Presence of β-cyanoalanine synthase in unimbibed dry seeds and its activation by ethylene during pre-germination

Evolution of HCN from both rice ( Oryza sativa ) and cocklebur ( Xanthium pennsylvanicum ) seeds increased during a pre-germination period and preceded the evolution of (C₂H₄). These two species were adopted as the representatives of starchy and fatty seeds, respectively. Ethylene promotes seed germination of many species. However, HCN evolution declined abruptly when the radicles emerged and before the peak in C₂H₄ evolution. More-over, both rice and soybean ( Glycine max ) seeds showed some activity of β-cyanoalanine synthase (CAS, EC 4.4.1.9) even in the unimbibed dry state. The activities of CAS in the lower seed of cocklebur and in soybean seeds increased rapidly after emergence of the radicle. However, the CAS of rice seeds, with high activity in the dry state, exhibited a bimodal change, gradually decreasing until radicle emergence had occurred, but then increaing. It is thus likly that HCN evolution during initial imbibition may be derived from cyanogenic reserves and controlled by both pre-existing and subsequently-developing CAS. The exogenous application of C₂H₄ stimulated the activities of CAS in both rice and upper cocklebur seeds and reduced their cyanogen contents. Therefore, the decline of HCN evolution after germination seems to be due to the increased activities of CAS by endogenously produced C₂H₄.     

C2H2 and Ar induce rapid declines in nitrogenase activity and CO2 evolution in nodules of Datisca glomerata

Preliminary studies have indicated that after addition of C₂H₂ there is a rapid decline in nitrogenase activity in the nodules of Datisca glomerata . The present work was undertaken to determine whether (1) there is also a decline in respiration and (2) the decline is associated with the cessation of ammonia production. The rates of C₂H₄ and CO₂ evolution by nodulated root systems of Datisca were measured as a function of time after exposure to C₂H₂. The peak rate of C₂H₄ evolution occurred at 30 s after C₂H₂ exposure, while the rate of CO₂ evolution started to decline at 60 s after exposure to C₂H₂. Incubation of nodules in a gas mixture containing Ar also caused a decline in CO₂ evolution. Further, pretreatment with Ar eliminated most of the C₂H₂-induced decline in nitrogenase activity and CO₂ evolution. These C₂H₂- and Ar-induced declines in Datisca nodules are more rapid than those reported in any other nodules. They are evidence that continued ammonia formation is essential for maintenance of normal nitrogenase activity in Datisca nodules.     

Characterization of cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Cyclic nucleotide phosphodiesterase (3',5'-cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, chromatography on 3',5'-cAMP-agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH₄)₂SO₄ pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca²⁺, Mg²⁺ and Mn²⁺, whereas NaF, PP_i and Fe³⁺ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An M_I of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3',5'-cAMP-agarose a phosphodiesterase was resolved that produced 5'-AMP as sole reaction product.     

Culture conditions affect the chemical composition of the exopolysaccharide synthesized by the fungus Aureobasidium pullulans

Aims:  To identify if culture conditions affect the chemical composition of exopolysaccharide (EPS) produced by Aureobasidium pullulans .
Methods and Results:  In batch airlift and continuously stirred tank (CSTR) reactors the EPS produced with low (0·13 g l⁻¹ N) initial NaNO₃ or (NH₄)₂SO₄ levels contained pullulan, with maltotriose as its major component, similar to that synthesized in the airlift reactor with high (0·78 g l⁻¹ N) initial NaNO₃ levels. EPS produced by CSTR grown cultures with high (NH₄)₂SO₄ levels contained little pullulan, possibly because of a population shift from unicells to mycelium. This chemical difference may explain why total EPS yields did not fall as they did with cultures grown under identical conditions with high NaNO₃ levels, where the pullulan component of the EPS disappeared. EPS synthesized in N-limiting chemostat cultures of A. pullulans changed little with growth rate or N source, being predominantly pullulan consisting of maltotriose units.
Conclusions:  While the EPS chemical composition changed little under N-limiting conditions, high initial medium N levels determined maltotriose content and/or pullulan content possibly by dictating culture morphology.
Significance and Impact of the Study:  These results emphasize the requirement of all studies to determine EPS chemical composition when examining the influence of culture conditions on EPS yields.     

