated after the light treatment. Gamma aminobutyrate was isolated from gluta- mine fed wheat leaves after both light and dark treatments. Glutamate metabolism of wheat leaves was investiga- ted by Naylor and Tolbert (26). Under anerobic conditions the major product was gamma aminobutyrate (GAB). However, When oxygen was supplied, glutamine and several organic acids were the major products. The large accumulation of 22 GAB at low oxygen conditions was a result of either increased decarboxylation of glutamate or reduced trans— amination of GAB to succinate semiadehyde. Synge (39) isolated GAB from perennial ryegrass, and found it to be present in quantities as high as 6% of nonprotein nitrogen. This indicates that GAB is a normal metabolite in a turfgrass species and not merely a product of abnormal amino acid metabolism as suggested by the above authors (29, 26). ' Glutamate carboxylase of wheat leaves was investi- gated by Weinberger and Clendenning (43). Greater quantities of the enzyme were isolated in the older leaves than in the young leaVes. Beevers (5) reported that the enzyme may be heat inactivated at temperatures above 30 C. In order to investigate the effect of heat stress on carbohydrate and nitrogen metabolism, it was necessary to review a wide range of literature. Final identification of the mechanism of growth steppage of cool season turf- grasses at supraoptimal temperatures may well involve a complex interaction of these metabolic processes. MATERIALS AND METHODS Establishment Procedures Mature sods of Toronto creeping bentgrass and Merion Kentucky bluegrass were obtained from the Michigan State University experimental turfgrass field laboratory. The bentgrass and bluegrass had been maintained at a cutting height of 0.6 cm and 3.8 cm, respectively. The Tifgreen bermudagrass, maintained at a 1.27 cm cutting height, was shipped from Florida (A. E. Dudek, Plantation Field Labora- tory, Ft. Lauderdale) as mature sod pieces approximately 25 x 25 cm. The sod was trimmed to a 2.0 cm soil depth and placed in greenhouse flats (36 x 25 x 10 cm) previously lined with plastic and filled to a 6 cm depth with a sandy loam soil mix. Drainage holes were punched in the plastic. All flats were placed in a 20 to 25 C greenhouse under an automatic irrigation system for two weeks prior to imposing the temperature treatments. New sod of creeping bentgrass and Kentucky bluegrass was used for each experiment. Cut- ting heights for bluegrass, bentgrass, and bermudagrass were maintained at 5.0 cm, 2.0 cm, and 2.5 cm, respectively, during the entire investigation. The cutting height of bermudagrass and creeping bentgrass was raised to provide greater leaf blade length for sectioning in the experi- ments involving radiocarbon uptake. 23 24 Growth Conditions The large environmental growth chambers (1.4 x 2.5 m) used for the experiments were maintained at a light intensity Of 24,000 lux. and at a day length Of 14 hours with day/night temperatures held constant. Following the greenhouse establishment period, the flats were trans- ferred to the chambers and watered daily and twice daily when under Optimal and supraoptimal temperatures, res- pectively. The samples were saturated twice weekly with a Hoaglands (l4) micro-nutrient solution having macro nutrients modified to a 4:1:2 ratio OfN, P, and K, res- pectively. An occasional dusting with malathion was 'necessary for insect control. One chamber was used throughout the experiment on aspects of intermediary metabolism and was initially ad- justed to a soil temperature Of 20 C. Six flats Of the three species were transferred to the chamber and the temper- ature was monitored with a thermometer placed at a 5 cm. soil depth. Following a two week acclimation period, the temperature was increased 5 C every two weeks through 35 C. Four replications