I” Hi WWW l \ WNW \\ W H ‘ £525 MIN N Tt-IW’SIS LIBRARY Michigan State University This is to certify that the thesis entitled DEVELOPMENT AND CHARACTERIZATION OF AN E VITRO SYSTEM RESPONSIVE TO l-TRIACONTANOL presented by Robert L. Houtz has been accepted towards fulfillment of the requirements for Master of Science degree in Horgiculture Q2; ma, Major professor Date 11-06-80 0-7 639 OVERDUE FINES: _ v I 25¢ per day per item ” fi‘n‘k\ L >- A ,4’ , RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulation records DEVELOPMENT AND CHARACTERIZATION OF AN IN VITRO SYSTEM RESPONSIVE TO l-TRIACONTANOL By Robert L. Houtz A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1980 ABSTRACT DEVELOPMENT AND CHARACTERIZATION OF AN IN VITRO SYSTEM RESPONSIVE TO l-TRIACONTANOL By Robert L. Houtz A system employing extracts from the leaves of rice (Oryza sativa L.) and corn (Zea mays L.) seedlings was developed for the study of the in vitro effects of tria- contanol (TRIA) on plant metabolism. The system is charac- terized by increases in total reducible nitrogen (total N) and reducing sugars in water extracts from TRIA treated leaves. Nitrate concentrations were not affected by TRIA in this system. Studies on the effect of pH, temperature, fractionation by centrifugation and incubation media established that the response is maximized in complete media at a pH of 6.0 and temperature of 25 to 35 C. The increases in total N due to TRIA in the crude extract of rice was doubled by centrifuga— tion at 4080 g. Starch phosphorylase activity in leaf segments from corn seedlings was increased approximately 40% over controls with- in 20 minutes after TRIA treatment. The in 3132 uptake of Pi was greater in TRIA treated rice seedlings 24 hours after treatment . ACKNOWLEDGEMENTS I would sincerely like to thank my major professor Dr. Stanley K. Ries for his help and advice during the completion of this thesis. The knowledge and techniques of scientificinvestigation that I have learned from Stan are invaluable and I am sure that I will benefit from them for years to come. I would also like to thank my committee members Dr. M. Zabik, Dr. D. Dilley, and Dr. K. Schubert for their advice and time that they contributed. Thanks also go to Mrs. Violet Wert whose expertise in analytical techniques helped me through many tough times during my program and to my parents who made it all possible. Finally I wish to express my thanks to Rick Knowles for his encouragement and advice during my program and to Pam Polhemus for her patience while I completed my thesis and especially her friendship. ii TABLE OF CONTENTS INTRODUCTION LITERATURE REVIEW . TRIA in Plant Cuticles Biological Activity of TRIA and other Lipids MATERIALS AND METHODS Plant Culture Chemical Treatment Applications Extraction Procedure Incubation Parameters Sampling and Analysis Methods Statistical Analysis RESULTS AND DISCUSSION Total N Response Studies Method of Treatment Centrifugation Experiments Incubation Media Studies Effect of TRIA on Reducing Sugars in vitro Effect of pH and Ratio of Extract to Incubation Media Incubation Temperature and Inhibitor Studies Studies on Starch PhOSphorylase Activity . CONCLUSIONS iii "U m (D \DOOCDm-PUJUOH h) to no r4 h‘ r4 h‘ F4 IA o~ U1 C) (p a) a» O\ r4 <3 30 33 35 4O LITERATURE CITED iv Table LIST OF TABLES Total N levels in crude extracts from TRIA treated leaves over time. Each treatment in each test contained 4 ml crude extract and 8 ml incubation media in 125 Erlenmeyer flask, after the appropriate times the extracts were quick frozen and dried at 65 C. After drying the entire residue was digested for total N determination. . . . . . . 16 Response of crude and supernatant fractions from rice leaves treated with TRIA (100 ug/l) . 21 Total N and nitrate levels in the supernatant fraction from corn leaves . . . . . . . . . . . 24 Effect of centrifugation force on total N increase in extracts form TRIA treated (lOO ug/l) leaves . . . . . . . . . . . . . . . 24 Effect of incubation media on total N increase in extracts from TRIA (lOO ug/l) treated leaves. The total N values for + incubation media have been corrected for the nitrogen in the incubation media. + and O incubation media were separate experiments . . . . . . . . . . . . . . . . . . 25 Effect of components of incubation media on the total N increase from TRIA (100 ug/l) treated rice leaves . . . . . . . . . . . . . . . 27 Total N and soluble carbohydrate levels in extracts from corn leaves treated with TRIA (100 ug/l) as affected by night temperature. 