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Hydroponic growth medium must be well buffered if it is to support sustained plant growth. Although 1.0 millimolar phosphate is commonly used as a buffer for hydroponic growth media, at that concentration it is generally toxic to a soybean plant that derives its nitrogen solely from dinitrogen fixation. On the other hand, we show that 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid, pKa 6.1, has excellent buffering capacity, and it neither interferes with nor contributes nutritionally to soybean plant growth. Furthermore, it neither impedes nodulation nor the assay of dinitrogen fixation. Hence, soybean plants grown hydroponically on a medium supplemented with 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid and 0.1 millimolar phosphate achieve an excellent rate of growth and, in the absence of added fixed nitrogen, attain a very high rate of dinitrogen fixation. Combining the concept of hydroponic growth and the sensitive acetylene reduction technique, we have devised a simple, rapid, reproducible assay procedure whereby the rate of dinitrogen fixation by individual plants can be measured throughout the lifetime of those plants. The rate of dinitrogen fixation as measured by the nondestructive acetylene reduction procedure is shown to be approximately equal to the rate of total plant nitrogen accumulation as measured by Kjeldahl analysis. Because of the simplicity of the procedure, one investigator can readily assay 50 plants individually per day.

Experiments were conducted to characterize the distribution of N compounds in the xylem sap of nodulated and nonnodulated soybean plants through development and to determine the effects of exogenous N on the distribution of N compounds in the xylem. Xylem sap was collected from nodulated and nonnodulated greenhouse-grown soybean plants (Glycine max [L.] Merr. "Ransom") from the vegetative phase to the pod-filling phase. The sum of the nitrogen in the amino acid, nitrate, ureide (allantoic acid and allantoin), and ammonium fractions of the sap from both types of plants agreed closely with total N as assayed by a Kjeldahl technique. Sap from nodulated plants supplied with N-free nutrient solution contained seasonal averages of 78 and 20% of the total N as ureide-N and amino acid-N, respectively. Sap from nonnodulated plants supplied with a 20 millimolar KNO(3) nutrient solution contained seasonal averages of 6, 36, and 58% of total N as ureide-N, amino acid-N, and nitrate-N, respectively. Allantoic acid was the predominant ureide in the xylem sap and asparagine was the predominant amino acid. When well nodulated plants were supplied with 20 millimolar KNO(3), beginning at 65 days, C(2)H(2) reduction (N(2) fixation) decreased relative to nontreated plants and there was a concomitant decrease in the ureide content of the sap. A positive correlation (r = 0.89) was found between the ureide levels in xylem sap and nodule dry weights when either exogenous nitrate-N or urea-N was supplied at 10 and 20 millimolar concentrations to inoculated plants. The results demonstrate that ureides play a dominant role in N transport in nodulated soybeans and that the synthesis of ureides is largely dependent upon nodulation and N(2) fixation.

From 10 to 60% of the nitrate present in plant tissue extracts and stem exudates of corn (Zea mays L.) was found to be reduced during Kjeldahl digestion, even in the absence of added reducing agents. This reduction is of particular concern in [15N]nitrate assimilation studies, because it results in an overestimate of nitrate reduction. To overcome this problem, a method was developed for removing nitrate prior to Kjeldahl digestion, thereby preventing nitrate reduction. The procedure utilizes hydrogen peroxide for partial oxidation of organic matter in order to minimize the nitration of organic compounds. The free nitrates are then volatilized as nitric acid from concentrated sulfuric acid at 95°C. When the proposed method was used as a pretreatment to Kjeldahl digestion, less than 0.5% of the applied nitrate was recovered in the reduced nitrogen fraction of plant tissue extracts and stem exudates.

The influence of nutrient nitrate level (0-20 millimolar) on the effects of NO(2) (0-0.5 parts per million) on nodulation and in vivo acetylene reduction activity of the roots and on growth and nitrate and Kjeldahl N concentration in shoots was studied in bean (Phaseolus vulgaris L. cv Kinghorn Wax) plants. Exposing 8-day old seedlings for 6 hours each day, for 15 days, to 0.02 to 0.5 parts per million NO(2) decreased total nodule weight at 0 and 1 millimolar nitrate, and nitrogenase (acetylene reduction) activity at all concentrations of nitrate. The pollutant had little effect on root fresh or dry weights. Shoot growth was inhibited by NO(2). The NO(2) exposure increased nitrate concentration in roots only at 20 millimolar nutrient nitrate. Exposure to NO(2) markedly increased Kjeldahl N concentration in roots but generally decreased that in shoots. The experiments demonstrated that nutrient N level and NO(2) concentration act jointly in affecting nodulation and N fixing capability, plant growth and composition, and root/shoot relationships of bean plants.

