( M 1 W M R : ,_. E——__’_ — , ______ , , 7 __ .- ,! r.__4—— MM 1 ‘l \|\ ‘. \H Hm; k I\14> M CDLOU'I THE EFFECTS O? HBGH ‘fEMPERATURE ON THE SOLUBLE METROGEN FRACT!QN OF KENTLICKY BLUEGRASS {PQA PFATEfiSES L} M "vr ‘fhesis for fine Dagma c? M. S. MSCHEGJKN STAKE UNWERSR‘Y fi‘iarian R. Stein ??56 ABSTRACT THE EFFECTS OE HIGH TEMPERATURE ON THE SOLUBLE NITROGEN FRACTION OF KENTUCKY BLUEGRASS (POA PRATENSIS L.) by Harlan R. Stoin The changes in the content and composition of the soluble nitrogen fraction of Kentucky bluegrass were studied in con- trolled environment chambers. Temperature variables were 21 C. and 35 C. during the 16 hour day with a common 16 C. at night. The high day temperature resulted in a 23% increase in total soluble nitrogen, an increase in the proportion of am- ides to free amino acids and increased ammonia. Aspartic and glutamic acids decreased at 35 C. while as- paragine showed an eleven-fold increase. Glycine, alanine, valine, isoleucine, and lysine increased at 35 C. Glutamine, leucine, and proline were highly variable. Cystine, methionine, phenylalanine, tyrosine, histidine, arginine, tryptophan, and hydroxyproline did not occur in meas- urable quantities. THE EFFECTS OF HIGH TEMPERATURE ON THE SOLUBLE NITROGEN FRACTION OF KENTUCKY BLUEGRASS (POA FRATENSIS L.) By Harlan R. Stoin A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crop Science 1966 K" ,‘1’ ACKNOWLEDGEMENTS The author wishes to express his appreciation to Dr. J. B. Beard for his guidance, criticism, and help during the course of this study and the preparation of the manuscript. The author also wishes to thank Dr. C. M. Harrison for reviewing the manuscript. ii TABLE OF CONTENTS INTRODUCTION REVIEW OF LITERATURE . MATERIALS AND METHODS RESULTS . . O 0 DISCUSSION OF RESULTS CONCLUSIONS BIBLIOGRAPHY O 0 iii Page 10 17 23 26 27 LIST OF TABLES Page TABLE 1. Buffers used for the amino acid analysis . . . 12 TABLE 2. Changes in total soluble nitrogen and to- tal free ammonia in Kentucky bluegrass after six days at 21 C. and 35 C. day tem- perature O O O O O O O O O O O C O 0' O O O O 0' 17 TABLE 3. Changes in the amide content of Kentucky bluegrass after six days at 21 C. and 35 C. day temperature . . . . . . . . . . . . 18 TABLE 4. Changes in the dicarboxylic amino acids and their amides in KentuCky bluegraSs after six days at 21 C. and 35 C. day tem- perature . . . . . . . . . . . . . . . . . . . 19 TABLE 5. Changes in glycine, alanine, valine, leu- cine, isoleucine, serine, threonine, ly- sine, and proline content of Kentucky bluegrass after six days at 21 C. and 35 0. day temperature . . . . . . . . . . . . 20 iv :FIGURE 1. :IEIGURE 2. IIEIGURE 3. fJEVIGURE 4. LIST OF FIGURES Page Changes in total soluble nitrogen, total amides, and total free ammonia in Kentucky bluegrass after six days at 21 C. and 35 C. day temperature . . . . . . . . . . . . . 21 Changes in the individual dicarboxylic amino acids and their amides in Kentucky bluegrass after six days at 21 C. and 35 C. day temperature. . . . . . . . . . . . 21 Changes in glycine, valine, leucine, iso- leucine, and lysine content of Kentucky bluegrass after six days at 21 C. and 35 C. day temperature. . . . . . . . . . . . 22 Changes in alanine, serine, threonine, and proline content of Kentucky bluegrass after six days at 21 C. and 35 C. day temperature. . . . . . . . . . . . . . . . . 22 INTRODUCTION The common grasses have been classified as warm— or cool- season depending on the optimum temperature for growth. Most of the grasses grown for forage, lawn, and recreational use in the Midwest are cool-season grasses. The hot continental summers frequently cause a reduction in growth of these grasses. This is especially true of Kentucky bluegrass, an important pasture and turfgrass of the Midwest. Reasons for this reduction in growth at temperatures above the optimum are not fully understood. many of the early work- ers attributed the reduction to carbohydrate depletion due to the higher temperature optimum for respiration than for photo- synthesis. Since temperature affects many plant processes, carbohydrate depletion is probably not the only reason for growth reduction. It has been suggested that an important site where envi- ronmental factors interact with metabolic processes is at the point of contact between carbohydrate and nitrogen metabolism. Specifically involved are the keto acids, amino acids, and amides. This study was initiated to examine the effects of high temperature on the free amino acids and amides of Kentucky bluegrass after a few days exposure to high temperature. These results would assist in elucidating the metabolic site or sites of high temperature growth reduction in cool-season grasses. Clarification of the metabolic processes