10 C and 16 C are separate experiments. . . . . 3O Nitrate, reducing sugars and total N levels in extracts from TRIA (100 ug/l) treated corn leaves. . . . . . . . . . . . . . . . . . . . . 31 Table Page 9 Reducing sugars, nitrate and total N levels as affected by time and ratio of extract incubation media in extracts from.TRIA (lOO ug/l) treated rice leaves . . . . . . . . . . . . . . . . . 32 10 Effect of pH on the increase in reducing sugars and total N in extracts from TRIA treated corn leaves. The total N and reducing sugar zero time values were 250 and 120 ug/ml, respectively . . 33 11 Effect of incubation temperature on reducing sugars and total N levels in extracts from TRIA (lOO ug/l) treated corn leaves. . . . . . . . . 34 12 Inhibition of increase in total N and reducing sugars by octacosanol in extracts from TRIA treated corn leaves . . . . . . . . . . . . . . 35 13 Inorganic phosphate, dry weight and water uptake in control and TRIA treated lZ-day-old rice seedlings after 24 h. . . . . . . . . . . . . . 38 vi LIST OF FIGURES Figure Page 1 Procedure for isolation of crude and super- natant fractions from rice or corn seedlings used for the study of the in vitro effects of TRIA . . . . . . . . . . . . . . . . . . . 13 2 Total N concentration with time in the super- natant fraction (4080's) from rice leaves treated with 0.1% Tween 20 or 0.1% Tween 20 + 100 ug/l TRIA. The F value is significant at the 0.1 level for the interaction of TRIA x linear time . . . . . . . . . . . . . . . . 23 3 Level of reducing sugars in supernatant frac— tion from rice leaves incubated at 35 C. * r value significant at .05 level for linear regression . 29 4 Starch phosphorylase activity from control and TRIA (100 ug/l treated corn leaves. ** r value significant at .01 level for linear regression . . . . . . . . . . . . . . 37 vii INTRODUCTION l-Triacontanol (CH3(CH2)28CH20H) (TRIA) is a 30-carbon straight chain primary alcohol with a molecular weight of 438.8 and a melting point of 88 C (Handbook of Chemistry and Physics, 57th edition, Chemical Rubber Publishing Company). TRIA is slightly soluble in ethanol, very soluble in ether 37 M as and the calculated solubility in water is 6.67 x 10- determined from the formula log M = -l.39 m + 5.53 where M equals molarity and m equals the number of carbon atoms in any primary alcohol. This formula is based on the solubility of n-aliphatic alcohols in water which was determined by surface tension measurements (23). The logarithm of the solubility is a linear function of hydrocarbon chain length. The effects of TRIA and some of the regulating para- meters have been well established on intact plants (3,8,24, 44,45). The most notable are increases in dry weight, soluble protein, water uptake and total reducible nitrogen (total N) along with an apparent regulatory role of COZin the atmosphere. Increases in free amino acids and total N have been measured within 40 to 80 minutes (8,24). Growth analysis, analytical studies of metabolism and some biochemical research failed to elucidate the mode or site of TRIA action. The ambiguous nature of these observations has prevented the development of a coordinated hypothesis. 2 The first effects of TRIA probably occur at a subcellular level and are manifested later in the ontogeny of the plant. These effects become self-limiting and may not be observed after they are delineated in whole plants. This hypothesis is supported by research with whole plants over short time periods. TRIA treated corn and rice plants had higher levels of soluble proteins, organic acids, amino acids and reducing sugars than control plants (8,3). Growth analysis of rice (Oryza sativa L.) seedlings treated with TRIA revealed an early increase in net assimilation rate (NAR) followed by a return to the control NAR (44). Studies conducted with rice leaf segments and whole plant suggest that carbohydrate pools within the plant are required for the TRIA response (14,3). The most substantial evidence for a biochemical effect at the molecular level is the rapid incorporation of deuterium oxide into the organic acid pool in TRIA treated rice plants (8). This study was conducted to develop and characterize an in vitro system responsive to TRIA with the hope of identi- fying a precedent biochemical effect. Through this approach it may be possible to define a mode of action or at least demonstrate a consistent in 32539 response similar to that observed in whole plants. LITERATURE REVIEW TRIA in Plant Cuticles. The plant cuticle contains a variety of wax components, the