The methodology, characteristics and application of the sensitive C(2)H(2)-C(2)H(4) assay for N(2) fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N(2)ase-catalyzed reduction of C(2)H(2) to C(2)H(4), gas chromatographic isolation of C(2)H(2) and C(2)H(4), and quantitative measurement with a H(2)-flame analyzer. As little as 1 mumumole C(2)H(4) can be detected, providing a sensitivity 10(3)-fold greater than is possible with (15)N analysis.A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C(2)H(2)-reducing activity in the field. Applications to field samples included an evaluation of N(2) fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N(2) fixation rate of 30 to 33 kg N(2) fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N(2) fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N(2) fixing activity of soybeans. The validity of measuring N(2) fixation in terms of C(2)H(2) reduction was established through extensive comparisons of these activities using defined systems, including purified N(2)ase preparations and pure cultures of N(2)-fixing bacteria.With this assay it now becomes possible and practicable to conduct comprehensive surveys of N(2) fixation, to make detailed comparisons among different N(2)-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N(2) fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N(2) fixation reaction.

This paper reports the results of determinations of the enzymes invertase, amylase, and ereptase ; analyses of total and amino nitrogen, reducing sugars and sucrose ; and the hydrogen ion concentrations, titratable acidity, and titration curves of the juices expressed from the leaves, stems, and roots of plants grown with varying amounts of potassium. Methods. Details of the care of the plants during growth and of the methods employed in harvesting them have been presented in the preceding paper (26). Invertase, amylase, peptase, and ereptase, and total and reducing sugars were determined by the methods used in the studies reported in 1929 (24). The titration method of Willstatter and Waldschmidt-Leitz (55) was also used for the detection of peptase. Total nitrogen was determined by the Kjeldahl method, while the Van Slyke method was employed for the estimation of amino nitrogen. The nitrogen determinations were performed by the Chemistry Department of this Station.

In previous studies on nitrogen fixation by yeast (2) it was noted that "according to the method of analysis used (Kjehldahl) there was a loss in nitrogen in the beginning and that actual gain in nitrogen was not apparent until after six or eight weeks. Moreover at the particular pH in which there was a maximum preliminary loss there was the maximum gain or fixation after the longer time interval/' It was stated that "this phenomenon has been observed many times in our work and after considering all phases of the matter, our hypothesis is as follows : Yeast is known to be rich in ring nitrogen compounds and in the early stages of growth the nitrogen may be thrown into a compound not to be analyzed by the usual methods. Later in the growth these compounds may be transformed into materials amenable to the analysis used." This communication deals with the development of a method by which larger percentages of nitrogen are obtained from yeast than are secured by the usual Kjeldahl procedure. Many modifications of the Kjeldahl method have been described by Hepburn (4). The use of hydrogen peroxide during digestion has been proposed by Berman (1), Heuss (5) and KleeMAN (6). The three general methods for nitrogen determination in organic compounds are

This report supplements the earlier report (13) which discussed the growth rates, water and mineral nutrient intake by the same group of plants. The data herein considered deal with the chemical composition of the leaf and stem tissues at different growth intervals of pineapple plants grown in nutrient solutions in the greenhouse and in soil under field conditions with different amounts of nitrate, potassium, and calcium. The plant tissues were analyzed for total nitrogen, soluble organic nitrogen, potassium, calcium, reducing and total sugars; the latter hydrolyzed by invertase. The methods for potassium and calcium were reported in a previous publication (9). Total nitrogen and soluble organic nitrogen were determined by the Kjeldahl procedure, using selenium oxychloride as catalyst in the oxidation of organic matter. The fractionation of total and soluble organic nitrogen was conducted according to technique reported previously (7, 8). Reducing sugars, total sugars, and sucrose, the latter by the difference between total and reducing sugars after hydrolysis with invertase, were determined according to the method of Quisumbing and Thomas (6). Starch was determined by the method of Pucher and Vickery (5).

Triacontanol (TRIA) increased fresh and dry weight and total reducible nitrogen (total N) of rice (Oryza sativa L.) seedlings within 40 minutes. Increases in total N in the supernatants from homogenates of corn (Zea mays L.) and rice leaves treated with TRIA for one minute before grinding occurred within 30 and 80 minutes, respectively. The source for the increase was investigated utilizing atmospheric substitution and enrichment and depletion studies with (15)N. The increase in total N in seedlings was shown to be independent of method of N analysis and the presence of nitrate in the plants. Automated Kjeldahl determinations showing apparent increases in N composition due to TRIA were shown to be correlated with hand Kjeldahl, elemental analysis, and chemiluminescent analysis in three independent laboratories. TRIA did not alter the nitrate uptake or endogenous levels of nitrate in corn and rice seedlings. Enrichment experiments revealed that the total N increases in rice seedlings, in vivo, and in supernatants of corn leaf homogenates, in vitro, are not due to atmospheric N(2). TRIA increased the soluble N pools of the plants, specifically the free amino acid and soluble protein fractions. No differences in depletion or enrichment of (15)N incorporated into soluble and insoluble N fractions of rice seedlings could be detected on an atom per cent (15)N basis. The apparent short-term total N increases cannot be explained by current knowledge of major N assimilation pathways. TRIA may stimulate a change in the chemical composition of the seedlings, resulting in interference with standard methods of N analysis.