involved in high temperature growth reduction would be a great aid in developing 1 2 better management practices for these grasses and in breeding varieties that are better adapted to high temperatures. REVIEW OF LITERATURE The Optimum temperature for growth has been defined as the temperature giving the maximum growth rate independent of the time factor (12). Since the effects of temperature are conditioned by other environmental factors, the optimum \ is actually a range rather than a single temperature. In gen— ? eral it is recognized that certain grasses grow better at cooli while others prefer warm temperatures. Mitchell (cited in 14) reported an optimum temperature of 20 C. for perennial ryegrass (Lolium perenne L.), orchard- grass (Dactylis glomerata L.), colonial bentgrass (Agrostis tenuis Sibth.), and velvetgrass (Holcus lanatus L.). The op- timum for Dallisgrass (Paspalum dilatatum Poir.), a warm-season grass, was near 30 C. The Observations were based on daily increase in dry weight. Above the optimum a rapid decline in growth occurred. Growth of the cool-season grasses ceased above 35 C. He also reported a decrease in new tillers above 28 C. Sullivan and Sprague (21) also reported an optimum near 20 C. for perennial ryegrass. Brown (4) and Harrison (6) re- ported an Optimum near 20 C. for top growth of Kentucky blue- grass. They found that the Optimum for root and rhizome growth} was near 16 C. I Darrow (5) grew Kentucky bluegrass at soil temperatures of 15, 23, and 35 C. while exposing the shoots to normal green- house temperatures. The best growth occurred at 15 and 23 C. with large reductions in growth at 35 C. High temperature 3 4 injury was more severe with ammonium than with nitrate nutri- tion. Brown (4) also reported soil temperature to be more 1 important than air temperature as far as causal factors were; concerned. This reduction in growth should be distinguished from direct high temperature injury. High temperature kill is some- times Characterized by a break in the time vs. temperature curve. Levitt (13) terms the reduction in growth as indirect injury and divides it into metabolic high temperature injury and transpirational high temperature injury. Several early workers observed a decrease in carbohydrates in grasses grown at higher temperatures. Sullivan and Sprague (21) grew perennial ryegrass under night—day controlled temperatures of 10-15.6, 15.6-21.1, 21.1- 26.7, and 26.7-32.2 C. The stubble and roots were analyzed quantitatively for sucrose, reducing sugars, and fructosan, an important reserve carbohydrate. Sucrose reached a low point in the stubble two weeks after clipping and then began to rise. The greatest rise occurred at 10.1-15.6 C. In the root tissue the decline of sucrose lasted four weeks and then rose only at the lowest temperature treatment. Reducing sugars fell more gradually after clipping and showed little response to temperature. Fructosan in the stubble made up about 25% of the dry matter at the time of clipping. Twenty-eight days after clipping, fructosan decreased to 18% at 10-15.6 C. and 7% at 26.7-32.2 C. The other temperature treatments were in— termediate. After 28 days fructosan began increasing at the two lowest temperature treatments but continued decreasing 5 at the two highest. The decrease of fructosan from the roots followed a similar pattern, although the original content was lower, 7%. Differences in dry weight changes of the tOps were noticeable two weeks after clipping. At that time the plants at the highest temperature treatment contained nearly 8% fruc— tosan in the stubble and 2% in the roots. Although the decline continued, complete exhaustion of carbohydrates was not observed at the termination of the experiment after 40 days. Brown (4) conducted a seasonal study of the carbohydrate changes in Kentucky bluegrass. Carbohydrate storage occurred in spring and autumn, but a net loss resulted during late spring and summer when temperatures were higher. Growth is a complex interaction of a number of interrelated physical and chemical processes. Many of these processes are affected by temperature as indicated in several reviews (10, 17, 23). Van Halteren (cited in 13) showed that metabolic proces— ses other than photosynthesis may be more heat sensitive than respiration. A temperature of 47 C. for the yeast Saccharo- myces for one hour lowered oxygen consumption by 40 to 60% but lowered nitrogen assimilation to zero. The longer the exposure to high temperature, the more slowly nitrogen assimi- lation recovered when the yeast was placed at normal tempera- tures and the lower the rate following recovery. Phosphate uptake was also stOpped for several hours after the high tem- perature exposure. Tissue culture studies have also shown