most common of which are ketones, diols, adehydes, secondary and primary alcohols (25). TRIA is an example of the latter category. TRIA was first isolated from the leaves of alfalfa (Medicago sativa L.) in 1933 (6). Since then TRIA has been found to be a constituent of the free alcohols in plant waxes in a number of species in quantities from 95% to less then 1% (26,54). The location of long-chain aliphatic plant cuticular compounds was at first believed to be only in the cuticle, but waxes and fatty acids of a similar nature have been found in chloroplasts and fat bodies within leaf cells (48). The function of the cuticle is the regulation of water loss, nutrient loss by leaching, and protection against insects and fungi by its physical nature (25). The growth regulating properties of the chemical constituents of the cuticle have received little attention. A growth promoting substance isolated from an alcoholic extract of tobacco (Nicotiana tabacum L.). 'Maryland Mamooth' leaves increased growth as measured by the oat internode bioassay (7). This same fraction demonstrated chemical properties characteristic of long-chain primary alcohols containing between 22 and 28 carbon atoms (56). A 3 4 chloroform extract from alfalfa meal was shown by Ries et al. (45) to increase the dry weight and water uptake of several plant species, and the biologically active component was identified as TRIA by mass spectrometry. Other long chain alcohols containing 8 to 11 carbon atoms inhibit or kill axillary and terminal bud growth in tobacco plants (5,50). Alcohols of longer carbon length (16-28) can selectively inhibit the increase in dry weight and water uptake ascribed to TRIA (19). Short chain alcohols ranging from methanol to butanol in concentrations less than 10-3 M exert a positive growth action on elongation of isolated wheat (Triticale hexaploide L.) roots (l3). Biological Activity 9f TRIA and other Lipids. There are several different classes of lipids, however, all can be characterized as water-insoluble organic mole- cules that can be extracted from cells and tissues by non- polar solvents. Some biological functions ascribed to lipids include components of membranes, sources of metabolic fuel, protective coating on the surface of organisms and hormonal activity. The latter function of lipids has been intensely studied and characterized in mammalian systems while research in plant systems leaves much to be uncovered about the pos— sible regulatory roles of lipids in plant growth and develop- ment. Fatty acid esters affect the response of excised pea (Pisum sativum L.) epicotyls to applied auxin (51). Methyl linoleate and methyl oleate in the pea bioassay apparently 5 increased the sensitivity of the pea sections to IAA, implicating a close relationship between auxin activity and lipid metabolism (51). Homologous series of alkyl chlorides, bromides and iodides enhanced auxin-induced pea stem elonga- tion at levels of 3 to 40 micromolar, the molecular length of these lipids was the determining factor for activity (52). In all the cases investigated using the pea stem elongation bioassay, lipids stimulated respiration in addition to augmenting cell elongation. Certain synthetic aliphatic hydroxycarboxylic acids have shown growth promoting activity on the root growth of lettuce (Lactuca sativa L.) seedlings (12) . Unsaturated fatty acids have a number of effects on isolated mitochondria and chloroplasts. Exogenous unsaturated fatty acids such as oleic or linolinic suppress the Hill reaction in isolated chloroplasts (35). This suppression was associated with a visable swelling of the chloroplast. Later investigation of the swelling suggested that the association of the fatty acids with the chloroplast membrane results in a decreased rigidity and polarity of the membrane (39,48). TRIA was shown in have growth regulating activity by Ries et a1. (45) in 1977. In experiments with several crop species, dry weight and water uptake were increased over con- trols by treatment with TRIA. Although the first studies were conducted over 7 to 8 days, later work indicated that the effect of TRIA occured within the first 24 h period after treatment (44). Increases in protein and leaf area were also observed but the most prodigious effect was a dry weight 6 increase within 6 h in the dark where controls typically lost dry weight. At first it was thought that the dry weight increase in the dark was due to TRIA stimulation of dark CO2 fixation. However, later studies by Bittenbender et al. (3) showed that there was no