HIHHIWEIN UL. ill WWW Ntms 3 1293 010882 This is to certify that the thesis entitled APPARENT INCREASES IN TOTAL PLANT NITROGEN FOLLOWING APPLICATIONS OF TRIACONTANOL presented by Norman Richard Knowles has been accepted towards fulfillment of the requirements for M . S . . Horticulture degree 1n 3%?) K Q2; 1 Major professor V/ZK/f/c' Date 0-7 639 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circalatton records 'eW . _ .-,.Q :w— - 2'1. >24”3333Rflfl;:' #53 W m “'~ " -32"! 1 - u r - 3‘ L£5$$3flwfl : ”a“ $183359? APPARENT INCREASES IN TOTAL PLANT NITROGEN FOLLOWING APPLICATIONS OF TRIACONTANOL by Norman Richard Knowles A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1980 x...) 1. 39 ,. c b .5 \ ABSTRACT APPARENT INCREASES IN TOTAL PLANT NITROGEN FOLLOWING APPLICATIONS OF TRIACONTANOL by Norman Richard Knowles Triacontanol stimulates an increase in growth and apparent Kjeldahl nitrogen (N) of rice seedlings (erza sativa L.) and N in supernatants of rice and corn (Zea maxs L.) within 40 minutes. The in_vitro N response is dependent upon atmospheric N which does not serve as sub- lsN2 enrichment experiments established that the apparent N increase in rice seedlings strate for the increase. Atmospheric substitution and was independent of atmospheric N. TRIA increased the soluble N pools of the plant, specifically the free amino acid and soluble protein fractions. No differences in depletion or enrichment of 15N incorporated into soluble and insoluble N fractions of rice could be detected. The apparent total N increases, therefore, appear to be an artifact of Kjeldahl analysis resulting from a TRIA stimulated change in the chemical composition of the seedlings. ACKNOWLEDGMENTS I would sincerely like to thank my major professor, Dr. Stan Ries, for presenting me with the opportunity of becoming a scientist and investing his time, money and confidence into my education. I would also like to thank Violet Wert who taught me practical laboratory techniques and gave me confidence throughout my research. I would like to thank Dr. P. Filner and Dr. Tiedje for the use of their mass spectrometers and the many helpful suggestions which they provided. In addition, I would like to thank Dr. D. R. Dilley for his suggestions and guidance throughout my research. I would finally like to thank Bob Houtz for the confidence and support, as well as constructive criticism, which he offered throughout my MS program, and my parents who made it all possible. ii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . iv LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . vi INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . 3 Genetic Selection . . . . . . .'. . . . . . . . . . . . . . . 3 Growth Regulation by Chemicals . . . . . . . . . . . . . . . 4 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . 8 Preparation of Plant Materials . . . . . . . . . . . . . . . 8 Preparation of Treatment Solutions . . . . . . . . . . . . . 9 Nitrate and Nitrate Reductase Analysis . . . . . . . . . . . 9 Growth Response Studies: Plant Analysis . . . . . . . . . . 10 Growth Response Studies: System Analysis . . . . . . . . . . 11 In Vitro Methods . . . . . . . . . . . . . . . . . . . . . . ll Afialysis of Total Nitrogen . . . . . . . . . . . . . . . . . 12 Atmospheric Substitution Procedures . . . . . . . . . . . . . 14 Generation of 15N2 . . . . . . . . . . . . . . . . . . . . . 15 Atmosphere Enrichment . . . . . . . . . . . . . . . . . . . . 18 Preparation of Samples for MS Analysis . . . . . . . . . . . 19 Depletion and Distribution Analysis . . . . . . . . . . . . . 20 Fractionation Procedures . . . . . . . . . . . . . . . . . . 21 Statistical Procedures . . . . . . . . . . . . . . . . . . . 22 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 24 Nitrate Reductase Experiments . . . . . . . . . . . . . . . . 24 Growth Response Studies: Plant Analysis . . . . . . . . . . 25 Growth Response Studies: System Analysis . . . . . . . . . . 26 Atmospheric Substitution . . . . . . . . . . . . . . . . . . 28 Atmosphere Enrichment . . . . . . . . . . . . . . . . . . . . 31 Depletion and Distribution Studies . . . . . . . . . . . . . 35 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . 4S LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . 46 iii TABLE 10. 11. 12. 13. LIST OF TABLES Effect of increasing concentration of TRIA on nitrite accumulation from corn leaves measured at various times after treatment with the ig_vitro assay . . . . . Effect of TRIA on NR activity in corn leaves measured 3 h after treatment with the in_vitro assay . . . . Growth response and total N content of TRIA treated rice seedlings . . . . . . . . . . . . . . . . . . . . Total N content of TRIA treated rice seedlings . . . . . Growth response and total N content of rice seedlings exposed to TRIA or TRIA + Tetracosanol . . . . . . . . . Total N content of the supernatant from TRIA (lOOO pg/L) treated corn leaves assayed as a function of time . . . Effect of atmospheric substitution on the total N content of the supernatant from TRIA (100 pg/L) treated rice leaves . . . . . . . . . . . . . . . . . Influence of an argon substituted atmosphere on the total N content of the supernatant from TRIA (100 pg/L) treated rice leaves . . . . . . . . . . . . . . . Acetylene test for the presence of nitrogenase in the supernatant of TRIA (100 ug/L) treated rice leaves . . . A comparison of methods for 15N2 atmosphere enrichment of TRIA treated rice seedlings . . . . . . . . . . . . . Dry weight change of TRIA treated rice seedlings in an 8.5% 15N2 enriched atmosphere . . . . . . . . . . . . Total N and 15N content of TRIA treated rice seedlings exposed to an 8.5% 15N2 enriched atmosphere . . . . . Growth response, total N and 15N content of TRIA treated rice seedlings exposed to an 8.5% 15N2 enriched atmosphere . . . . . . . . . . . . . . . . . . . . . . . iv Page 24 25 26 27 27 29 29 30 31 32 32 33 33 TABLE 14. Total N and 15N content of the supernatant from TRIA (1000 g/L) treated corn leaves exposed to a 13% 15N2 enrichment atmosphere . . . . . . . . . . . . . . . . . 15. Growth response and total N content of TRIA treated rice shoots in a normal and argon substituted atmosphere 0 O O O O O O O O O O O O O I O O O O O O O O 16. Growth response, total N and 15N content of TRIA treated rice seedlings grown on KlsNO3 (5.3 atom %) for 4 days prior to treatment . . . . . . . . . . . . . 17. Total N, 15N and reducing sugar content of the super- natant from TRIA (1000 pg/L) treated, corn leaves incubated in media containing KISNO3 (5.3 atom °o) for 2 h . . . . . . . . . . . . . . . . . . . . . . . . . 18. Growth response, N and water insoluble 15N content of rice seedlings cultured on KISNO3 (5.3 atom °o) for 20 h prior to treatment with TRIA . . . . . . . . . . . 