that factors other than carbohydrate depletion are involved. White (24,25) grew 6 excised wheat (Triticum vulgare Host.) and tomato (Lycopersicum esculentum L.) roots in a nutrient medium containing dextrose, a carbohydrate source. He found a typical temperature vs. growth curve with an optimum near 30 C. Burstrom (cited in 17) studied cell division and elongation OI wheat roots with ex- ium ani with intact (D 5.1.. H) cised root tips in a glucose-contbining mv roots in the dark. The growth behavior of both groups of roots was similar, increasing in length and number of cells to a maximum near 25 C. and then decreasing rapidly at 30 C. and above. A recent review of the biochemical aspects of temperature response by Langridge (10) listed five possible causes for high temperature effects. All ultimatelv involved blocéage of syn- thesis or acceleration of breacdown of soqe essential metabo— . I“ .W,‘ ..... a ,.,.- ;,‘"1"J-'7’."':: -’-‘.O\'CA" ---L._.-S._- . . i U .1" l lite. He listed a number of exam lee O; 1132 deficisncies in micro—organisms that were overcome by the ad— dition of certain metabolic substances. In many cases a single substance was sufficient. Within a species different substances were sometimes found limiting at different temperatures. The organic compounds most frequently required were glutamic acid, thiamin, and biotin. High temperature growth deficiencies have been tenmed "climatic lesions" by Bonner (3). Ketellapper (7) reported several instances in higher plants where the appl'cation of certain metabolites would partially or completely overcome the effects of supraoptimal temperatures but had no effect at th optimum temperature. Vitamin C was partially effective on peas (Efigsum sativa L.) and broad beans (Vicia faba L.). A mixture 7 of vitamin C, several B vitamins, several ribosides, and casein hydrolysate was slightly more effective on broad beans. A vitamin B mixture was effective on Lupinus nanus Dougl. Sucrose prevented injury to peas at 23 C. but could not completely pre- vent injury at 26 C. Varietal differences within species were also noted. Langridge and Griffing (9) reported similar results with Arabidopsis thaliana Heynh. Forty-three races were grown at constant temperatures. Five races were depressed in growth and morphologically abnormal at 31.5 C. The addition of biotin to the growth medium overcame the injury in two races, cytidine was partially effective in a third, and two races did not re— spond to supplements. From the foregOing it may appear an impossible task to understand the effects of temperature on the growth of the whole organism. However, Langridge (10) has stated, "The as- sumptions necessary to make physicochemical deductions from the growth response of the whole organism to temperature are often so numerous as to cause many scientists to deprecate such attempts as wholly unrealistic. However, at temperature extremes, the rate of growth may be limited by the velocity of a single reaction, and, to the extent that this is so, inter— pretation in molecular terms appears to be valid." Sullivan and Sprague (21) found an increase in the soluble nitrogen content of perennial ryegrass after clipping. The .largest increase occurred at the highest temperature treatment._ This large increase occurred after the first four days. Beard (2) conducted a seasonal study of the changes in 8 total nitrogen, non—protein nitrogen, glutamine, asparagine, total amide, and total free ammonia in bentgrass (Agrostis palustris Huds.). A linear multiple regression-correlation analysis of the environmental parameters involved showed that temperature was the major environmental factor influencing the seasonal variations in all six nitrogen fractions. Total nitrogen increased with soil temperature through 27 C. and then showed a marked decrease. Non-protein nitrogen increased 1 slightly with higher 3 but decreased above 27 C. ~- ‘- (D C ‘WNj '1 4 L. \/-l (\1 L1 '0 g 1" . A (U u) Total free ammonia showed little response to environment except for increased variability at high temperatures. Glutamine showed a marked reduction as temperatures increased, decreas— ing from .26% to .O1% of the dry weight. Asparagine also was reduced but to a lesser extent than glutamine. Steward (20) summarized a number of instances where the environment caused changes in the amount and composition of the soluble nitrogen fraction. With the mint plant (Mentha piperita L.), daylight, long days, and high K to Ca ratio in the nutrient medium, tended to promote protein synthesis and glutamine accumulation in the leaves, especially with low night temperatures. On the other hand, darkness, high Ca to K ratio, short days, and high night temperatures tended to favor aspar- agine. In studies with the banana (Mgsa species) variety Gros Michel, the fruit that matured in July had more amide nitrogen with a higher proportion of glutamine, while fruit that matured in December had predominantly asparagine. These differences ‘were attributed primarily to night temperature. In tulip (Tulipa gesneriana) leaves high temperatures caused an increase 9 in serine/glycine, asparagine, glutamine, gamma—methyleneglutamic acid, and ammonia and a decrease in aspartic and glutamic acids. In peas grown under long days, the content of asparagine in— creased with high night temperatures. MATERIALS AND METHODS PLANT SAMPLES. The experimental material was obtained from a three-year-old, irrigated turf of Merion Kentucky blue- grass cut at 1% inches. On May 19,1965 plugs 7 inches in di- ameter and 2 inches thick were placed in one gallon earthen- ware containers filled with a mixture of equal parts of sand, soil, and peat. The bluegrass was grown in the greenhouse for 29 days to permit rooting and clipped periodically to one inch. Temperature treatments of 21 and 35 C. day temperature were begun in two controlled environment chambers on June 17, 1965, one day following clipping. During the 8 hour night period the temperature dropped to 16 C. in both chambers. Light intensity in both chambers was 2200-2400 f.c. All treat- ments were given complete nutrient solution the day after treat- ments were begun. The plants were watered daily. To minimize variation due to location in the chambers, the containers were rearranged at random daily. After 6 days, two containers from each chamber were har— vested at 3:00 P.M. The plants were clipped to one inch, the tissue collected, immediately frozen between blocks of dry ice, and freeze-dried. The freeze—dried tissue was ground with a Wiley mill using a 40—mesh sieve. Subsequently the samples were stored in air-tight containers at ~10 C. until analyzed EXTRACTION PROCEDURES. The ground samples were extracted With 70% ethanol as follows: 10 ml. of 70% ethanol was added to 8.500 mg. plant sample, shaken, and allowed to sit overnight. Tile following day the mixtures were shaken for several hours, 10 11 centrifuged, and the supernatant collected. Then another 10 ml. of 70% ethanol was added to the plant material, the mixture shaken for at least 4 hours, centrifuged, and the supernatant added to the previous extract. The process was repeated two more times. The collected extract (Solution A) was made up to 100 ml., filtered, and stored at 4 C. until analysis. For the amino acid analysis, 50 ml. of Solution A was evaporated to dryness under vacuum at 65 C. using a flash evap- orator. The residue was taken up with 5 ml. of Buffer 1 (see Table 1) in two portions. Two one ml. aliquots of this solution (Solution B) were used in the amino acid analysis. When Solution B was analyzed by column chromatography (see procedure below), the amides asparagine and glutamine overlapped threonine and serine. Therefore, the amides were hydrolyzed to their respective amino acids aspartic and glu- tamic. A hydrolyzed sample was then rerun on the 150 cm. col- umn using only the first buffer, since aspartic and glutamic acids both emerge early. The amides were then determined by the aspartic and glutamic acid differences of the regular and the hydrolyzed samples. Hydrolysis was accomplished by mixing 1 ml. of 4N HCl with 1 ml. of Solution B in a screw cap vial and heating for 4 hours in boiling water. One ml. of the hydrolyzed mixture was used on the 150 cm. column. AMINO ACID, AMIDE, AND FREE AMMONIA ANALYSIS. The free amino acids, amides, and free ammonia were separated by the ion exchange column chromatography method of Moore, Spackman, and Stein (16) using sulfonated polystyrene resins. A 150 cm. column was used for the neutral and acidic amino acids and 12 TABLE 1. Buffers used for the amino acid analysis. Citric acid NaOH HCl Final Buffer pH 'HZO conc. Vol. G. G. ML. L. 1 3.25:0.01 210 82 106 10 2 4.25:0.02 210 82 47 10 3 5.28:0.02 123 72 34 10 Notes on buffers: 1. Phenol (10 g.) was added to the final volume of each buffer. 2. The buffers in 10 l. quantities were stored at 4 C. L0 3. Thiodiglycol GEES ml./l.) was added to Buffers 1 and 2 to 2 liter aliquots shortly before used. 4. BRIJ 35* (6.5 ml./l.) was added to all three buffers to 2 liter aliquots short- ly before used. The BRIJ 35 solution was prepared by dissolving 50 g. of BRIJ 35 in 150 ml. of hot water. 