net dark CO2 fixation in response to TRIA treatment but C02 played a regulatory role rather than substrate in the TRIA response. Increase in respiration, soluble and insoluble Kjeldahl-N and soluble carbohydrates were also reported. It was postulated that this increase in dry weight was due to the metabolic incorporation of water through hydrolysis of some stored products within the plant. TRIA increased the growth of cell cultures of several crop species apparently by increasing the cell number (14). Tests with octacosanol [CH3(CH2)26CH20H] suggested that a specific chain length may be required for activity (45). This hypothesis was substantiated by Jones et al. (19), when the activity of several analogs of TRIA were tested for growth promoting effects on rice seedlings. Perhaps more important was the finding that all the analogs and other long chain hydrocarbons applied in combination with TRIA at equimolar concentrations inhibited the TRIA response in rice and tomato seedlings. Analysis of 2H incorporation with deuterium oxide into plant metabolites in control and TRIA treated rice seedlings using a stable isotope tracer technique and metabolic pro— filing, indicated rapid increases in the pools of succinate and some a-amino acids in TRIA treated plants (8). The re- sults insinuate that the effect of TRIA is related to the 7 control of the level of organic nitrogen available for pro- tein synthesis in the leaves. The carbon used to increase the a-amino acid pools in 10 min was believed to arise from glucose metabolism rather than from photosynthesis (8). To date only one enzyme has been shown to be affected by TRIA. Both dark and light-grown lettuce leaf tissue exhibited greater polyphenol oxidase activity than controls when treated with TRIA (16). In studies on seed germination and early growth in 15 species, TRIA had no effect on germin- ation or morphology (17). Plant age and concentration of TRIA have been shown to be important factors in obtaining a response (47). The growth of several vegetable and field crops was increased under greenhouse conditions by applications of TRIA to the foliage, soil, or seed (41). However, in the field only foliar sprays increased the yields of 7 out of 10 crOps tested. The most recent work on the TRIA response in rice seedlings has shown that TRIA treatment results in an apparent increase in Kjeldahl-N that can be observed in as little as 15 40 min (24). However, studies with N-enriched and depleted 15N. It plants revealed that no change occurred in atom % was concluded that the increase in total N was an artifact of Kjeldahl analysis unique to TRIA treated plants, TRIA there- fore must have initiated a gross change in the plants chemistry altering the Kjeldahl analysis (24). These conclusions were reached after the majority of the studies that follow had been completed. MATERIALS AND METHODS Plant Culture. Rice seed, 'IR-8', 'ESD 7—1' and 'Starbonnet', were surface sterilized with 0.1% (w/v) HgCl2 and germinated in 77 ml plastic cups containing vermilculite and turface (Wyandotte Chem. Co., Detroit, MI). The plants were grown as previously described (43). Field corn 'Pioneer 3780' was planted in 18 cm clay pots in a 1:1:1 (v/v/v) sterilized mix of peat, sand and sandy loam soil. Greenhouse conditions were maintained at 25 C night temperature. Seven days after sowing, the seed- lings received 250 m1 of a 20-20—20 soluble fertilizer at 1.0 g/l twice weekly. Chemical Treatment Applications. Pure synthetic TRIA was used for all treatments (American Cyanamide, Princeton, New Jersey). TRIA treatments were pre- pared from either a TRIA-Tween 20 (polyoxyethylene sorbitan monolaurate) or a TRIA-chloroform stock solution, 0.1 to 1.0 mg/ml or 1.0 mg/ml respectively. The stock solutions were diluted with double distilled deionized water to give a solu- of the desired concentration. Treatment solutions made up from the chloroform stock were placed on a warm stirring plate until the chloroform droplets had disappeared. Those solutions 8 9 made up from TRIA-Tween 20 stock contained 0.1% (w/v) Tween 20. All solutions used in controls were prepared exactly as the TRIA solutions minus the TRIA. The fourth and fifth leaves from 18 to 22 day old rice plants or the third and fourth leaves of 8 to 10 day old corn plants were excised and weighted. The leaves were immediately placed in an Erlenmyer flask containing 250 ml of treatment or control solution per g fresh weight of leaves and shaken for one min. After removal of the leaves from the treatment solution they were