Carbon and nitrogen limitations on symbiotically grown soybean seedlings (Glycine max [L.] Merr.) were assessed by providing 0.0, 1.0, or 8.0 millimolar NH(4)NO(3) and 320 or 1,000 microliters CO(2)/liter for 22 days after planting. Maximum development of the Rhizobium-soybean symbiosis, as determined by acetylene reduction, was measured in the presence of 1.0 millimolar NH(4)NO(3) under both levels of CO(2). Raising NH(4)NO(3) from 0.0 to 8.0 millimolar under 320 microliters CO(2)/liter increased plant dry weight by 251% and Kjeldahl N content by 287% at 22 days after planting. Increasing NH(4)NO(3) from 1.0 to 8.0 millimolar under 320 microliters CO(2)/liter increased total dry weight and Kjeldahl N by 100 and 168%, respectively, on day 22. Raising CO(2) from 320 to 1,000 microliters CO(2)/liter during the same period had no significant effect on Kjeldahl N content of plants grown with 0.0 or 1.0 millimolar NH(4)NO(3). The maximum CO(2) treatment effects were observed in plants supplied with 8.0 millimolar NH(4)NO(3), where dry weight and Kjeldahl N content were increased 64% and 20%, respectively. An increase in shoot CO(2)-exchange rate associated with the CO(2)-enrichment treatment was reflected in a significant increase in leaf dry weight and starch content for plants grown with 1,000 microliters CO(2)/liter under all combined N treatments. These data show directly that seedling growth in symbiotically grown soybeans was limited primarily by N availability. The failure of the CO(2)-enrichment treatment to increase total plant N significantly in Rhizobium-dependent plants indicates that root nodule development and functioning in such plants was not limited by photosynthate production.

The relative effects of water stress on growth parameters of subterranean clover (Trifolium subterraneum L. cv. Woogenellup) dependent on either N(2) or 8 millimolar NH(4)NO(3) for N were examined. Whole-plant carbon exchange rate (CER), acetylene reduction (AR), dry matter production, and Kjeldahl N accumulation were measured on uniform, intact swards of clover that were maintained under adequately watered conditions or were subjected to three cycles of water stress (leaf water potential/=-30 bar) over an 18-day period. In the absence or presence of water stress, growth rate, net N accumulation rate, and total N concentration of plants dependent on N(2) were 25 to 26, 45 to 50, and 20 to 21% less, respectively, than plants supplied with 8 millimolar NH(4)NO(3). The water stress treatment produced less than a 50% decrease in CER regardless of plant N source, a 90% inhibition of AR in plants dependent on N(2), and a 41% decline in dry matter production on both N sources. Water stress decreased reduced N accumulation 55% in N(2)-dependent plants and 50% in NH(4)NO(3)-dependent plants. Changes in growth and N accumulation caused a 10 to 11% decrease in total plant N concentration of water-stressed plants compared to adequately irrigated controls, but water stress decreased the N concentration of tissue synthesized during the 18-day treatment period in N(2)-grown plants more than in plants supplied 8 millimolar NH(4)NO(3). Thus, the relative effect of water stress on growth under the two N regimes was similar, but N accumulation by N(2)-dependent clover was inhibited to a slightly greater extent (P/= 0.001) than in NH(4)NO(3)-dependent plants.

The number of nodules produced per clover seedling inoculated with Rhizobium trifolii 0403 can be increased almost 2-fold by the addition of penicillin or mecillinam. Two-day-old dutch white clover seedlings grown in 250 milliliter boston round jars containing agar-solidified plant growth medium were inoculated with exponentially growing Rhizobium trifolii 0403 cells. Penicillin or mecillinam (100 micrograms per milliliter) were added immediately or after 24 hours. Following 42 days growth, 10 replicate sets of 5 plants for each treatment were assayed for nodule number, plant dry weight, and Kjeldahl nitrogen. Both antibiotics increased nodule number, plant dry weight, and Kjeldahl nitrogen. Increases in nodule number and dry weight were statistically significant. The range of values in Kjeldahl nitrogen was so extensive as to make the data insignificant at the P < 0.05 level, however nodule number, plant dry weight, and Kjeldahl nitrogen displayed a significant correlation with each other. There were no significant differences in treatment with either antibiotic or with time of treatment. Nodule number increased by about 85%, and plant dry weight and nitrogen increased by about 30%.

The micro-Kjeldahl method for nitrogen determination has been widely used in organic chemistry laboratories in recent years. The value of this method, developed through the brilliant researches of Pilch, Bang, Parnas and Wagner, Pregl, and co workers, has justified its application to industrial and clinical problems. Plant physiologists have not generally adopted quantitative micro-methods for nitrogen analysis. The purpose of the present study was to test the applicability of the micro-Kjeldahl method for the determination of nitrogen in plant materials.