were harvested twice weekly for analysis Of growth and nitrogen content. Three replications of fresh leaf tissue were sampled for the radiocarbon study during 1 the final three days of the 25 C and 35 C growth periods. Sampling occurred after two hours of light duration. In the carbohydrate metabolism experiment, six flats Of both Merion Kentucky bluegrass and Toronto 25 creeping bentgrass were placed in each Of two growth chambers adjusted to a constant day/night temperature of 20 and 35 C. After three weeks, four replications were harvested fOr the nitrogen, protein, and carbohydrate analyses. Three replications of fresh leaf tissue were sampled for the radiocarbon study during the final three days of the experiment. The effect Of light duration was achieved by sampling just as the light period began (0 hours) and again just prior to the dark period (14 hours of light). When the temperatures during incubation were adjusted to 20 and 35 C, samples were taken after two hours light duration. One growth chamber was used in the glutamine metabolism experiment. Six flats of Merion Kentucky blue- grass were grown at 26 C for three weeks. The chamber was then adjusted to 32 C for another three week period. Three replications of fresh leaf tissue were sampled following two hours of light duration during the final three days of both growth periods. Sample Preparation For growth, nitrogen, protein, and carbohydrate measurements, the tissue samples were frozen immediately in dry ice and carried to the laboratory where they were freeze-dried, ground in a Wiley mill, and stored in air- tight bottles. 26 In the radiocarbon studies, fresh tissue was collected, placed in an airtight plastic bag containing a moistened paper towel, and chilled to 0-3 C. A special cutting apparatus having three parallel razor blades at a 0.5 cm spacing was used to cut sections of fresh leaf blade tissue which were quickly transferred to a flask con- taining an uptake buffer (to be described in Incubation Procedures). Sixty sections Of bluegrass and bermuda- grass, and 80 sections of bentgrass were cut for each flask. This represented approximately 60 mg fresh tissue per flask. Radioisotope Materials Glucose, glutamine and leucine were Obtained as 50 uCi each of uniformly labeled carbon-14. Ten uCi of acetyl-CoA were labeled on the carbonyl carbon Of the acetyl group. Each incubation flask received an aliquot containing 0.5 uCi (0.404 ug) glucose dissolved in 10 ul 85% ethanol, 0.5 uCi (0.334 ug) glutamine dissolved in 10 ul water, 0.5 uCi (0.268 ug) leucine dissolved in 10 ul Of 0.005 N HCl, or 0.2 uCi (3.26 ug) acetyl-GOA dissolved in 10.0 ul water. Incubation Procedures Warburg flasks with sidearm were used for the radio- active uptake studies. Two ml Of chilled 0.05 M phosphate buffer (pH 6.4) were placed in the bottom of each flask. The 27 leaf blade sections were placed immediately in the buffer solution. Ten ul of the radioactive chemical were placed in the uptake buffer and mixed thoroughly. Each flask was immediately covered with a rubber septum fitted with a glass tube inserted just below the buffer surface. The flasks were then placed in a temperature controlled water- bath maintained at 30 C throughout all experiments except when the effect of temperature during incubation was being studied. An aluminum foil cover was placed over the water- bath to insure a dark respiration measurement. The sidearm Of the flask was connected to a vacuum apparatus containing 14 a C02 trap. The C02 evolved from the tissue was trapped and counted. Flow rates of air through the flask were maintained at 30 ml per minute. In the intermediary metabolism and carbohydrate metabolism experiment, incubation was allowed to continue for a period of two hours, after which the leaf sections were rinsed in a Buchner funnel and blotted dry. Half of the sections were transferred to a preweighed sample wrapper and placed in a 80 C oven. The other half were placed in a 10 ml teflon grinder, frozen with dry ice and placed in a freezer. Glucose was the only radioactive chemical used in the carbohydrate metabolism experiment. In the glutamine metabolism experiment, glutamine was the only radioactive chemical used. All leaf sections_ were frozen immediately after a one hour incubation period. 