extracted. For the starch phosphorylase (EC 2.4.1.1) studies a similar procedure was used except corn leaves were cut into 1 cm segments after treatment and incubated in 20 mM potassium phosphate buffer (pH 7.0) for 20 min before extraction of the enzyme. Extraction Procedure. Treated rice leaves were extracted at 4 C with cold 20 mM potassium phosphate buffer (pH 7) containing 1.0 mM B-mer- captoethanol, in the ratio of 7 ml buffer/g fresh weight. Corn leaves were extracted with the same buffer and condi- tions except 4 ml/g fresh weight was used. Rice leaves were homogenized in a cold room with an automatic mortar and pestle (Torsion Blaance Co., Clifton, New Jersey). Corn leaves were homogenized by hand with a cold mortar and pestle held on ice. The crude extract was used for the initial in yiggg test or centrifuged at 4080 g at 4 C for 20 minutes to obtain the supernatant fraction. The supernatant suspension was poured 10 into a cold beaker held on ice and stirred. Aliquots were pipetted from this solution. Starch phosphorylase was extracted from corn leaves with cold 10 mM maleate-KOH buffer (pH 6, 4 ml/g fresh weight) by grinding in a cold mortar and pestle (4 ml/g fresh weight). The extract was filtered through four layers of cheesecloth and centrifuged at 20,000 g for 20 min. The supernatant fluid was then used as the enzyme source. Incubation Parameters. The crude extract or the supernatant fraction was added to cold incubation media in a ratio of 1:4 (v/v) in 50 ml or 125 ml Erlenmyer flasks on ice. Incubation media was made up fresh the day of the experiment with the same buffer used for extraction and contained the followed components: NADPH 1.3 mM, NADH 0.13 mM, ATP 0.18 mM, MgC12° 6 H O 0.28 mM, 2 oxaloacetic acid 0.13 mM, OEketoglutarate 0.12 mM. All of the above reagents except magnesium chloride hexahydrate were obtained from Sigma Chemical Co., St. Louis, Missouri, the MgC12-6 H20 was analytical grade. Initial ig.yi££g experiments with the crude extracts were carried out with a reciprocating shaker that was placed in a growth chamber and held at 25 C for the duration of the experiment. Light conditions were the same as that used for the growth of the rice seedlings. Incubation of the super- natant fraction was conducted in a reciprocating water bath shaker held at 25 C on a lab bench. 11 After addition of the extract to the cold incubation media, samples were removed for zero time analysis and the remainder placed on the shaker. Additional samples were removed at the designated time for analysis. A simplified schematic is shown (Figure l) depicting the extraction and incubation parameters used. Starch phosphorylase activity was determined by measur- ing the rate of Pi production. The reaction mixture consisted of 10 mM maleate-KOH buffer, 0.3% soluble starch (w/v) (Difco Laboratories, Detroit, MI), 50 mM glucose-l—P (Sigma) and enzyme in a total volume of 2 ml. The reaction mixture without enzyme was placed in a water bath shaker held at 37 C and after addition of the enzyme the Pi liberated was measured over 10 to 20 min intervals by the method of Taussky and Shorr (53). Sampligg and Analysis Methods. Three ml samples were used for all total N determina- tions and 1 ml samples for all other analyses. The samples for total N were pipetted into 50 to 125 ml Erlenmyer flasks and frozen with a mixture of dry ice and acetone. After freezing the samples were either lyophylized, dried at 65 C in a forced air over or digested immediately with the diges— tion mixture used for Kjeldahl digestion. All total N was determined by an automated micro-Kjeldahl procedure (10). Samples for total N determination were digested with a solution containing 1800 ml concentrated H2804, 40 ml 70% HC104, 160 ml distilled water and 6 g Se02. Four mls of the 12 Figure 1. Procedure for isolation of crude and supernatant fractions from rice or corn seedlings used for the study of the in vitro effects of TRIA. l3 LEAVES FROM RICE OR CORN SEEDLINGS 9 7 TREAT FOR 1 MIN WITH 2.28 x 10’ M TRIA GRIND AT 4 C FOR 20 MIN IN 20 mM POTASSIUM PHOSPHATE BUFFER (pH 7.0) PLUS 1 mM B-MERCAPTOETHANOL CRUDE FRACTION CENTRIFUGE AT 4080 g FOR 20 MIN AT 4 C I \ DISCARD PELLET SUPERNATANT I ADD TO INCUBATION MEDIA AT A RATIO OF 1:4 Tliy REMOVE ZERO TIME SAMPLE INCUBATE AT 25 C AND FREEZE IMMEDIATELY, FOR VARIOUS INTERVALS FOLLOWED BY APPROPRIATE OF TIME ANALYSIS REMOVE SAMPLE, FREEZE, FOLLOW BY APPROPRIATE ANALYSIS l4 digestion mixture were added to each dried 3 ml sample from the in vitro experiments or directly added