19. Total N and1 5N in the water soluble and insoluble fractions of rice seedlings grown on KISNO3 (5.3 atom °o) for 20 h prior to treatment with TRIA . . . . . . . 20. Distribution of N and 15N in the water soluble and in- soluble fractions of rice seedlings enriched with 15 K NO3 treatment with TRIA . . . . . . . . . . . . . . . in the nutrient solution for 20 h prior to Page . 34 36 . 37 38 . 41 . 42 . 44 LIST OF FIGURES FIGURE Page 1. In_vitro methods for assaying the N content of TRIA treated corn extracts over time . . . . . . . . . . . . . . 13 2. A N gas generating apparatus as modified from . . . . l7 Rittenburg (37) . . . . . . . . . . . vi INTRODUCTION Evidence of the growth-promoting effects of long Chain alcohols can be traced back to 1959 when Crosby and Vlitos (6) demonstrated growth enhancement in Avgna coleoptiles. Recent work with triacontanol (TRIA), a naturally occurring, 30-carbon, aliphatic, primary alcohol [CH3(CH2)28CH20H], has shown that it stimulates an apparent total N increase when applied to rice and corn at concentrations ranging from 1 to 1000 ug/L (36). Total Kjeldahl nitrogen (N) increases have been found to be linear with time, over short time intervals, and can be measured within 10 min in corn seedlings and 40 min in rice seedlings (Ries, unpublished data). Hangarter et al., (17) demonstrated that TRIA significantly increases the protein content of haploid tabacco cell cultures. The response was dependent upon light and involved an increase in cell number indicating a stimulatory effect on the rate of protein synthesis. This study was initiated in an attempt to reveal the source, fate and distribution of the reduced N responsible for the rapid increase in Kjeldahl N in_vivg, in TRIA treated rice seedlings, and in_vitrg_in corn extracts. The source of the N was investigated by nitrate analysis, nitrate reductase assays and atmospheric substitution experiments. Mass spectrometric techniques, utilizing an isotope ratio mass spectrometer, have been employed to detect the movement of heavy N (ISN) into, as 2 well as within, the plant. Relative levels of 15N in soluble and in- soluble N pools of treated and control plants were compared. LITERATURE REVIEW Current increases in energy costs, coupled with an ever expanding world population, necessitates more efficient production of important food crops. The expenses involved in production and application of N fertilizers remain the single most critical input for obtaining higher crop yields (12). These costs can be kept minimal and yields could be be increased per unit area to help meet present demands by increasing the efficiency of N utilization by plants. This discussion briefly summarizes two approaches to achieving these goals: genetic selection and growth regulation by chemicals. The pertinent literature on TRIA as a possible stimulator of N metabolism in crops is also reviewed. Genetic Selection Genetic enhancement of N acquisition and assimilation by plants can be achieved through breeding for high protein content and increased nitrate uptake and reduction. In rice, nitrate absorbed by the roots is translocated to the leaves where it is reduced to ammonia and in- corporated intocxketo acids by reductive amination (30). Similarly, ”'high levels of nitrate reductase (NR) have been reported for the leaf tissue of corn indicating that this is the primary location of nitrate assimilation (41). Attempts have been.made to utilize levels of the enzyme NR as a criterion for genetically selecting high grain protein varieties. Early 3 4 studies on corn revealed direct relationships between soluble leaf pro- tein and NR activity. However, grain yields showed no such relation (15,45). In wheat, positive correlations between NR activity and grain N are only evident when sufficient supplies of nitrate are available to the plant (7,11). Deckard et al., (8) explained that since nitrate up- take shows diurnal fluctuations, high NR genotypes would not be expressed under low nitrate regimes, due to the fact that their outputs of mid-day reduced N would be significantly less than indicated in enzyme assays. Recent work has revealed that genotypes differing in nitrate ab- sorption, nitrate content and NR activity do not always display varying protein levels (22). Other physiological parameters such as amounts of protein reserves and rates of translocation of N from vegetation to the maturing grain play important roles in determining final protein content and must be considered in screening for high protein genotypes (22). Growth Regulation by Chemicals Stimulation of N utilization by chemical means has been an area of relatively intense research the past two decades. Wray et al., (43) proposed that the efficiency of protein accumulation could be enhanced by hastening synthesis or slowing natural breakdown processes. The use of growth-regulating chemicals to accomplish this might result in more efficient N utilization, higher protein content and possibly improved protein quality. Ultimately, an increase in grain protein would be achieved, perhaps resulting in enhancement of the nutritional status of populations dependent upon cereals as major protein sources (22). In environments where nitrate is not limiting, the rate of assimi- lation of N into reduced organic forms is largely dependent on the 5 activities of nitrate and nitrite reductase. Nitrate reductase, a substrate inducible enzyme, has been found to be stimulated by sub- lethal doses of several herbicides. Ries et al., (34) reported that simazine, a triazine herbicide, significantly accelerated the rate of protein accumulation in rye and peas. It was confirmed that, at least in rye and oats, simazine exerted its effect by stimulating NR activity and the increased protein which resulted contained no new protein types. Enhanced NR activity in maize and cucumber (Cucumis sativus L.) following treatment with 2,4-dichloro-phenoxyacetic acid (2,4-D) has been reported by Beevers et al., (1). Sub-lethal doses of 2,4-D have been found to significantly increase yields of sugar beet (Beta vulgaris L.), potatoes (Solanum tuberosum L.) and other crops in field trials (42). A summary of the published data indicates that 2,4-D influences the distribution of plant protein while increasing the over- all total N content, possibly through enhanced NR activity (29). Total N content of plants can also be increased by non-herbicidal compounds. In 1975, it was shown by Ries et al., (35) that coarsely ground alfalfa hay (Medicago sativa L.) significantly increased the yield and total N content of several commercially important vegetable species. Realization of the fact that the observed increases with alfalfa were greater than those achieved with equivalent amounts of fertilizer led to an extensive search