28 Total l4C-uptake Determination The leaf sections that had been placed in the oven, were dried for 24 hours and weighed. The weight of the sections was doubled and used as a dry weight measure- ment for each sample. The sample was then combusted in an oxygen atmosphere and 20 ml of a mixture of ethanolamine and ethanol (1:2) was added to absorb the 14C02. A 2 ml aliquot was removed and placed in a scintillation vial with 10 ml scintillation solution for counting. Total uptake was expressed as dpm/mg dry weight/hour, and was determined by totaling the dpm Of 14C02 and combustion. In the glutamine metabolism experiment, the 14C- compounds were extracted in One ml Of cold methanol-water (50:50). The extract was filtered through a previously weighed Whatman No. 29 black sample wrapper. The cell wall debris was oven dried, weighed and combusted to 14C02 to determine the radioactivity. The filtrate was stored in a cold chamber. The weight of the cell wall debris was used to ad- just the data to a per mg dry weight basis. The dpm of combustion, of C02 evolved, and of the extract was summed to provide a measurement Of total uptake (dpm/mg dry wt/hr). Protein-14C Analysis Procedure The leaf sections placed in the freezer were ground in the teflon grinder with a phosphate buffer described 29 by WilkinsOn and Beard (45). Grinding was complete in two minutes without heating and the sample was poured into a centrifuge tube using an additional 2 ml Of buffer to rinse the grinder. The protein extract was separated at 15,000 x g for 20 minutes in a refrigerated centrifuge. The supernatant was decanted into a test tube and placed in an ice bath. A 0.6 m1 aliquet was transferred to another test tube and 2.0 ml of 10% TCA was added. Protein precipitation was allowed to occur for a minimum of 10 minutes. The samples were removed from the ice bath and filtered through a 0.45 um millipore filter. This filter was placed in a scintillation vial and was completely dis- solved by the 10 ml scintillation solution. The scintillation solution for all samples con- tained: 0.10 g POPOP, 5.0 g PPO, 380 ml pedioxane, 380 ml toluene, and 240 m1 absolute ethanol. Counting efficiency was always in the range of 45 tO 55 percent. All data from the radioisotOpe investigations were reported as total up- take of the radioactive chemical, percent Of total uptake 14 .evolved as C02 and percent of total uptake incorporated 14 into protein C. Thin Layer Chromatography Techniques Two dimensional thin layer chromatography (TLC) was used in the separation Of amino acids in the extract. A 0.5 mm layer Of cellulose was spread on a 20 x 20 cm glass plate and allowed to dry for a period of 24 hours. 30' A 0.1 ml aliquot of the extract was spotted in the lower lefthand corner Of the plate and was allowed to dry thoroughly before being chromatographed. The sol- vent system for the first direction was chloroform- methanol-l7 percent ammonia (40:40:20). The second direction was phenol-water (75:25). To insure accurate identificatiOn Of the amino acids and radioactive spots, a mixture Of Silica Gel G and cellulose (50:50) was substituted as the thin layer, rand the solvent system for the first direction was re- placed by butanol-acetic acid-water (60:20:20). It was determined that the radioactivity was being emitted from the amino acids. Twenty-one amino acid