to 3 ml liquid samples. The samples were heated until the solution cleared and cooled, followed by addition of 4 or 1 ml distilled water for dried or liquid samples respectively. The samples were then placed on an Auto-Analyzer (Technicon Instruments Corporation, Tarrytown, NY) for sampling and automated total N analysis by reacting the NH4+ ions in the acid mixture with alkaline phenol and NaOCl. The reaction generates a blue\color with maximum absorbance at 632 nm. Standards were prepared from ground wheat (2.566% N) and all data cal- culated from a regression line computed from the wheat standards. The percent nitrogen in the ground wheat was calibrated using the nitrogen standard for plant material from the National Bureau of Standards, Washington, D. C. (standard reference material 1571). One ml samples from which nitrate, reducing sugars, soluble carbohydrates, soluble protein and free amino acids were quantified were removed from the incubating extracts at various times, placed in l x 10 cm test tubes and quick frozen. The samples were then held at -70 C for not more than 48 h until the appropriate analysis could be performed. All spectrophotometric determinations were carried out using tri- plicate samples from the thawed extracts. For nitrate analysis 20 ul samples were removed from the rice extracts or 10 ul samples from corn extracts and added to 0.5 ml of 0.1 M K-succinate buffer at pH 6.8 in l x 10 cm test tubes. The nitrate was then determined by the method of 15 Lowe and Hamilton (30). The concentration of nitrate in the sample was calculated from a linear regression line generated from a set of standards prepared from a stock KNO3 solution (12.4 ug/ml). Samples of 50 ul each for corn and rice extracts were used for determination of reducing sugars. The samples were placed in 2 x 15 cm test tubes containing 1 ml deionized distilled water and the reducing sugars determined by the method of Nelson and Somogyi (38,49). Standards were prepared from an 0c-D(+) glucose solution (1 mg/ml) and all data cal- culated from a linear regression line on the standards. Free amino acids were determined by a modified ninhydrin colorimetric procedure developed by Rosen (46), using 50 ul samples from both corn and rice extracts. All data was cal- culated from a linear regression line using leucine as a standard (1 umole/ml). Total soluble carbohydrates analysis was carried out on 50 ul samples from the rice and corn extracts by the phenol- sulfuric acid method as described by Hodge and Horfreike (l8) and all data obtained from a linear regression line on a set of standards prepared from a 0C-D(+) glucose solution (1 mg/ml). Soluble protein determinations were carried out on 10 ul samples from both corn and rice extracts using a modification of Lowry (31). The modification is as described by Bensadoun and Weinstein (1) and effectively removes many of the sub- stances present in plant extracts that interfere with the nor- mal Lowry procedure. Bovine serum albumin was used as the 16 standard (1 mg/ml) and all data calculated from a linear regression line on the standards. For starch phosphorylase activity of the Pi production in the reaction mix was measured by removing 100 pl samples over 10 or 20 min intervals and precipitating the protein with 4 ml of 10% (w/v) trichloracetic acid. The mixture was well agitated and then centrifuged at 1000 g for 30 min. The supernatant fluid was removed and 3 ml used for the determination of Pi° Standards were prepared from a stock potassium phosphate solution (1.0 mg/ml) and all data cal- culated from a linear regression line on the standards and blanks consisted of samples from the reaction mix minus enzyme. Blanks were also prepared from reaction mix minus soluble starch so that any increase in Pi due to phosphatase activity could be subtracted from the starch phosphorylase activity data. Protein levels in the extracts used for this assay were determined by the same modification of Lowry described earlier. Statistical Analysis. Several designs were used. Most often a randomized com- plete block was used blocking for position in the incubator. Where it was appropriate trend analysis was conducted. All data was subjected to analysis of variance and the means com- pared by use of the least significant difference except where there was only one degree of freedom for the treatment. In these instances the F value from the analysis of variance was used for comparison of means. Starch phosphorylase data was l7 analyzed by linear regression. There was very little