Upon treatment of whole filaments of Anabaena variabilis with a French pressure cell, the activity of sucrose synthase (UDP glucose; D-fructose 2-α-ᴅ-glucosyl transferase, EC 2.4.1.13) was found to be associated almost exclusively with the vegetative cells. The sucrose-cleaving enzyme, an alkaline invertase (β-ᴅ-fructofuranoside fructohydrolase, EC 3.2.1.26), appears to be localized solely within the heterocysts. The implications of these findings, in the light of relevant data from the literature, are summarized in a model representing the physiological differentiation and molecular exchange between vegetative cells and heterocysts.

The principal initial product of metabolism of ['3N]N2 and '3NH4' by five diverse cyanobacteria is glutamine. Methionine sulfoximine inhibits formation of [l:JN]glutamine except in the case of Gloeothece sp., an organism with a thick sheath through which the inhibitor may not penetrate. Thus, glutamine synthetase appears to catalyze the initial step in the assimilation of N2-derived or exogenous NH4' by these organisms. ['3N]Glutamate is, in all cases, the second major product of assimilation of "IN-labeled N2 and NH4+. In all of the N2-fixing cyanobacteria studied, the fraction of "N in glutamine declines and that in glutamate increases with increasing times of assimilation of ['3N]N2 and l NH4', and (Gloeothece again excepted) methionine sulfoximine reduces incorporation of`N into glutamate as well as into glutamine. Glutamate synthase therefore appears to catalyze the formation of glutamate in a wide range of N2-flxing cyanobacteria. However, the major fraction of [I:JN]glutamate formed by Anacystis nidulans incubated with "NH4' may be formed by glutamic acid dehydrogenase. The formation of ['3N]alanine from ":3NH4+ appears to be catalyzed principally either by alanine dehydrogenase (as in Cylindrospermum licheniforme) or by a transaminase (as in Anabaena variabilis). Experiments using the radioisotope '3N haveshown that in the heterocyst-forming cyanobacterium Anabaena cylindrica the major enzymatic pathway for the asimilation of NH4', whether derived from N2 or supplied exogenously, consists of glutamine synthetase (L-glutamate:ammonia ligase [ADP forming], EC 6.3.1.2) and glutamate synthase (L-glutamate:ferredoxin oxidoreductase [transaminating], EC 1.4.7.1) (11,19). Glutamic acid dehydrogenase and alanine dehydrogenase also function in the assimilation ofexogenously supplied NH41 in this cyanobacterium, but at a much lower rate even in the presence of relatively high levels of NH4+ (11). In this respect, A. cylindrica differs from some heterotrophic, dinitrogen-fixing bacteria in which N2-derived NH4+ is assimilated principally by the glutamine synthetase/ glutamate synthase pathway, whereas, during growth in the presence of high exogenous concentrations of NHEV, the NHE is assimilated via glutamic acid dehydrogenase (6).When A. cylindrica is grown aerobically, t Preent address:

When detached soybean Glycine max (L.) Merr. cv. Hark, nodules assimilate 113NIN2, the initial organic product of fixation is glutamine; glutamate becomes more highly radioactive than glutamine within 1 minute; 13N in alanine becomes detectable at I minute of fixation and increases rapidly between I and 2 minutes. After 15 minutes of fixation, the major 13N-labeled organic products in both detached and attached nodules are glutamate and alanine, plus, in the case of attached nodules, an unidentified substance, whereas 113Niglutamine comprises only a small fraction of organic 13N, and very little 13N is detected in asparagine. The fixation of 113NIN2 into organic products was inhibited more than 99% by C2H2 (10%, v/v). The results support the idea that the glutamine synthetase-glutamate synthase pathway is the primary route for assimilation of fixed nitrogen in soybean nodules.Ammonium is the initial product of dinitrogen fixation by symbiotic (1) and free-living (19, 21) prokaryotes and by nitrogenase in vitro (16). Activities of the ammonium-assimilating enzymes glutamic acid dehydrogenase and glutamine synthetase, as well as glutamate synthase, have been detected in bacteroid fractions isolated from root nodules of soybean (2, 3) and lupin (2, 9).Nevertheless, free-living N2-fixing Rhizobiumjaponicum and bacteroids of R. japonicum isolated from soybean release 90 to 94% of the products of fixation of '5N2 into the medium as ammonium (1, 13). Dilworth and Brown (2) suggested that ammonium produced by bacteroids is assimilated in nodules through either the glutamine synthetase-glutamate synthase or glutamic acid dehydrogenase pathways, and favored the latter. In serradella, the major amino acids formed from '5N2 were glutamate and glutamine, but the initial organic product, and thereby the primary assimilatory route, was not determined (8 Pathology, Michigan State University, was maintained on YM agar (1.5%) slants and transferred every 3 months. Liquid cultures, started from slants in 20 ml of YM medium, were transferred to 500 ml of YM medium in 1-liter flasks, and were aerated by agitation on a reciprocating shaker. Cells from 2-day-old cultures were harvested by centrifugation at 6,000g for 10 min, washed and resuspended in ("N-free") legume nutrient medium free of combined nitrogen (6), and used as inoculum.Glycine max (L.) Merr. cv. Hark seeds were surface-disinfected for 20 sec with I % NaOCl, washed in running tap water, and planted in a Vermiculite-Perlite bed. The bed was inoculated with the Rhizobium suspension. Aerated N-free medium was circulated through the bed of germinating seeds for I week. The beds were illuminated with two 40-w Gro-lux fluorescent lamps (Westinghouse

Several in vitro model systems have been described in which ethylene is formed from methionine through a reaction involving free radicals (6, 9, 13). In our attempts to elucidate the pathway of ethylene biosynthesis, we investigated whether compounds which are known to quench free radicals inhibited ethylene generation in plant tissues. Among the substances tested were selenoamino acids which have been reported to act as protectants against freeradical attack (12). We found that selenomethionine and selenoethionine greatly enhanced ethylene formation in senescing flower tissue and in pea stem sections treated with IAA. Our results indicate that in these tissues both selenoamino acids are better precursors of ethylene than is methionine. MATERIALS AND METHODSPlant Material. Morning glory plants (Ipomoea tricolor Cav., cv. Heavenly Blue) were grown as described before (2). Rib segments were prepared from flower buds according to Kende and Hanson (5). The following designations are used to describe the age of the flower tissue: day 0, day of opening and fading of the flower; day -1, 1 day before flower opening; day -2, 2 days before flower opening.Pea seeds, Pisum sativum L., cv. Alaska (Vaughan's Seed Co., Downers Grove, Ill.), were imbibed for 5 hr or overnight in aerated tap water. They were sown in Vermiculite and grown in the dark at 25 C for 6 to 9 days. Pea stem sections were isolated between 4:00 and 6:00 PM, and batches of 12 sections were floated in 6-cm Petri dishes on 2 ml of distilled H20 or a solution of the compound to be tested. The dishes were kept overnight in the dark at 25 C. The stem sections were removed from the Petri dishes between 8:00 and 9:00 AM the next morning. They were rinsed with distilled H20, blotted dry, and transferred to 25-ml Erlenmeyer flasks containing 1 ml of distilled H20 or the appropriate test solution with or without 0.1 mM IAA. Each flask was flushed with ethylene-free air for 2 min, closed with a serum-vial cap and incubated in darkness at 27 C.The pH of all test solutions was adjusted with 0.1 N NaOH to 6.5 to 7.0. Each experiment was repeated at least three times with similar results.Determination of Ethylene Formation. Ethylene production was measured by gas chromatography as described earlier (5).Determination of Specific Radioactivity of Ethylene. The specific radioactivity ofethylene was determined according to Hanson and Kende (3) RESULTS Effect of Selenoamino Acids on Ethylene Synthesis in FlowerTissue. When rib segments excised from flower buds of I. tricolor were incubated continuously from the afternoon of day -I through the evening of day 0 on solutions containing selenomethionine, ethylene production on day 0 commenced earlier and proceeded at a higher rate than in the control tissue (Fig. 1). The total amount of ethylene produced on day 0 by rib segments treated with selenomethionine was usually three times higher than that produced by rib segments incubated on water. This result was surprising since methionine, the precursor of ethylene in mor...

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