standards were chromatographed for the determination of Rf values. Following development of the chromatogram, the plate was allowed to dry for 15 minutes, placed in a spray chamber and coated with-a layer of Ninspray (ninhydrin) and . transferred to an 80 C chamber for four minutes. After the oven treatment, the plate was allowed to cool two hours as the spots develOped. The average density of each indi- vidual spot was recorded with a Photovolt densitometer. Through the use of chromatographed standards Of known con- centrations Of amino acids, an estimate Of the quantity Of' amino acids isolated from.the extract, was determined, and reported as ug amino acid/mg dry weight. Following the densitometer measurements, the spots were scraped from the plate and placed in liquid 3'1 scintillation vials for counting. The radioactivity isolated from each amino acid was recorded as percent Of total dpm. NO areas of the TLC plate, with the exception of the origin and the five amino acid spots, were found to contain detectable radioactivity. Dry Weight Production The weight of the freeze-dried tissue was total- ed for each temperature period and reported as mg dry weight/sq decim/wk. Nitrogen Analysis A modified micro Kjeldahl technique (2) was used in determining the total nitrogen content of the freeze- dried tissue. A 50 mg sample was used for bentgrass and bluegrass and a,75 mg sample was used for bermudagrass. Data were reported as percent nitrogen on a dry weight basis. Protein Analysis Fifty mg of the dry weight sample were placed in a Virtis blender with 50 ml of 0.2 M phosphate buffer (pH 7.0) containing 10 mM Naethylenediamine tetraacetic acid. The extract was centrifuged at 15,000 xg for 20 minutes and the supernatant decanted for analysis. The extract was kept cold (0 to 3 C) throughout the analysis. Onehalf ml extract was assayed for protein using the Lowry protein test (19). Optical density was determined 32 with a Perkin-Elmer spectrophotometer adjusted at 660 mu and compared to a series Of standards prepared with bovine serum albumen. Data were reported as percent protein on a dry weight basis. Carbohydrate Analysis A 50 mg sample was placed in a large test tube with 10 ml Of 85% ethanol. The tube was stoppered and placed on an automatic shaker for one hour. Approximately 1 g activated charcoal was added to remove the chlorophyll, and the extract was filtered in a Buchner funnel through Whatman NO. 42 filter paper. The ethanOl was removed in a flash evaporator. The filtrate was transferred to a 100 ml volumetric flask and diluted to volume. One ml of extract was used for analysis. Analysis was based on the pro- cedure described by Martin (20) utilizing the color reaction Of the anthrone reagent with the carbohydrates contained in the sample. Optical density was determined with a spectrophotometer adjusted at 620 mu. A standard curve was provided by analyzing a series of known concentrations of glucose. Statistical Analysis Statistical significance of all data was deter- mined with Duncan's multiple range test after Obtaining an analysis of variance with a significant F test. RESULTS Intermediary Metabolism Experiment The influence Of temperature on the dry matter production and the nitrogen content of the leaves is Shown on Table 1. Maximum dry matter production Occurred TABLE 1.