5. All buffers were boiled before used, and placed in the buffer reservoirs while hot to prevent air bubble formation in the columns. A layer of mineral oil was placed on top of the hot buffers and fresh boiled buffer added with a funnel through the oil layer. * Atlas Powder 00., Wilmington, Delaware 13 amides and a 15 cm. column for the basic amino acids and free ammonia. Both columns were operated at 50 C. by means of wa- ter jackets and a circulating water bath. The large column was Operated under 4-6 lbs. pressure. A tank of compressed nitrogen was connected through a pressure regulator to the buffer reservoir attached to the tOp of the column. This gave a flow rate of 12 i 2 ml. per hour. The 15 cm. column was operated under 2—3 lbs. pressure with a flow rate of 25-30 ml. per hour. Resin of 56 i 9 microns particle size was used for the 150 cm. column. Particles of this size were fractionated from Amberlite CC 120 resin (200-400 mesh) by the hydraulic flow method of Hamilton outlined in (16). It was necessary to ad- just the flow rate specified by Hamilton to obtain the proper sized resin particles. This adjustment was probably due to the high Ca and Mg content of the water used. The 15 cm. col- umn was prepared with a commercially separated resin, Aminex- MS, Fraction C (Calbiochem, Los Angeles). The columns were prepared as outlined in (16). Samples were eluted through the columns with sodium cit- rate buffers made up as shown in Table 1. Buffers 1 and 2 were used for the 150 cm. column and Buffer 3 for the 15 cm. column. The eluate was collected in 2 ml. portions on an au- tomatic linear fraction collector. The following procedure was used to operate a column: 1. Water at 50 C. was circulated through the water jacket for at least one hour before applying a sample. 2. The buffer above the resin surface in the column was 14 then removed with a pipette, avoiding disturbance of the resin surface. A one ml. sample was carefully placed on the resin surface with a pipette and allowed to run into the resin under pressure. The sample was then washed into the resin with two 1 ml. portions of the starting buffer, each being allowed to run into the surface under pressure before the next was added. The column above the resin was next filled with start- ing buffer, the buffer reservoir attached, and pres— sure applied. The buffer was changed on the 150 cm. column after elution with 260 ml. of the first buffer. A proce- dure similar to Step 4 was used. The first buffer was removed and two 1 ml. portions of the new buffer were washed in before attaching the new buffer res- ervoir. The sample was completed at an effluent volume of 480 ml. for the 150 cm. column and 160 ml. for the 15 cm. column. To regenerate a 150 cm. column the column was eluted overnight with 0.2N sodium hydroxide containing BRIJ 35 (5 ml./l.). The column was then eluted with Buf- fer 1 until the visible front had moved at least half-way through the column. Each change to sodium hydroxide or buffer was performed as in Step 6. The 15 cm. column required no regeneration. However, 15 when new buffer was added to the reservoir, 80 ml. was eluted through the column before a new sample was run, since differences in ammonia content of dif- ferent lots of buffer would cause a plateau before or after the ammonia peak. 10. Cheesecloth soaked in citric acid was placed on the fraction collector platform during a run in order to prevent the uptake of ammonia by fractions in the tubes. COLOR DEVELOPMENT. After elution from the columns, the amino acids were determined colorimetrically using a Coleman Jr. spectrophotometer. Ninhydrin color was developed by the method of Rosen (18) with several minor modifications sugges- ted by Lawrence and Grant (11). Ninhydrin concentration was raised to 5% since 2 ml. samples were used. Cyanide and ace— tate components of the developing buffer were mixed just be- fore use and used within 4 hours. The procedure was as fol- lows: Reagents: 1. Stock NaCN:0.01M (49o mg./l.) 2. Acetate buffer: 2700 g. NaOAc.3H20 + 2 l. H O + 500 ml. glacial HOAc. Make up to 2 7.5 l. with H20. Its pH should be 5.3- 5.4. 3. Acetate-Cyanide: 2.0 ml. of Soln. 1 made to 100 ml. with Soln. 2. Used within 4 hours. 4. Ninhydrin: 5% in Methyl Cellosolve (eth- ylene glycol monomethyl ether), peroxide- 16 free. 5. Diluent: iSOpropyl alcohol-water, 1:1. Method: One-half ml. of acetate-cyanide buffer and e ml. 5% ninhydrin solution in Methyl Cellosolve were added to a 2 ml. sample and heated 15 min. in a boiling weter bath. Im— mediately after removal from the water bath, 5 ml. of isopropyl alcohol—water diluent was rapidly added, and the tubes vigorously shaken. The tubes were allowed to cool to room temper- ature and read with a spectrOphotometer at 570 mu. (proline at 440 mu.). If the color density was too high, further 5 ml. portions of diluent were added. A standard curve was prepared with known concentrations of leucine dissolved in Buffer 2. The concentration of the other amino acids was based on their known color yield com- pared to leucine as 100% (18). The location of emerging