for the responsible compound(s). Isolation of the growth-promotor in crystalline form was achieved from a chloroform extract of alfalfa meal. It was identified as l-triacontanol via mass spectrometry (33). TRIA was first identified in alfalfa by Chibnall (5) and was later shown to be a constituent of the cuticular waxes of many plant species 6 (26). The mechanism by which this compound influences plant growth remains obscure, however, the magnitude of the levels needed to produce a response (1 to 1000 pg/L) suggest a hormone-like activity. Jones et al., (24) demonstrated that a chain length of 30 carbons coupled with a terminal hydroxyl group was specific for TRIA's growth- enhancing activity. Tests of the growth-promoting activity of TRIA analogs varying in chain length from 16 to 32 carbons proved negative. In fact, these compounds resulted in inhibition of the TRIA response when applied simultaneously (24). Gross (14) has shown that several aliphatic alcohols with chain lengths from 9 to 11 carbons significantly inhibit bud growth. Recent tests on 15 weed, crop and horticultural species have shown no effect of TRIA on germination and early growth, however, an inhibi- tion of axis elongation was apparent in three of the test species (20). TRIA was shown to enhance growth of Great Lakes lettuce (Lactuca sativa L.) roots in both light and dark grown seedlings (19). Enhanced poly- phenol oxidase activity following TRIA application has been demonstrated in lettuce leaf tissue (19). Both TRIA isolated from alfalfa, and synthetic TRIA (Analabs, North Haven, Conn.), will stimulate increases in dry weight, leaf area and N content when applied to rice and corn at concentrations ranging from 1 to 1000 ug/L (36). In rice, the response proved to be independent of light conditions and CO2 concentration appeared to play a regulatory rather than a substrate role (3). This was supported by the fact that TRIA induced dark responses could be eliminated by removing atmospheric CO2 (36). Six h dark responses were characterized by increases in dry weight, soluble and insoluble Kjeldahl N and soluble carbohydrates. 7 Metabolic profiling results, utilizing rice grown in nutrient media containing deuterium oxide and harvested at intervals after treat- ment, showed that TRIA somehow increases the incorporation of carbon into most tricarboxylic acid intermediates (9). Both carbon and N incorporation into the a-amino acids were also found to increase over control plants within 10 min. It has been hypothesized that TRIA either increases the mobilization of N from the roots or somehow in- creases amino acid pools making them available for increased protein synthesis (9). MATERIALS AND METHODS Preparation of Plant Materials Rice seed, 'IR-8' or 'ESD 7-1' (Calif.), was sUrface sterilized with a 0.1% (w/v) solution of mercuric chloride. The seeds were planted in 77 ml plastic cups containing turface (Wyandatte Chemical Company, Detroit, MI) and watered with distilled water to the point of saturation. Growth conditions were maintained on a 16 h photOperiod at 30°C and an 8 h night at 25°C with 7.0 uW/cm2 in the phytosynthetically active region as measured from the top of the canopy. At the 7 to ll-day-old stage the seedlings were transplanted into 220 ml plastic cups containing 180 ml of quarter-strength Hoagland's solution (pH 4.5) having 3 mM nitrate N (21). Four seedlings were suspended in the solution by a foam rubber disc in the top of the cup. The cups were wrapped in alumi- num foil to exclude light. Nutrient solutions were renewed every 2 to 3 days thereafter with half-strength Hoagland's containing 6 mM nitrate N. Field corn, 'Pioneer 3780', was sown in 18 cm clay pots (8 seeds/ pot) containing a sterilized soil mix of equal volumes peat, sand and sandy loam soils. The pots were placed in the greenhouse with the night temperature maintained at 25°C and a day temperature averaging 30°C. At the 7-day-old stage a fertilization program was initiated with the seed- lings receiving a soluble 20-20-20 fertilizer twice a week at a concen- tration of 1 g/L. The N in the fertilizer was composed of 5.61% nitrate, 3.96% ammonia and 10.43% urea. All plant materials received fertilizer 8 9 or fresh nutrient solution the night before an experiment took place. Preparation of Treatment Solutions Treatment solutions for the NR experiments were prepared from stocks of pure TRIA (American Cyanamid, Princeton, NJ) dissolved in chloroform and added directly to the infiltration media (see below). Solutions for all other experiments were prepared from stocks of 0.1 to 1.0 mg/g TRIA-Tween 20 (polyoxyethylene sorbitan monolaurate) emulsions. The amount of stock added to glass distilled water was ad- justed to achieve a final concentration of 0.1% (w/v) Tween 20 and 100 to 1000 ug/L TRIA. Nitrate and Nitrate Reductase Analysis The NR activity of corn leaf tissue was measured by both an in_vivg (l6) and an ig_!i£rg_(39) colorimetric assay. The methods of Lowe and Hamilton (28) were employed to analyze free nitrate in rice tissues. For the in_!i!g experiments, freshly harvested corn leaves were cut into segments (2 to 5 mm) or discs (No. 3 cork borer) and placed into 50 ml Erlenmeyer flasks containing cold (3°C) infiltration media and TRIA (10 to 100 pg/L). The infiltration media consisted of 300 mM KNO and 1 mM potassium phosphate (pH 7.5). The flasks were stoppered 3 and repeatedly evacuated (3S mm-Hg for 30 5) until the segments were visibly wetted. Aliquots of 0.1 or 0.2 ml were removed at timed inter- vals for determination of nitrite. In all tests, variation due to dif- ferences in fresh weight and positions on the leaf from which the seg- ments were taken, was accounted for using a randomized complete block design. 10 For the in 31:32 experiment, corn was germinated in 18 cm clay pots containing soil in the greenhouse and transferred to pots containing vermiculite at the 3-day-old stage. Two days later, the seedlings were suspended in 220 ml plastic cups containing 180 ml of full-strength Hoagland's solution with a double concentration of minor elements. The cups were placed in a growth chamber with continuous aeration under con- ditions previously described. The seedlings were treated at the 7-day- old stage by dipping in a 1000 pg/L TRIA, 0.1% (w/v) Tween 20 solution. Nitrate reductase activity was measured 3 h after treatment with the reduced NADH methods of Sanderson and Cocking (39). Growth Response Studies: Plant Analysis Rice seedlings were sorted and blocked for size. Six blocks were utilized in a randomized complete block design with three randomly assigned treatments. The treatments consisted of a zero—time harvest, i.e. seedlings were harvested at the outset of the experiment, a control in which the seedlings were inverted and dipped in 100 ml of a 0.1% (w/v) Tween 20 solution and a TRIA treatment in which the seedlings were dipped in a similar solution containing 100 ug/L TRIA. The treat- ment solutions were changed after each replicate and the roots of each seedlings were rinsed three times in distilled water before being placed in 25 mm x 200 mm, open test tubes (two seedlings/tube). The tubes contained 10 ml of half-strength Hoagland's (pH 4.5) with 1 mM N as (NH4)2SO4. Plants were placed in a growth chamber at 30°C for 40 min after which they were harvested by separating roots from shoots, weighing the shoots, and immediately digesting the shoots for automated Kjeldahl analysis (13). Fresh weight and N content were analyzed. 