varia- tion in these tests indicated by range of coefficients of variations of 0.5% to 2.0% for all of the experiments. Error due to pipetting could account for only 2.6 pg nitrogen/ml while the detection limit of the automated micro Kjeldahl is approximately 4 ug nitrogen. RESULTS AND DISCUSSION Total N Response Studies. One of the most rapid and significant changes in TRIA treated plants is the increase in total N (8,24)° This response can be detected within 40 min (8, unpublished data, Ries). The results of a preliminary test with a crude extract from TRIA treated rice leaves revealed that a similar total N response could be observed in vitro (Table 1, Test 1). The total N in the TRIA treatment increased from zero time to 320 min. There was no change in total N in controls treated with a solution of 0.1% Tween 20. In a second test the extract from TRIA treated leaves again demonstrated an increase in total N over an 320 min interval, but the ex- tract from leaves treated with 0.1% Tween 20 showed no change in total N over time (Tafile 1, Test 2). Method pf Treatment Experiments were conducted to determine if TRIA could be added directly to the extracts or through the extraction buffer during extraction and the same response still be observed. Addition of the extract to incubation media con— taining all components plus 1 mg/l TRIA had no significant effect on the total N content in the extract. In this 18 19 Table 1. Total N levels in crude extracts from TRIA-treated rice leaves over time. Each treatment in each test contained 4 ml crude extract and 8 ml incuba- tion media in 125 Erlenmeyer flask, after the appropriate times the extracts were quick frozen and dried at 65 C. After drying the entire residue was digested for total N determination. Each mean is the average of 10 replicates. Treatments Test 1 Test 2 Time TRIA Nitrogen (min) (100 ug/l) (mg/flask) 0 0 2.09 0 + 1.87 1.97 20 0 1.98 20 + 1.99 2.08 80 0 1.98 80 + 2.05 2.10 320 0 1.96 320 + 2.05 2.15 L.S.D. at .01 level 0.15 0.12 20 experiment with 6 replicates the average zero time and 80 min total N values in the extract were 836 and 832 ug N/ system i 2.88, respectively. Addition of TRIA (1 mg/l) via the extraction buffer also was ineffective in eliciting an increase in total N. The average zero time and 80 min total N value from 8 replicates in this experiment were 754 and 755 pg N/system i 2.55 respectively. These experiments suggest that the integrity of the plant tissue must be maintained during treatment with TRIA for an ip yiggp effect to be observed, but once TRIA has reached its active site the initiation of the response is achieved and disruption of the tissue from this point does not result in a loss of activity. Centrifugation Experiments. Centrifugation of the crude extract at 4080 g doubled the observed difference in total N between the zero time and 80 min treatments (Table 2). The augmentative effect of centrifuging the extract could have been due to the removal of impurities or inhibitors of the factor(s) responsible for the total N increase. The response in this supernatant fraction was found to be linear over time up to 80 min, a control supernatant suspension indicated no change in total N as had been observed earlier with the crude extracts (Figure 2). A similar experiment with the supernatant fraction from leaves of 9 day-old corn plants revealed a trend similar to 21 Table 2. Response of crude and supernatant fractions from rice leaves treated with TRIA (100 ug/l). Each mean is the average of 8 replicates. . Time Total N Extract Fraction (min) (mg/ml) Crude 0 .318 80 .333* Supernatant (4080 x g's) 0 .212 80 .245** *,** F value for comparison with zero time significant at .05 and .01 levels, respectively. that observed in the rice extracts (Table 3). In this experi- ment nitrate nitrogen was also determined and the possibility that the ip vitro reduction of nitrate by nitrate reductase was responsible for the total N increase was rejected. Lat- ter experiments with rice extracts will support the same con- clusion for rice. This data agrees with studies conducted on the effect of TRIA on the enzymatic reduction of nitrate nitrogen (24). Dry weights of the supernatant were obtained at the beginning of the experiment and all subsequent total N and nitrate data expressed per g dry weight. Further investigation of the effect of centrifugation on the total N response in rice and corn extracts from TRIA treated leaves revealed no increase in the magnitude of the response with increasing centrifugation force (Table 4). The increased response to TRIA from centrifugation at 4080 g with no enhanced activity from further centrifugation Figure 2. 