amino acids was determined by running a known mixture of amino acids through the columns and comparing with the results of Moore, Spackman, and Stein (16). RESULTS Total Soluble Nitrogen. The changes in total soluble nitrogen content after 6 days are shown in Table 2 and Figure 1. High temperature resulted in an increase from 17.1 to 21.0 mg./g. dry weight in total soluble nitrogen content, a 23% increase. On a percent dry weight basis it rose from 1.71 to 2.10%. At 21 0. this fraction was composed of 84% amino acids and 16% amides but 74% amino acids and 26% amides at 35 C. Total Amides. Asparagine and glutamine were the only amides found in measurable quantity. This fraction showed a 95% increase from 21 to 35 0. Almost all of this change was due to asparagine. These changes are shown in Tables 2 and 3 and in Figure 1. TABLE 2. Changes in total soluble nitrogen and total free ammonia in Kentucky bluegrass after six days at 21 C. and 35 C. day temperature. Fraction Sample Day temperature ”0' 21 C. 35 C. 21 C. 35 C. 21 C. 35 C. mg./g.D.W. % dry weight % of total soluble N Total sol. N 1 16.6 20.4 1.66 2.04 ... ... 2 17.6 21.7 1.76 2.17 ... ... Av. 17.1 21.0 1.71 2.10 ... ... ug./g.D.W. Total free NH3 1 303 387 0.030 0.039 1.8 1.8 2 240 384 0.024 0.038 1.4 1.8 Av. 272 386 0.027 0.038 1.6 1.8 Total amides 1 2487 5093 0.25 0.51 15.0 25.0 2 3036 5698 0.30 0.57 17.3 26.3 Av. 2762 5396 0.28 0.54 16.2 25.7 17 TABLE 3. Changes in the amide content of Kentucky bluegrass after six days at 21 C. and 35 0. day temperature. Compound Sample Day temperature NO. 21 6. 35 C. % of total amides Asparagine 1 15.4 55.2 2 5.7 58.0 Av. 10.1 55.7 Glutamine 1 84.6 44.8 2 4.3 42.0 AV. 89.9 4303 Tgtgl F322 Ammonia. The free ammonia content increased at the high temperature, increasing from .027% to .038% of the total dry weight (Table 2). Individual Amino Acids And Amides. The influence of tem- perature on the individual free amino acids and amides is shown in Tables 4 and 5. 0f the amino acids regularly occurring in ,protein, cystine, methionine, phenylalanine, tyrosine, histi- dine, arginine, tryptophan, and hydroxyproline were not pres- ent in measurable quantities. The changes in the individual amino acids and amides were not unidirectional. Important changes occurred in the dicarboxylic amino acids and their amides. Aspartic acid and glutamic acids both de- creased in content and in percent of the total soluble nitro- gen at 35 C. Glutamine was highly variable among samples, and no trend due to temperature could be determined. However, the change in asparagine at 35 C. was quite striking. An eleven- fold increase in asparagine content occurred with an increase from 1.6% of the total soluble nitrogen to 14.5%. Glycine and valine content doubled on a dry weight basis. 19 Alanine, serine, and threonine showed smaller increases at 35 C. Isoleucine was present in only a trace amount in one sample at 21 0., and lysine was not detectable at the low tem- perature. Both were present in fair quantities at 35 0. Leu- cine and proline were quite variable and no changes associated with temperature could be determined. The amino acid composi- tion expressed as per cent of the soluble nitrogen fraction is shown in Table 5. Glycine, valine, isoleucine, threonine, and lysine percentages of the total soluble nitrogen increased while alanine and leucine percentages decreased. Serine and proline remained constant. TABLE 4. Changes in the dicarboxylic amino acids and their amides in Kentucky bluegrass after six days at 21 C. and 35 0. day temperature. Compound Sample Day temperature Day temperature N°° 21 C. 35:6. 21 C. ‘3‘5"C'."'""' ug./g.D.W. % of total soluble N Aspartic acid 1 1344 972 8.1 4.8 2 1477 1065 8.4 4.9 Av. 1410 1018 8.2 4.8 Glutamic acid 1 6678 5384 40.3 26.4 2 6561 5825 37.4 26.9 Av. 6620 5604 38.8 26.6 Asparagine 1 383 2814 2.3 13.8 2 172 3302 1.0 15.2 Av. 278 3058 1.6 14.5 Glutamine 1 2104 2279 12.7 11.2 2 2864 2396 16.3 11.1 :D < [\D .p ml 4:. N \N \Nl (I) —8 ‘pl U1 J TABLE 5. 20 Changes in glycine, alanine, valine, leucine, iso- leucine, serine, threonine, lysine, and proline con- tent of Kentucky bluegrass after six days at 21 C. and 35 0. day temperature. Amino acid Sample Day temperature Day temperature NO' 21 C. 35 c. 21 CT 35 C. ug./g.D.W. % of total soluble N Glycine 1 120 173 0.72 0.85 2 20 240 0.51 1.10 Av. 105 206 0.62 0.98 Alanine 1 2129 2334 12.8 11.5 2 2058 2388 11.7 11.0 Av. 2094 2361 12.2 11.2 Valine 1 129 398 0.78 2.0 2 211 422 1.20 1.9 Av. 170 410 1.00 2.0 Leucine 1 315 210 1.9 1.00 2 223 118 1.3 0.54 Av. 269 164 1. 