11 Growth Response Studies: System Analysis The system analysis experiments entailed a simple procedure in which the entire plant culture system, (nutrient solution and seedling), was digested for automated Kjeldahl analysis. Individual seedlings were cultured, sorted, treated and placed in a growth chamber for 40 min as described previously. Harvesting involved removing the plants from the growth chamber and freezing them in a dry-ice and acetone bath. The nutrient solutions, containing 1 mM N as (NH4)2504, were frozen in a similar manner and lyophilized along with the seedlings. The freeze- dried plants were weighed and combined with the nutrient solutions in which they grew for estimation of total N. Treatment solutions were prepared by dissOlving TRIA or tetracosanol plus TRIA in chloroform (10 mg/ml) and adding determined amounts to distilled water containing 0.1% (w/v) Tween 20 to achieve a final concentration of 100 ug/L of each alcohol. In Vitro Methods The in_vitrg_methods described in this thesis were developed by H Robert L. Houtz (Department of Horticulture, Michigan State University). For corn, the youngest one to three leaves were treated with a 1000 pg/L TRIA solution and ground in a cold mortar and pestle (4°C) with 4 m1 grinding media for each 3 fresh weight of tissue. The grinding media consisted of 20 mM potassium phosphate (pH 7.2) containing 70 uL/L 8' mercaptoethanol. Rice was treated with a 100 ug/L TRIA solution and ground in 5 ml grinding media for every 2 g of leaf tissue. The result- ing crude homogenates were squeezed through 4 layers of cheese cloth and centrifuged for 20 min at 8000 g for corn or 5000 g for rice. The 12 supernatant solution was added to cold incubation media (1 ml for every 2 ml of media) and kept on ice until the initiation of an experiment (Figure l). The incubation media was prepared by combining the follow- ing solutions shortly before the leaves were to be extracted; NADPH (Na salt) 50 mg/60 ml, NADH (Na salt) 42 mg/60 m1, ATP (Na salt) 44 mg/ 80 ml, MgC12'6 H20 25 mg/125 ml, oxaloacetic acid 10 mg/60 ml and a- ketoglutarate 10 mg/60 ml. Each component solution was prepared in grinding media. An experiment began by pipetting predetermined volumes of the extract into 25 mm x 200 mm test tubes or 50 to 125 ml Erlenmeyer flasks which served as replicates. At the start of a test, a 3 ml sample was taken for N analysis from each replicate and the tubes were stoppered and placed in a water bath shaker with gentle shaking at 25 to 30°C. Similar samples were harvested from the replicates at 60, 80 or 120 min incubation. Samples were freeze-dried or digested directly for N analysis. All experiments were analyzed utilizing a completely randomized design. Analysis of Total Nitrogen Total N was measured utilizing the automated Kjeldahl procedure of Ferrari (13). Digestion of plant samples was accomplished by addition of a sulfuric-perchloric acid, selenium dioxide mixture com- posed of 40 ml perchloric acid, 1800 ml sulfuric acid and 6 g SeO2 made up to 2 L with distilled water (4 ml acid for every 20 mg dry wt of rice). The samples were solubilized by heating until the solutions cleared. After cooling, the samples were diluted with distilled water (6 ml water for every 4 ml of acid) and poured into plastic cups which were placed in a sampling tray. The tray was attached to an Auto-Analyzer 13 EXCISED CORN LEAVES TREATED WITH TRIA Po;3 buffer pH 7.2 extraction at 4°C (1:4, w/v) CRUDE HOMOGENATE Filter through 4 layers cheesecloth centrifuge 8000 g for 20 min I ’1 SUPERNATANT PELLET (discard) Combine with incu- bation media (1:2, v/v) EXPERIMENTAL EXTRACT Incubate at 25 to 30°C ZERO-TIME SAMPLE; TWO-HOUR SAMPLE; -Kjeldahl analysis -Kjeldahl analysis 15 . lS . - N analySIS - N analySIS Figure l. In_vitro methods for assaying the N content of TRIA treated corn extracts over time. l4 (Technicon Instruments Corporation, Tarrytown, NY) for automated Kjeldahl analysis. Nitrogen was detected by an alkaline-phenol Color reaction with maximal absorbance at 632 nm. Standards, consisting of 10 to 50 mg of ground wheat (4.34% moisture, 2.566% N) were analyzed with each experiment for calibration purposes. Percentage N in the ground wheat was established utilizing orchard leaves obtained from the National Bureau of Standards, Washington, D.C. (standard reference material 1571). Modifications of the procedure involving addition of ' salicylic acid to include estimates of nitrate N and other high valence forms were not employed (31). Hence, total N refers to the total Kjeldahl detectable N utilizing the above procedures. Atmospheric Substitution Procedures For the in_vitro atmosphere experiments, N2, argon, oxygen and carbon dioxide were mixed with the aid of a proportioner and admitted to the experimental flasks through serum stoppers. The gasses were proportioned to achieve an 80% argon or N atmosphere while 0 and CO 2 2 2 remained at their normal levels (20% O 0.033% C02). The gas mixtures 2. were admitted at a flow rate of 12 ml/s, for 30 5, through a disposable syringe needle puncturing the serum bottle stopper. A vent needle was also utilized during the gassing procedure to allow flushing of the flasks. Similar methods were used to change the atmosphere around ex- cised rice shoots which were treated as previously described. The shoots were placed in 35 mm x 300 mm test tubes and gassed for 30 3 through serum bottle stoppers at a flow rate of 16 mI/s. Acetylene was generated by the Kipp reaction which involves the addition of water to calcium carbide pellets. The resulting acetylene 15 was stored over a saturated solution of NaZSO4. In the acetylene test for nitrogenase activity (18), in_vit£9_rice extracts added to 35 ml gas bottles were sealed with serum bottle stoppers. The bottles were flushed with an 80% Ar, 20% O and 0.033% CO mixture for 30 s at a 2 2 flow rate of 17 ml/s. Three ml of acetylene was injected into the appropriate treatments by first withdrawing 3 ml from the bottles and then injecting the acetylene. This created approximately a 13% ace- tylene atmosphere in