22 Total N concentration with time in the supernatant fraction (4080 g's) from rice leaves treated with 0.1% Tween 20 or 0.1% Tween 20 + 100 ug/l TRIA. The F value is significant at the .01 level for the interaction of TRIA x linear time. Each mean is the average of 10 replicates. TOTAL N(Ug/ml) 216" 212-1 208‘ 204‘ 23 ZOO 196‘ 4O TlME(min) 80 24 suggest that the site of TRIA action is in the soluble fraction of the plant cell. The insoluble portion at most Table 3. Total N and nitrate levels in the supernatant fraction from corn leaves. Each mean is the average of 6 replicates Treatments Nitrogen (mg/g dry weight) TRIA Time Total ' (100 ug/l) (min) (Kjeldahl) Nitrate + 0 74.70 3.7 + 30 81.45** 3.8 + 60 81.80** 3.7 + 120 82.93** 3.7 + 1380 75.93 3.8 ** F Value for comparison with zero time significant at .01 level. Table 4. Effect of centrifugation force on total N increase in extracts from TRIA treated (100 ug/l) leaves. Each mean is the average of 9 replicates. Total N (pg/m1) Time Centrifugation force (min) (xg) Rice Corn 0 328 297 60 4000 334** 305** 0 305 286 60 5000 3II** 294** 0 289 277 60 16000 296 286** 25 Table 4 (cont'd) ** F value for comparison with zero times significant at .01 level. ' plays a minor role in the TRIA response IE.Vitr0° Incubation Media Studies Experiments were conducted in order to test the neces- sity of the incubation media for the TRIA response. After centrifugation of the crude extract, at 4080 g's the super- natant solution was added to buffer at a ratio of 1:2, zero time samples removed and the extract incubated at 25 C. After 60 min, 3 ml samples were removed and the total N determined as described under Materials and Methods. Removal of the components of the incubation media from the extract eliminated the increase in total N for corn and rice leaves (Table 5). Further investigation into the role of the Table 5. Effect of incubation media on total N increase in extracts from TRIA (100 ug/l) treated leaves. The total N values for + incubation media have been cor- rected for the nitrogen in the incubation media. + and 0 incubation media were separate experiments. Each mean is the average of 10 replicates Total N (Hg/ml) Time (min) Incubation Media Rice Corn 0 + 205 274 60 + 220** zgzaa 0 0 220 276 60 0 220 279 26 Table 5 (cont'd) ** F value for comparison with zero time significant at .01 level. components of the incubation media was conducted with the supernatant from rice leaves. The incubation media compo- nents were removed separately in groups of similar chemical and metabolic nature. Treatments were included where none or all of the components were removed. Removal of any one group eliminated in the response (Table 6). In some tests the removal of d-ketoglutaric acid and oxaloacetic acid had no effect on the total N increase, so the necessity of the two carbon skeletons still remains obscure even though on the average they are apparently necessary. Apparently for TRIA to elicit a total N increase ip vitro reducing power and biological energy are required in the form of reduced pyri- dine nucleotides and adenosine 5'-triphosphate. Effect pf TRIA pp Reducing Sugars ip vitro. Since several earlier studies on the effect of TRIA in whole plants implicated the involvement of carbohydrate metabolism in the TRIA response (3,8,14), experiments were conducted to determine if a similar response was occurring in vitro. When extracts from TRIA and control treated rice leaves were incubated at 35 C, the level of reducing sugars measured at 0, 10, 20 and 40 min increased linearly with both control and TRIA treatments (Figure 3). However, the rate of increase in reducing sugars with TRIA treatment was almost twice as large as the rate with control treatments. These 27 Table 6. Effect of components of incubation media on the total N increase from TRIA (100 ug/l) treated rice leaves. Each mean is the average of 10 replicates. Time Total N Component removed (min) (pg/ml) None 0 278 60 284** NADH, NADPH O 280 60 274 ATP, MgCl2 0 281 60 277 0AA, CcKETO 0 281 60 284 ALL 0 300 60 301 ** F value for comparison with zero time significant at .01 level. 28 Figure 3. Level of reducing sugars in supernatant fraction from rice leaves incubated at 35 C. Each mean is the average of 4 replicates. A r value significant at .05 level for linear regression REDUCING SUGARS(ug/ml) 160‘ 150* 140‘ 130‘ 120- 110i 29 ' Qeae ’/ I - CONTROL(SIOpe = .67) o ~TRIA