--The effect Of temperature on the growth and nitrogen content Of three turfgrass species. , Temperature Growth Nitrogen SpeCies C ' (mg dry wt/sq decim/wk) (% dry wt) Kentucky 20 370 e 4.41 h bluegrass 25 510 g 4.26 g 30 437 f 3.98 ef 35 103 a 3.52 d Creeping 20 226 c 4.21 g bentgrass 25 272 d 3.77 e 30 252 cd 4.36 g 35 150 b 4.16 fg Bermuda- 20 83 a 3.00 c grass 25 184 b 2.89 c 30 391 e 2.67 b 35 503 h 2.38 a Values with the same letter within vertical columns are not significant at the 5% level (Duncan's Multiple Range Test) at the 25 C growth temperature for Kentucky bluegrass and at 25 C to 30 C for creeping bentgrass. Significant reduc- tions of dry matter were found as the growth temperature 33 34 was increased to 35 C. Maximum dry matter production occurred at 35 C for bermudagrass, while statistically significant reductions were noted at lower temperatures. The nitrogen content decreased with increasing temperatures in bluegrass and bermudagrass. In creeping bentgrass the highest nitrogen content was found at 30 C and the lowest at 25 C. These are the temperatures where the growth rate was highest. The effect of temperature on the nitrogen content was inversely related to the effect of temperature on growth in bermudagrass and directly related in Kentucky bluegrass. Uptake studies of four radiocarbon materials were conducted at maximum (25 C) and minimum (35 C) growth temperatures for the two cool season species. Supra- optimal growth temperatures reduced the uptake Of glucose- 14C in Kentucky bluegrass, increased uptake in creeping bentgrass, and did not affect uptake in bermudagrass 14 (Table 2). Increased temperature reduced the percent C02 evolved, and increased the percent protein-14C synthesized in all three species. 14C was used as the source, total When glutamine- uptake was increased with temperature in creeping bentgrass (Table 3). Uptake in Kentucky bluegrass and bermudagrass were not affected. Increased temperature during growth in- 14 creased the percent C02 evolved in Kentucky bluegrass. In creeping bentgrass, the percent 14C02 evolved was reduced 35 TABLE 2.--The effect of temperature during growth on the 4C—uptake, 1 C02 evolved and protein- C synthesized from glucose-14C by leaf sections Of three turfgrass species. Growth Total uptake Species temperature (dpm/mg dry 14C02 Protein-14C (C) wt/hr) (% total) (% total) Kentucky 25 6394 b 24.4 b 1.14 b bluegrass 35 2681 a 19 4 a 1.30 c Creeping 25 6939 b 30.1 c 1.91 d bentgrass 35 10508 c 25.8 b 3.54 e Bermuda- 25 1701 a 40.4 d 0.68 a grass 35 2451 a 30.5 c 1.88 d Values with the same letter within vertical columns are not significant at the 5% level (Duncan's Multiple Range Test) TABLE 3.--$he effect of temperature during grow on the C-uptake, 4C02 evolved an? protein- C synthesized from glutamine- C by leaf sections of three turfgrass species. Growth Total uptake 14 . Species temperature (dpm/mg dry C02 Protein-14C C wt/hr) (% total) (8 total) Kentucky 25 2032 a 37.4 a 0.74 a bluegrass 35 1859 a 45.1 b 1.84 c Creeping 25 3381 b 58.8 c 0.78 a bentgrass 35 9249 c 50.6 b 2.15 c Bermuda- 25 1572 a 64.7 d 0.40 a grass 35 2035 a 69.3 d 1.41 b Values with the same letter within vertical columns are not significant at the 5% level (Duncanfs Multiple Range Test) 36 by supraoptimal temperatures while bermudagrass was un- affected. Synthesis Of protein-14C was increased signifi- cantly with increasing temperatures in all three species. Leucine is an amino acid which enters into relatively few metabolic reactions. The total uptake Of leucine-14C was in the same general range as that found for glutamine, but the percent evolved as 14C02 was reduced (Table 4). Supraoptimal temperatures TABLE 4.-—The effect of temperature during growth on the C-uptake, C02 evolved and protein-14C synthesized from leucine-14C by leaf sections of three turfgrass species. Growth Total uptake 14 Species temperature (dpm/mg dry C02 Protein—14C (C) wt/hr) (% total) (% total) Kentucky 25 3438 b 7.9 b . 