each bottle. After taking 1 ml, zero-time gas samples, the bottles were placed in a water-bath shaker with gentle shaking at 28°C. Final gas samples were analyzed at the end of a 70 min incubation period and compared with the zero-time samples. Analysis of ethylene in all samples was accomplished utilizing a Varian model 3700 gas chromatograph with a 2 m Porapak-N column. Optimal sensitivity was achieved with an injection temperature of 120°C and an N flow rate of 30 ml/min. 2 Generation of 15N2 A N gas generating apparatus was constructed as modified from Rittenburg (37) by David R. Dilley (Professor of Horticulture, Michigan State University) (Figure 2). Ammonium sulfate containing a 99.9% 15N label was obtained from Monsanto Research Corporation (Mound Laboratory, Miamisburg, OH) and 1.754 g was added to reservoir A with stopcock l in the closed position (Figure 2). A minimal amount of water was admitted for complete dissolution of the (NH4)ZSO and 2 4 or 3 drops of 5% (w/v) H2504 was added for acidification. With stop- cock 2 in the closed position, reservoir B and gas trop C were evacu- ated (30 mm-Hg) by opening the three-way stopcock (3) which was then 16 Figure 2. A37? gas generating apparatus as modified from Rittenburg 18 closed to maintain the vacuum in the apparatus. The (NH4)2SO4 solution in reservoir A was de-gassed of N2 with helium or argon and slowly ad- mitted to reservoir B. Approximately 1.5 ml of de-gassed water was used to rinse the sides of A and was similarly admitted to B preserving as much of the vacuum in the apparatus as possible. Lithium hypobromite solution, prepared by adding 2 ml of reagent grade bromine to 60 ml of a cold (0 to 5°C) 10% (w/v) solution of lithium hydroxide, was slowly admitted to reservoir B until the reaction had visibly stopped and no more could be added. At this point, gas trap C was immersed in liquid N2 which created a negative pressure in the apparatus. The remainder of the LiOBr solution was added (approximately 75 ml) until all of the (NH 804 had reacted. The liquid N2 was removed and the solution in 4)2 reservoir 8 was drained into a waste container via stopcock 2. Reservoir A was filled with a saturated NaZSO4 solution for displacing 15N2 from the apparatus. 15 To sample the N the liquid N was placed Under the gas trap 23 and a maximum amount of NaZSO4 was added to B. The gas was drawn through the septum with a disposable syringe while simultaneously ad- mitting an equal volume of Na2504. saturated Na2S04 solution and found to be 98.98atom5% on a Varian MAT GD-lSO isotope ratio mass spectrometer. Labelled gas was stored over a 15N as determined Atmosphere Enrichment The atmosphere enrichment experiments consisted of exposing treated rice seedlings and in_vitro corn extracts to various levels of 15N2. Plants were treated with test solution and placed in 35 mm x 300 mm test tubes, (or 250 ml Erlenmeyer flasks for the ip_vitro extracts), 19 containing half-strength Hoagland's solution (pH 4.5) with 1 mM N as (NH4)ZSO4. The amount of Hoagland's solution was adjusted so that the available gas space within each tube was 154 ml. The tubes were clOsed with rubber stoppers, bored to accommodate septums, through which gas samples could be exchanged. Atmosphere enrichment was accomplished by replacing 10 to 25 ml of the atmosphere surrounding the plant or ex- tract with an equivalent volume of 15N2. Plants were harvested after 40 min incubation by freezing in dry-ice and acetone and lyophilizing. Dried plant material was ground in a Wiley mill (40 mesh screen) with aliquots taken for total N analysis and 15N analysis utilizing a VG Micromass MM-622 isotope ratio mass spectrometer. The experimental sequence of the ig_vi£rg experiment appears in Figure 1. Three replicates were used in a completely randomized design. Heavy N gas was admitted to the flasks containing the experimental ex- tracts after taking the zero-time samples. Samples were taken for both total N and mass spectrometric (MS) analysis. Total N samples were digested immediately and samples for MS analysis were dried at 72°C in preparation for micro-Kjeldahl digestion. Preparation of Samples for MS Analysis The micro-Kjeldahl procedures of Black (4) were employed for the digestion and distillation of plant samples for MS analysis. Catalyst, consisting of a 1:10:100 mixture of 5e02, CuSO4 and K2804, was added to previously ground plant material (1 g catalyst for each mg total N in sample). Sulfuric acid was added to the plant-catalyst mixture (3 ml for each mg total N) and the flasks were placed on burners for digestion. Samples were digested for approximately 6 h during which time the or- ganic N was converted to (NH4)ZSO4. 20 The NH; was liberated by steam distillation following addition of 10 N NaOH to the digestion mixture. Eighty percent ethanol was run through the distillation apparatus between samples to prevent cross 4. 4 HCl and dried at 72°C resulting in pure NH4C1 crystals. The MS analysis of 15N was accomplished by the addition of LiOBr solution to the crystals contamination. The resulting free NH was trapped in 10 m1 of 0.1 N and admitting the resultant gas into the mass spectrometer. All samples for MS analysis contained from 2 to 4 mg total N. Depletion and Distribution Analysis Nitrogen-15 depletion experiments involved culturing rice seedlings on KISNO3 (5.3 atom %) for 20 to 96 h prior to treatment with TRIA. Plants were sorted for size and suspended in 220 ml plastic cups containing 180 ml of half-strength Hoagland's solution with 6 mM KISNO3 as the sole source of N. For the 20 h experiments, 12 to 16 cups, each con- taining four to six seedlings of uniform size, were placed in a growth chamber for the duration of the prelabelling period. Following the 20 h interval, the seedlings in each cup were sorted again, randomized and treated by dipping in the appropriate test solution. Treated plants were placed in 25 mm x 200 mm test tubes containing 6 to 10 ml of. labelled or unlabelled Hoagland's solution. After 80 min incubation in a growth chamber, the plants were harvested by separating roots from shoots, freezing both in dry-ice and acetone, and lyophilizing or oven drying at 72°C. Seedlings in blocks I through IV, V through VIII and IX through XII were combined yielding a total of three blocks and three treatments with four plants/treatment. This was necessary in order to assure 21 enough plant material for fractionation procedures. The dried shoots were ground in a Wiley mill. Aliquots were taken for total N, soluble protein, free amino acid, nitrate and 15N analysis. In most experiments the shoot material alone was analyzed. However, in the soluble-insoluble fractionation experiment 20 mg aliquots of the shoot material were com- bined with proportionate amounts of root