0.77 Isoleucine 1 00 131 0.00 0.64 2 55 118 0.22 0.54 Av. 20 124 0.11 0.59 Serine 1 932 1105 5.6 5.4 2 888 1322 5.1 6.1 Av. 910 1214 5.4 5.8 Threonine 1 1793 2799 10.8 13.7 2 2014 2885 11.5 13.3 Av. i904 2842 11.2 13.5 Lysine 1 00 809 0.0 4.0 2 09 483 0.0 2.2 Av. 00 646 0.0 3.1 Proline 1 35 563 2 2 I\) It} \10‘xm mix] #4 00\ \N o o 0 mg wlwm o OWCD 3:. <1 U1 \N 2O 15 mg./g. D.W. 10 ‘ 5 Total soluble N FIGURE 1. Changes in total soluble nitrogen, six days at 21 C. 71 61 51 E E 4. a: mg.4g. j_ Do“. 34 2 . 1 Aspartic Glutamic FIGURE 2. (W -> Total amides llllllllllllllllIlllllllllllllllllIlllllllllllllllllllllllllllllfllll. Total U14 total amides, CO 0 9 free ammonia and total free ammonia in Kentucky bluegrass after and 35 0. day temperature. Asparagine Glutamine and their amides in Kentucky bluegrass after six days at 21 C. and 35 0. day temperature. 21 Changes in the individual dicarboxylic amino acids 0.61 21 C. 35 C. 0.5 0.4 0.3 0.2 Glycine Valine Leucine Isoleucine Lysine FIGURE 3. Changes in glycine, valine, leucine, isoleucine, and lysine content of Kentucky bluegrass after six days at 21 C. and 35 0. day temperature. 3.0 1 2.5 . mg./g. D,W, 105 1 1.0 . 3; O. 5 '27:. w.— .. 7-. . .- . "A“ ., Alanine Serine Threonine Proline FIGURE 4. Changes in alaniie, serine, threonine, and proline content of Kentucmy’tfliuxyraes after six days at 21 C. and 35 3. day ta perrture. 22 DISCUSSION OF RESULTS The large increase in asparagine and free ammonia plus the decrease in aspartic acid at 35 C. may have been due to an increased breakdown of proteins resulting in a release of ammonia, since asparagine can be synthesized from ammonia plus aspartic acid. This would alsoaccount for increases in the other amino acids as products of protein breakdown. Although glutamine and asparagine are similar compounds, they have different metabolic functions (15,19). Asparagine is often associated with adverse conditions for growth or with conditions where (1) protein synthesis has ceased, (2) protein breakdown occurs, and (3) the storage of soluble nitrogen is in excess. 0n the other hand, glutamine is generally associ- ated with conditions favorable for growth. An exception is the formation of glutamine during the breakdown of protein in excised barley (Hordeum vulgare L.) leaves. Species differ— ences in the relative importance of the two amides have also been reported. Ammonia may have reached toxic levels at the high temper— ature. Studies have shown that ammonia can uncouple photo- phosphorylation (8). High levels of ammonia have also been associated with reduced photosynthesis (1). Ammonium toxicity would also explain the increased high temperature injury to Kentucky bluegrass when grown under ammonium vs. nitrate nu— trition (5). An increase in amides occurred during the assimilation of ammonium nitrogen by green algae (Chlorella vulgaris 23 24 Beijerinck) and by barley leaves (22,26). Labeling studies showed that these amides were synthesized primarily through glutamic acid. Therefore, if tne increase in asparagine found in this study were caused from an increase in ammonia as a protein breakdown product, a shift in reactions from those utilizing glutamic acid to ones utilizing aspartic acid or to some other pathway for the formation of asparagine might be involved. Asparagine and glutamine are not always formed by the same pathways. Asparagine may be formed by a reworking of the breakdown products from protein rather than directly from sugars (19). A shift in reactions due to environmental influences from ones favoring the formation of C5 keto acids to reactions fa- voring C4 keto acids might also be involved in the changes in the dicarboxylic amino acids and amides (2o). Studies with the mint plant have shown tnat long days tend to foster the formation of alpha—ketoglutaric acid, especially at low tem- peratures, while short days tend to impose a trend toward the C4 acids, especially at high temperatures. In tobacco (Nigg- 1 tiana tabacum L.) leaf discs, 0 4 labeled proline and 014 la- beled urea were converted to glutamine in the light and to asparagine in the dark. In view of these findings one would be tempted to look for a light- and temperature-sensitive re— action or series of reactions which could cause a shift in metabolism from favoring 05 compounds to C4 compounds and WhiCh might also be associated with increased catabolic activities, possibly a decarboxylation reaction. The increase in soluble nitrogen compounds found in this 25 study would also be expected with a blockage of or decrease in protein synthesis. This would occur, for example, if car- bohydrates were exhausted. However, the changes observed in this study occurred very early and carbohydrate depletion would not be expected. Evidence has shown that the soluble amino acids are not always the immediate precursors of protein but that early products of photosynthesis provide the carbon skel- etons for protein amino acids (19). If this were the case, one might expect a reworking of the contents of the soluble amino acid pool to a form or ferms readily accessible to the site of protein synthesis, possibly one of the amides. If protein synthesis were blocked, this compound would accumulate. CONCLUSIONS Important changes occurred in the soluble nitrogen frac- tion of Kentucky bluegrass after 6 days exposure to a day tem- perature of 35 0. compared to 21 C. Total soluble nitrogen, total amides, total free ammonia and the proportion of amides to free amino acids increased at the high temperature. Aspartic and glutamic acids decreased at 35 C. while aspar- agine showed an eleven—fold increase. Glycine, valine, alanine, serine, threonine, isoleucine, and lysine content increased at the high temperature. Glutamine, leucine, and proline were all highly variable, and no definite trend due to temperature could be determined. Cystine, methionine, phenylalanine, tyrosine, histidine, arginine, tryptophan, and hydroxyproline did not occur in mea- surable quantities. 26 10. 11. 12. 13. 14. BIBLIOGRAPHY Barker, A. V., Volk, R. J., and Jackson, W. A. Effects of ammonium and nitrate nutrition on dark respiration of excised bean leaves. Crop Sci. 5:439-444. 1965. Beard, J. B. The independent and multiple contribution of certain environmental factors on the seasonal variation in amide nitrogen fractions of grasses. PhD. Thesis. Purdue University. 1961. Bonner, J. The chemical cure of climatic lesions. Eng. Brown, E. M. Seasonal variations in the growth and chem- ical composition of Kentucky bluegrass. Missouri Agr. Exp. Sta. Res. Bull. 360. 1943. Darrow, R. A. Effects of soil temperature, pH, and nitro- gen nutrition on the development of Poa pratensis. Bot. Gaz. 101:109-127. 1940. Harrison, 0. M. Response of Kentucky bluegrass to varia- tions in temperature, light, cutting and fertilizing. Plant Physiol. 9:83—106. 1934. Ketellapper, H. J. Temperature-induced chemical defects in higher plants. Plant Physiol. 38:175—179. 1963. Krogman, D. W., Jagendorf, A. T. and Avron, M. Uncouplers of spinach chloroplast photosynthetic phosphorylation. Plant Physiol. 34:272—277. 1959. Langridge, J. and Griffing, B. A study of high tempera- ture lesions in Arabidopsis thaliana. Australian Jour. of Biol. Sci. 12:117-135. 1959. Langridge J. Biochemical aspects of temperature response. Annual Rev. Plant Physiol. 14:441-462. 1963. Lawrence, J. M. and Grant, D. R. Nitrogen mobilization in pea seedlings. II. Free amino acids. Plant Physiol. 38:561-566. 1963. Leitch, I. Some experiments on the influence of temper— ature on the rate of growth of Pisum sativum. Ann. Botany 30:25-46. 1916. Levitt, J. The Hardiness 9f Plants. Academic Press, Inc. 1956. McCloud, D. E., Bula, R. J. and Shaw, R. H. Field plant physiology. Adv. in Agron. 16:1-58. 1964. 27 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 28 McKee, H. S. Nitrogen Metabolism in Plants. Clarendon Press, Oxford. 1962. Moore, S., Spackman, D. H.. and Stein, W. H. Chromatog- raphy of amino acids on sulfonated polystyrene resins, an improved system. Anal. Chem. 30:1185-1190. 1958. Richards, S. J., Hagan, R. M., and McCalla, T. M. Soil temperature and plant growth. In: Soil Physical Condi- tions and Plant Growth. B. T. Shaw, Ed. Academic Press, Inc. 1952. Rosen, H. A modified ninhydrin colorimetric analysis for amino acids. Arch. Biochem. Biophys. 67:10-15. 1957. Steward, F. C. and Bidwell, R. G. S. The free nitrogen compounds in plants considered in relation to metabolism, growth and development. In: Amino Acid Pools. J. T. Holden, Ed. Elsevier Pub. Co. 1962. Steward, F. 0. Effects of environment on metabolic pat- terns. In: Environmental Control 9; Plant Growth. L. T. Evans, Ed. Academic Press. 1963. Sullivan, J. T. and Sprague, V. G. The effect of temper- ature on the growth and chemical composition of the stub- ble and roots of perennial ryegrass. Plant Physiol. 24:706-719. 1949. Syrett, P. J. The assimilation of ammonia and nitrate by nitrogen-starved cells of Chlorella vulgaris. III. Dif- ference of metabolism dependent on the nature of the ni- trogen source. Physiol. Plant. 9:28-37. 1956. Went, F. W. The effect of temperature on plant growth. Annual Rev. Plant Physiol. 4:347-362. 1953. White, P. R. Seasonal fluctuations in growth rates of excised tomato root tips. Plant Physiol. 12:183-190. 1937. White, P. R. Influence of some environmental conditions on the growth of excised root tips of wheat seedlings in liquid media. Plant Physiol. 7:613-628. 1932. Yemm, E. W. and Willis, A. J. The respiration of barley plants. IX. The metabolism of roots during the assimila— tion of nitrogen. New Phytologist 55:229-252. 1956. .7411‘351451ATE UNIVERSITY LtfiRfiFCIES «1,; s n 1 I ‘ I I 31 6 0557 I 293031 7.1 I t