8.40 c bluegrass- 35 2616 b 4.9 a 11.41 d Creeping 25 2972 b 16.5 c 6.71 b bentgrass 35 8710 c 7.0 b 15.71 e Bermuda- 25 1234 a 20.6 d 1.34 a grass 35 1230 a 20.9 d 9.43 c Values with the same letter within vertical columns are not significant at the 5% level (Duncan's Multiple Range Test) during growth reduced the percent 14C02 evolved from the cool season species but had no effect on the warm season species. Synthesis of protein-14C from leucine was found in very high percentages compared to that from other 37 radioactive substrates. Increasing temperatures during growth from 25 to 35 C resulted in statistically signif- icant increases in synthesis of protein-14C in all three species. Total uptake Of the relatively large acetyl-CoA molecule was apprOximately one-tenth of the other radio- I carbon sybstrates (Table 5). Differences in uptake due to species and temperature, however, were similar to those found for glucose and glutamine. Supraoptimal temperatures increased the percent evolved as 14C02 from Kentucky blue- grass and creeping bentgrass, but reduced the percent evolved from bermudagrass. Temperature increased the per- 14 cent protein- C synthesized in all three species. Carbohydrate Metabolism Experiment The effect of light duration on the percent ethanol-soluble carbohydrates was determined to see if there was any effect on the uptake Of radioactive glucose (Table 6). Carbohydrate levels were greater after 14 hours light when Kentucky bluegrass and creeping bentgrass were grown at 20 C. However, when grown at 35 C, the diurnal variation in carbohydrate content was moderated. Therefore, if carbo- hydrates were measured early in the morning, a statistically significant increase was found with increasing temperature, but if carbohydrates were measured in the evening, a slight decrease was found for Kentucky bluegrass. 38 TABLE 5.--The effect Of temperature during growth on the C-uptake, l4C02 evolved and protein-14C synthesized from acetyl-CoA-14C by leaf sections of three turfgrass species. . Growth Total uptake 14 14 SpeCies temperature (dpm/mg dry C02 Protein- C (C) wt/hr) (% total) (% total) Kentucky 25 261 a 18.7 a 2.55 a bluegrass 35 156 a 21.7 b 5.57 c Creeping 25 495 b 22.4 b 2.56 a bentgrass 35 855 c 36.2 d 4.09 b Bermuda— 25 127 a 37.1 d 2.97 a grass 35 131 a 27.7 c 6.21 c’ Values with the same letter within vertical columns are not significant at the 5% level (Duncan's Multiple Range Test). TABLE 6.--The effect Of temperature and light duration on the nitrogen, protein and carbohydrate content of two cool season turfgrass Species. Growth Light Content (% dry wt) Species temperature duration . (C ) (hrs ) NitrOgen Protein Carbohydrate Kentucky 20 0 5.31 s 14.2 d 4.0 a bluegrass . 14 4.95 d 13.8 d 17.1 f 35 0 4.54 be 12.9 bc 11.8 d 14 4.27 ab 12.5 b 14.6 e Cree in 20 0 4.50 bc 10.5 a 3.1 a bentgrags 14 4.07 a 9.8 a 9.8 c 35 0 4.92 d 13.4 cd 6.1 b 14 4.66 cd 12.5 b 9.1 c Values with the same letter within vertical columns are not significant at the 5% level (Duncan's Multiple Range Test). 39 The nitrogen content decreased in Kentucky bluegrass and increased in creeping bentgrass with increasing temper- atures. This response was similar to that reported in Table 1. Increasing growth temperatures reduced the protein content of leaf tissue of Kentucky bluegrass and increased the protein content in creeping bentgrass. These changes, however, were not of the magnitude Observed for nitrogen with the same temperatures. A fraction of the nitrogen content other than protein was being altered by supra- optimal temperatures. After 14 hours Of light, the nitrogen content of both species grown at 20 C was reduced, but no effect was found when grown at 35 C. The protein content was not affected by light except in creeping bentgrass grown at 35 C where the content was reduced. Table 7 indicates the effect Of temperature and light duration on the uptake, percent 14C02 evolved and percent protein-14C synthesized from radioactive glucose by leaf sections Of Kentucky bluegrass and creeping bentgrass. Light was found to significantly reduce the amount of glucose-14C uptake at 20 C and 35 C for creeping bentgrass. Light did not affect uptake in Kentucky blue- grass. Supraoptimal temperatures reduced uptake in Kentucky bluegrass and increased uptake in creeping bent- grass regardless Of light duration. TABLE 7.