material as determined on the basis of the original shoot to root dry weight ratio. The 96 h experiment necessitated the addition of labelled Hoagland's solution to the cups twice over the 4-day interval. Six blocks and three treatments (four plants/treatment) were utilized in a randomized complete block design. Seedlings were treated and incubated in 220 ml cups con- taining unlabelled Hoagland's solution for 80 min. Plants were harvested by freezing and lyophilizing. For the in vitrg_depletion experiment, 73 mg of KISNO3 (5.3 atom %) was added to 158 ml of incubation media which was combined with super- natant solution of corn leaf extracts yielding replicates (Figure l). Zero-time aliquots for total N and 15N analysis were sampled and the extracts were placed in a water-bath shaker (25 to 30°C) for the duration of the 120 min incubation period. All samples were dried at 72°C prior to N analysis. Fractionation Procedures The N distribution experiments involved separation of the plant material into various N fractions followed by 15N analysis of each fraction. The 17 to 19-day-old rice seedlings were prelabelled and treated as discussed in the previous section. An aliquot of the dried plant material, containing 2 to 4 mg of water soluble N, was extracted 22 in a mortar and pestle with glass distilled water (1 ml for every 10 mg of plant material). The resulting extract was centrifuged at 10,000 g for 30 min and the supernatant solution was set aside. The pellet was washed with an equivalent volume of water and recentrifuged. Both supernatants were recentrifuged (10,000 g for 30 min) and the pellets were combined by transferring to 125 m1 Erlenmeyer flasks with 25 m1 of distilled water. The resulting supernatant solutions were also transferred to 125 ml flasks and all fractions were dried at 72°C. Separation of the plant material into water soluble and water insoluble N fractions was thus achieved. Lyophilized plant material was utilized in experiments involving MS analysis of soluble and insoluble N fractions. Separation into soluble and insoluble portions was achieved as discussed previously and the soluble protein was precipitated with 20% trichloroacetic acid (TCA). Isolation of the protein was accomplished by centrifugation (10,000 g for 30 min) and the resulting protein pellet was transferred to 125 ml Erlenmeyers with 20% TCA and dried at 72°C. Quantitative determinations of soluble protein N, free amino N and nitrate N were made by either assaying the supernatant solutions in the regular fractionation sequence or utilizing aliquots of the dried plant material specifically for this purpose. Soluble protein N was determined by a modification of the Lowry procedure (2). The methods of Hyman Rosen (23) were used in assaying free amino N levels. Statistical Procedures In all rice seedling experiments, a randomized complete block design was utilized to remove variance due to differences in plant 23 size. Prior to the initiation of a test, the seedlings in each block were assigned to a particular treatment utilizing a random number table. In most tests, six blocks and three treatments were employed and coeffi- cients of variation ranged from 1 to 6%. Orthoganol comparisons were utilized in separating treatment effects from controls. F tests and LSD's were used to compare means where appropriate. Analysis of variance for the in_vitrg_experiments was accomplished using a completely randomized design. Replicates were formed by pipet- ting aliquots of extract into labelled Erlenmeyer flasks containing incubation media. Samples were harvested at intervals for analysis of N. Since the extract had been previously treated with TRIA, the effect of time on N content was being tested, i.e. time served as treatment. Coefficients of variation in these tests ranged from 1 to 4%. Where appropriate, orthoganol comparisons were utilized. RESULTS AND DISCUSSION Nitrate Reductase Experiments It was hypothesized, that the apparent total N increase in both corn and rice, was caused by a TRIA induced stimulation of NR activity. This would result in enhanced reduction of nitrate within the leaf tissue and ultimately a larger pool of total N would be available for detection by Kjeldahl. In rice (44), measurements were restricted to endogenous nitrate levels which indirectly revealed the role of NR in the TRIA response. These results will become apparent in later sections. Direct measurements of NR activity in corn was accomplished by employing both 12 give and in_zitrg_procedures. The results of a corn leaf NR experiment, utilizing the in 3119 assay methods, appear in Table 1. No significant effect on NR could be demonstrated over the times or concentrations of TRIA employed. Table 1. Effect of increasing concentration of TRIA on nitrite accumu- lation from corn leaves measured at various times after treatment with the in_vivo assay. Nitrite Accumulation (mumol NOZ/h/g dry wt) TRIA (mg/L) 30 min 60 min 120 min 0.00 4,470 11,042 15,226 0.01 3,050 11,827 15,557 0.05 3,482 11,077 14,663 0.10 3,400 8,878 11,937 24 25, Other in_vivg_tests were conducted utilizing similar incubation times and TRIA concentrations with results showing no effects on enzyme activity due to TRIA. To further substantiate these results, an experiment employing the in_!itrg methods of NR analysis was conducted. Corn seedlings, grown in nutrient culture, were treated at the 7-day—old stage and NR activity was measured 3 h after treatment. Once again, no significant effect on NR activity was achieved with the TRIA treatment (Table 2). Table 2. Effect of TRIA on NR activity in corn leaves measured 3 h after treatment with the in vitro assay. TRIA NR Activity (0.1 mg/L) (mumol NOZ/h/g fresh wt) - 6,093 + 5,919 Many more tests, utilizing both assay methods, were conducted with the results supporting the null hypothesis. The conclusion that TRIA does not enhance the activity of corn NR was, therefore, accepted. Growth Response Studies: Plant Analysis Attention was turned toward experiments designed to characterize the total N increases at the whole plant level. A 40 min experiment comparing the fresh weight and N content of control and treated rice shoots indicated that the TRIA treated seedlings significantly gained in fresh weight and total N when compared with controls (Table 3). The apparent N increase paralleled the increase in fresh weight with no change in N concentration evident. Hence, the total N increase was due to the larger plants resulting from TRIA treatment. 