--The effect Of temperature and light duration on the 14C02 evolved and protein-14C synthesized from glucose-14C by leaf sections of two cool season turfgrass species. . 14 Growth dLight Total uptake (:02 Protein-14C Species temperature urat on . . (C) (hrs) (dpm/mg dry wt/hr) (% total) (% total) Kentucky 20 0 8166 b 21.7 b 1.41 a bluegrass 14 5942 b 24.5 cd 1.37 a 35 0 1996 a 18.7 a 2.40 d 14 2780 a 25.4 d 1.77 bc Creeping 20 0 10197 c 26.2 d 1.55 ab bentgrass 14 7342 b 22.5 be 1.31 a 35 0 19916 d 23.0 be 1.93 c 14 10006 c 22.4 bc 2.52 d Values with the same letter within vertical columns are not significant at the 5% level (Duncan's Multiple Range Test). TABLE 8. --The effect of temperature during grOwth and incubation on the 14C-uptake, 14C02 evolved and protein-14C synthesized from glucose-14C by leaf sections of two cool season turfgrass species. 5 ci . G owzfimisrzguiion Total uptake 1€002 Protein-14C Pe es r (C) c“; (dpm/mg dry wt/hr) (s total) (4 total) Kentucky 20 20 2545 b 13.3 c 1.80 ha bluegrass 35 4004 c 18.5 e 1.10 a 35 20 697 a 6.9 a '2.54 d 35 2572 b 11.0 b 2.00 bed Creeping 20 20 3817 c 12.7 c 2.47 cd bentgrass 35 5035 d 1 7 d 1.46 ab 35 20 3342 c 10.8 b 3.75 e 35 7617 s 17.3 de 2.66 d Values with the same letter within vertical columns are not significant at the 5% level (Duncan's Multiple Range Test). 41 4 After 14 hours of light the 1 C02 evolved in 14 Kentucky bluegrass was increased. The protein- C synthesi- zed in Kentucky bluegrass was decreased by maximum light duration at 35 C. In creeping bentgrass the 1.4C02 evolved was decreased at 20 C and the proteineldc increased at 35 C with increasing light duration. The higher tempera- ture reduced the percent 14C02 evolved for both species at 0 hours light duration. No effects Of temperature on 14C02 were found after 14 hours light duration. Supra— optimal temperatures stimulated protein-14C synthesis in both species at both 0 and 14 hours of light. The effect of temperature during growth and 14C02 evolved, and 14 incubation on the uptake, percent protein-14C synthesized from glucose- C is found in Table 8. The higher temperature for growth ~significantly reduced the amount of uptake in Kentucky bluegrass, while the higher temperature during incubation increased uptake. The higher temperature for incubation increased uptake in leaf sections of creeping bentgrass. The higher temper- ature for growth increased uptake at 35 C incubation and did not affect uptake at 20 C. The percent 14 C02 evolved was always increased at the high temperature during incubation which is due to normal enzyme response to temperature. SupraOptimal temperature during growth increased respiration in Kentucky 42 bluegrass at both temperatures during incubations and in creeping bentgrass incubated at 20 C. Supraoptimal temperatures for growth stimulated protein-14C synthesis regardless of the temperature during incubation. It should be noted however, that more rapid synthesis of pro- tein-14C occurred when the temperature during incubation was 20 C rather than 35 C. Glutamine Metabolism Experiment Thirteen Spots were isolated by the solvent systems of which nine spots could be accurately iden- tified through the use Of chromatographed standards (Figure 1). Five of the spots that were identified con- 14C-labeled amino acids. They were identified as tained glutamine (GLM), glutamate (GLA), gamma aminobutyrate (GAB), aspartate (ASA), and asparagine (ASN). Threonine, alanine, glycine, and serine were also identified, but contained no detectable radioactivity. Of the five amino acids which contained radio- activity, glutamine was present in the largest quantity (Table 9). As temperature during incubation was increased above 26 C, a significant accumulation Of GLM occurred when grown at 26 C. At 32 C, increasing temperatures during incubation resulted in a significant reduction of 43 I I I I I I I I I I I I l I E- < 3 I —'I E I I I I I I I I I I I I *Spots could not be positively identified. Figure 1I--Position of thirteen amino acids isolated on a 0.5 mm layer of cellulose by thin layer chromatography. 