26 Table 3. Growth response and total N content of TRIA treated rice seedlings. Time TRIA Fresh wt Total N (min) (100 pg/L) (mg/shoot) (mg/shoot) 0 0 215 1.98 40 - 223 1.97 40 + 252** 2.20** **F value for comparison with controls significant at the .01 level. A definitive explanation of the data was complicated by the fact that the increase in total N was larger than the amount of N provided to the plant through the nutrient solution (10 ml of 1 mM N as (NH4)2804). It thus appeared that treating the seedlings with TRIA enabled them to acquire N other than that directly provided via the nutrient culture. Growth Response Studies: System Analysis In an effort to further define the role of the nutrient solution in the N increase, experiments were conducted where the total N in both plant and nutrient culture was analyzed. The data was expressed as total system N (mg/system). After 40 min incubation, a significant 21 and 24 percent increase in total system and total plant N occurred, respectively with the TRIA treatment (Table 4). An increase in concen- tration of N was evident from the significant rise in system N. Once again, the increase was too large to be attributed to N provided to the plants through the nutrient solution. 27 Table 4. Total N content of TRIA treated rice seedlings. Time TRIA Total N (min) (100 pg/L) (mg/system) (mg/plant) 0 0 1.69 1.53 40 - 1.73 1.57 40 + 2.10** l.94** **F value for comparison with controls significant at the .01 level. A similar experiment was conducted, however, a treatment of TRIA combined with tetracosanol was added. Single rice seedlings were ex- posed to foliar applications of the appropriate test solution and in- cubated in a growth chamber for 40 min prior to system analysis. As discussed previously, tetracosanol has been shown to be an effective inhibitor of TRIA (24). No significant change in dry weight could be detected in any of the treatments after the 40 min growth period (Table 5). The total N content of the TRIA treated plants and plant- nutrient culture systems was 12 and 7 percent higher, respectively than the other two treatments. Tetracosanol resulted in complete in- hibition of the TRIA induced N accumulation effect confirming earlier research performed in this laboratory (24). Table 5. Growth response and total N content of rice seedlings ex- posed to TRIA or TRIA + Tetracosanol. Treatment Dry wt Total N @ (100 pg/L) (mg/plant) (mg/system) (mg/g dry wt) Control 42.7 1.70 34.6 TRIA 41.2 1.83** 39.1** TRIA + Tetracosanol 43.0 1.71 34.9 @mg plant N/g dry wt of plant (Hoagland's N subtracted). **F value for comparison with controls significant at the .01 level. 28 Atmospheric Substitution Since the total system N increased, it was hypothesized that the source for the rapid accumulation of N in TRIA treated plants was the atmosphere. This becomes feasible when considering the possibility that TRIA may be inducing N2 fixation through its influence on phylloplane bacteriacnrblue-green algae in close association with the roots. Estimates of the rates of N2 fixation by phylloplane bacteria and blue-green algae are available. Bacteria in the leaves of Douglas Fir (Pseudotsuga menziensii Franco) fixed a maximum of 5.2 mg N/g leaf N over a one month period (25). Similarly, Lovett and Sagar (27) demon- strated the presence of free-living N2 fixing bacteria in the leaves of Comelina sativa (L.) Crantz. Rates for blue-green algae have been determined by Stewart (40) as ranging from 10 to 78 kg N fixed/ha for 2 a rice crop on an annual basis. The apparent increases in total N content of plants due to TRIA are on the order of 1000 times these values. However, the above estimates resulted from research performed in natural environments. These workers did not attempt to maximize rates of fixation through optimizing growth conditions or chemical stimulation. Therefore, the decision was made to test the possibility of atmospheric N2 being directly involved in the TRIA induced, total N accumulation effect through these types of symbiotic relationships. Both the in_vitrg_and whole seedling systems were utilized in testing the role of atmospheric N2 in the TRIA response. The apparent total N content of an ig_vitro corn system, measured at various in- tervals over a 2 h period, was increased by TRIA (Table 6). 29 Table 6. Total N content of the supernatant from TRIA (1000 ug/L) treated corn leaves assayed as a function of time. Time Total N (min) (mg/g dry wt) 0 74.70** 30 81.45 60 81.80 120 82.93 **F value for comparison with treatments significant at the .01 level. Similar N responses were observed in rice extracts (unpublished data). If the gain in total N within the extracts required the presence of atmospheric N2, then inhibition of the response should be achievable by replacing N2 in the air, surrounding the extract, with argon. An appro- priate experiment was constructed utilizing rice extracts and the re- sults appear in Table 7. Table 7. Effect of atmospheric substitution on the total N content of the supernatant from TRIA (100 pg/L) treated rice leaves. Time Closed@ Total N (min) Atmosphere System (pg/system) 0 -- 0 506 60 Normal - 504 60 Normal + 520* 60 Ar replacing N2 + 505 60 Total N2 + 507 @Incubation tubes were either stoppered (+) or left open (-). * F value for difference from all other treatments significant at the .05 level. Argon appeared to inhibit the N increase characteristic of the TRIA treatment in which normal amounts of N2, CO2 and 02 were supplied to 30 the extracts. To confirm an earlier observation, that the in_!itrg response was optimized when all incubation tubes were stoppered, an open—tube system was included. The tube was left open for the duration of the experiment resulting in no net N gain. The anaerobic treatment (total N2 atmosphere) showed no gain in N. This supported the research of Bittenbender et al., (3) that CO2 is required for the response. In a similar in_vitrg_rice experiment, Ar was substituted for N2 in a normal atmosphere. A significant increase in total N occurred over the zero-time and argon treatments (Table 8). Table 8. Influence of an argon substituted atmosphere on the total N content of the supernatant from TRIA (100 pg/L) treated rice leaves. Time TRIA Total N (min) (100 ug/L) Atmosphere (mg/system) O + —- 2.01 80 + Ar replacing N2 2.08 80 + Normal 2.30** **F value for comparison with controls significant at the .01 level. These tests were indicative of the involvement of atmospheric N2 in the in_vitrg TRIA response. Dinitrogen may be utilized indirectly via an activation role or directly through acting as substrate for fixation via nitrogenase provided through bacterial or algal contamina- tion. To test for the presence of nitrogenase in the in_vitrg_rice assay a 13% acetylene atmosphere was introduced to the flasks containing the rice extracts. Gas samples were taken at zero-time and 70 min after incubation for analysis of ethylene. The rice extracts produced very little endogenous ethylene (Table 9). 