44 .amumm gauchscec can we c0euoouoc Mom meow ocean mo mowuwuccso ucowowumsmsfl on mac mum mosac> ascend. .Aumoa omcmm mamwuasz m.cmocsov HO>OH we ocu um ucmowmwcmwm uos one mascaoo amowuuo> cwcufi3 Houuoa mean on» suds mosac> « II c mm.~ m mm.o m mm.m m mo.q mm mm o mm.m o mm.v m mo.a c mm.m o mated mm mm o om.¢ o mm.o I II c mo.m o on.om mm mm a om.e an mm.n a mo.e a oa.m a m~.e~ mm mm o mo.v n mh.v m oH.H m mm.m o mm.m~ mm mm m ma.a o mv.h c om.o m mo.m o mm.ma em mm sawmcucmmd oucuucmmd oumuwusm oumEmuoHo oceamusao cOeucnoocH nu3ouo Ocfiad MEEMU D3 mun mE\ms ADV ousumuomEOB .mmmumoan axosucmm gowns: mo mcoauoom mama Eouu coucaomw moflom ocHEm o>flw mo ucoucoo man so cowumosoca one £u3oum mcwuso ousumucmfiou mo poommo oneII.m mqmde 45 the GLM content of the leaf tissue. No differences in the quantity of glutamate were detected. The gamma aminobutyrate content of the tissue was small but detectable. The values in Table 9 are not significantly different from each other. The ninhydrin spray was unable to detect less than 0.90 ug Of GAB. Therefore, the absent value could range anywhere from 0 to 0.9 ug GAB. Significant reductions in the aspartate content oc- curred as temperatures during incubation increased above 26 C at both temperatures during growth. When grown at 32 C, another reduction occurred at 38 C. NO significant differences were found due to temperatures during growth. The asparagine content of the leaf sections grown at 26 C increased significantly when the temperature ‘ during incubation increased above 26 C. When grown at 32 C however, the highest ASN content was found at the 26 C and 32 C temperatures during incubation. The absent value was due to the fact that the ninhydrin spray could not detect less than 1.10 ug ASN. The effect Of temperature on uptake of glutamine- 14C is included in Table 10. The higher growth temperature and increasing temperatures during incubation above 26 C reduced total uptake. 46 .mbOLw accomfloc EOuw EQU wo COHDSHOmOu ucmHonwomcfl ou wot me osac> ucomo<¢ .Aumoe omcmm sameness m.ccocsov Hm>ma ea one on ucmonecmHm Doc mum mcEdHoo acceuum> Cenuez nouuwa seam ecu zue3 mOSHm> o m.m m n.v o o.m o m.am o n.am m a.mH m Hood mm mm on m.a m m.v m m.m o m.na p N.ov c ©.vH m nmma . mm mm a I o m.m o H.v m ©.oH o m.0m o n.vm Q mmmm mm mm o H.N Q m.m o m.m n m.ma c a.ma o v.0m on Hoom mm mm o o.H o o.m n ©.m a a.ma Q ©.hm o a.mm to comm mm mm c o.H c m.v o m.m c m.oa to m.nm D m.mm O homm mm mm mumu>usm OUHXOHQ Q Q . . daemons m4 mucous m4 OcHE< mEEmo conumu oumEcusHo mcHEMDSHo mxmumb Hmuoe COADMQDOCH :u3ouu excuma Hmuoe mo assumed Au£\u3lfi;ooE\EmUv ADV aboucummsoe .mmmumODHQ wxosucmx cofluwz mo mcofluomm mama >n UVHIOCHEcuSHm Eoum cowemmcuc>m mmueaoocumEIUvH com mxmuQSIU H wow so coflumosoce cam £u3oum mcflusc wusucummEmu mo pommmm omelr.oa mqma mma om mm.e G.HN Hem om «ouuesumoa Hm.m m.v swam om N>.m H.> nmmm om oceosoq NH.H m.mm emmm om vo.a a.mm oama om OCHEmuus ma.H o.am Nwov om mmmuoOSHb mo.a v.mm mvmm om omoooaw >xosucmx Ov Aamuou wv Aamuou wv Au£\u3 wuo mE\Eoov A a Q saw One) N o m a: O seduces Emu condom moaoo m .0 mm can om um ozonm mmmumucon mnemoouo oucouoe can mmmum Iooab wxosucmm coflumz wo mcofluoom mama an woousom conucooflcmu v Eoum Ummflmwcucwm U «a Icflmuoum cam cm>ao>m NOU .o m moi ea x p U VH HMUOBII.M mqmfie XHQmemd. w I Elllivt 1293 02585 5705