31 Table 9. Acetylene test for the presence of nitrogenase in the super- natant of TRIA (100 ug/L) treated rice leaves. Acetylene Ethylene (pl/1) Atmosphere Initial 70 min -- 0.0000 0.0560 13% 0.3065 0.2837 The acetylene treatment showed no increase in CZH at the end of the 4 incubation period. The relatively high initial C2H4 concentration in the zero-time sample represents C produced in the generation of 2H4 acetylene. No measurable amount of nitrogenase existed in the in_vitro rice system, therefore, the TRIA induced response may not be attributed to phylloplane bacteria. The role of rhizosphere bacteria and blue- green algae in the intact seedling assays can not be ruled out. Atmosphere Enrichment (To test the hypothesis that the N response in intact plants directly involved atmospheric N2, the atmosphere surrounding the seed- lings was enriched with 15N2. Analysis for enrichment of plant material was accomplished by MS analysis. An experiment was performed to determine the optimal method of enriching the atmosphere surrounding rice seedlings. Replacing a gas sample, taken from a tube containing the seedling, with an equivalent volume of 15N2 resulted in maintaining the TRIA induced, total N response. When the 15N2 sample was injected with no previous gas withdrawal the response was inhibited (Table 10). 32 Table 10. A comparison of methods for 15N atmosphere enrichment of TRIA treated rice seedlings. 15 Treatment . N TRIA Total N Enrichment method (100 ug/L) (mg/plant) (mg/g dry wt) Replace vol of atm - 3.08 39.4 with eq vol 15N2 + 3.60** . 43.0** Addition of 15N2 - 3.01 39.3 to atm . + 3.08 40.7 **F value for comparison with control significant at the .01 level. The slight positive pressure imposed upon the seedling with the latter method may have caused the inhibition. The replacement procedure was utilized in all atmosphere enrichment experiments of this type. The first enrichment experiment consisted of three treatments and six blocks in a randomized complete block design. Nineteen day-old rice seedlings were handled and gassed as previously described. A highly significant increase in dry weight was evident after 40 min incubation (Table 11). Table 11. Dry weight change of TRIA treated rice seedlings in an 8.5% 15N2 enriched atmosphere. Time TRIA Dry wt (min) (100 pg/L) (mg/plant) 0 0 59.4 40 - 59.3 40 + 62.2** **F value for comparison with controls significant at the .01 level. 33 To obtain sufficient sample for MS analysis the replicates were combined ”into three blocks. Although the apparent increase in total N due to TRIA was 14%, the increase in 15N was only 4% and neither difference was significantly higher than the controls (Table 12). Table 12. Total N and 15N content of TRIA treated rice seedlings ex- posed to an 8.5% 15N2 enriched atmosphere. 15 Time TRIA Total N' N (min) (100 pg/L) (mg/plant) (mg/g dry wt) (atom %) 0 0 2.10 35.3 0.375 40 - 2.28 38.2 0.374 40 + 2.59 41.3 0.390 The previous experiment was repeated and the samples were sent to another lab for 15N analysis. After 40 min incubation, the TRIA treated plants gained in dry weight and total N over controls (Table 13). The increase was characterized by a gain in plant N (mg/plant) rather than concen- tration N (mg/g dry wt). Table 13. Growth response, total N and 15N content of TRIA treated rice seedlings exposed to an 8.5% 15N2 enriched atmosphere. (Analysis conducted by R. P. Hauck, Division of Agricultural Department, Tenn. Valley Authority, Muscle Shoals, AL 35660). 15 Time TRIA Dry wt Total N N (min) (100 ug/L) (mg) (mg/plant) (mg/g dry wt) (atom %) O O 60.3 2.23 36.9 0.362 40 - 59.3 2.21 37.3 0.362 40 + 65.6** 2.39* 36.5 0.361 *,**F value for comparison with controls significant at the .05 and .01 levels, respectively. 34 If the apparent 7.5% increase had come exclusively from N2 in the en- riched atmosphere then the atom% 15N in the TRIA treated plants should have increased to 0.978% from natural abundance. No increase in atom%; 15N was evident in any of the treatments (Table 13). It was apparent that atmospheric N2 did not provide the N in the TRIA induced N re- sponse of intact seedlings. Earlier results with Argon replacing N2 revealed the need for the presence of NZ to maintain the in_xitrg_total N response. Therefore, an enrichment experiment was constructed utilizing corn extracts (Table 14). Table 14. Total N and 15N content of the supernatant from TRIA (1000 ug/L) treated corn leaves exposed to a 13% 15N enrich- ment atmosphere. 2 Time Total N 15N (min) (mg/system) (mg/g dry wt) (atom %) 0 28.19 86.5 0.365 120 29.51** 90.5** ‘ 0.366 **F value for comparison with control significant at the .01 level. The concentration of N in the TRIA extracts increased significantly over the 2 h incubation period. Had this increase come directly from N2 in the enriched atmosphere, the 2 h sample should have increased to 0.930 atom % 15N. However, this sample remained at natural abundance. The atmospheric substitution and enrichment experiments did not account for N dissolved in the solutions. Due to the high solubility, of N2 in water (17.8 mg/L), the possibility remained that there was sufficient N2 dissolved in these solutions to contribute directly or indirectly in the TRIA response. 35 To test this possibility, a 40 min atmospheric substitution experi- ment was constructed. The shoots from treated 18-day-old rice Seedlings were placed in test tubes containing half-strength Hoagland's solution. The tubes were stoppered and flushed with the appr0priate gas mixtures. As in previous experiments, the concentration of CO2 and 02 were kept. at normal levels while argon replaced N at an equivalent concentration. The concentration of N within the TRIA treated seedlings increased signi- ficantly regardless of the presence of atmospheric N2 (Table 15). Since the experiment involved shoots only, the role of dissolved N in the 2 response was probably negligible. Depletion and Distribution Studies Depletion experiments were undertaken to test the hypothesis that TRIA was hastening the metabolism of contaminant sources of N. These compounds could affect both the atmosphere or nutrient solution. For example, N from nitrous oxide (N20) is readily metabolized by, and incorporated into, reduced N fractions of plants (38). The reactivity of N20 in aqueous solutions results in the formation of nitrate and nitrite ions. Although normal atmospheres contain very little N20 (0.2 pg/L to 0.5 mg/L) it is possible that the TRIA treated plants are utilizing it in combination with other unknown N-compounds possibly arising as a result of microbial activity within the nutrient solution. Depletion experiments permit the detection of any N utilization during an experiment on the basis_of dilution of 15N prelabelled plant material. Both the in_!itrg_and whole plant systems were utilized in these tests. 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