Role of growth regulators in the senescence of Arabidopsis thaliana leaves

A homozygous, dominant, C₂H₄-resistant line of Arabidopsis thaliana (L.) Heynh (cv. Columbia; er ) was selected from ethylmethylsulfonate-mutagenized seed, and used to test the role of C₂H₄ and other growth regulators in senescence of mature leaves. Chlorophyll (Chl) loss from disks excised from leaves of er was much slower than that from wild-type (WT) disks, whether they were held in the light or in the dark. C₂H₄ accelerated Che loss from WT disks but had no effect on the yellowing of mutant disks. C₂H₄ biosynthesis was higher in disks from the mutant plants, particularly in the light. In the dark, treatment with the cytokinin, 6-benzyladenine (BA), reduced Chl loss from wild-type disks, but had no effect on mutant disks. In the light, BA treatment stimulated chlorophyll breakdown in both wild type and mutant disks. Treatment with abscisic acid (ABA) stimulated chlorophyll loss in wild-type and mutant disks, whether they were held in the light or the dark. C₂H₄ production was stimulated in ABA-treated disks, but they still yellowed even when C₂H₄ production was inhibited by application of aminooxyacetic acid (AOA). These data indicate that C₂H₄ is only one of the factors involved in leaf senescence, and that the promotion of senescence by ABA is not mediated through its stimulation of C₂H₄ production.     

l-Myo-inositol-1-phosphate synthase from lower plant groups: Partial purification and properties of the enzyme from Euglena gracilis

A survey for the enzyme L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) has been conducted among various members of the lower plant groups, mainly algac, bryophytes and fungi; some properties of the partially purified enzyme from Euglena gracilis Z . are presented. The enzyme was detected in Chloropycean algae, Marchantiales and the Basidiomycetous fungi. The enzyme from Euglena had a pH optimum at 7.5. The Km for glucose-6-P was 2.1 m M and for NAD⁺ 80 μ M . When assayed in the absence of added NAD⁺, the enzyme showed a basal activity suggesting the presence of bund NAD⁺ in the system. NH₄Cl increased the enzyme activity two-fold, altough the enzyme was inactivated by (NH₄)₂SO₄.     

Associative cellulolysis and N2 fixation by co-cultures of Trichoderma harzianum and Clostridium butyricum: the effects of ammonium-N on these processes

Mixed cultures of the cellulolytic fungus Trichoderma harzianum with the anaerobic diazotroph Clostridium butyricum were shown to co-operatively degrade cellulose and utilize the degradation products for N₂ fixation. Cellulose degradation and N₂ fixation were stimulated by small (0.1 mg/ml) additions of (NH₄)₂SO₄. The (NH₄₂SO₄ stimulates cellulolysis thereby increasing the supply of cellulose degradation products to the diazotroph. In aerobic environments the anaerobe depends on the respiration of the aerobe to create anaerobic microsites. The N source increased O₂ uptake by the fungus increasing the number of sites suitable for the development of the anaerobe. Stimulation in the growth of T. harzianum by (NH₄₂SO₄ resulted in increased growth and N₂ fixation by Cl. butyricum.     

Utilization of nitrogen from storage and current-year uptake in walnut spurs during the spring flush of growth

[¹⁵N]-depleted (NH₄)₂SO₄ applied to the soil in 1985 resulted in residual labeling of about 16% of the storage nitrogen (N) pool of mature walnut ( Juglans regia L. cv. Serr) trees in 1987. Application of [¹⁵N]-depleted (NH₄)₂SO₄ fertilizer to a different set of mature walnut trees in 1987 allowed monitoring of the kinetics and utilization of N from current year uptake in 1987 and resulted in >20% labeling of fruit N following completion of leaf expansion. Redistribution of storage N to the new growth predominated during the spring flush of growth although N derived from the soil during current-year uptake contributed increasingly during leaf expansion. Labeled N from current year uptake accumulated preferentially in the leaves as compared with reproductive organs during leaf expansion but subsequent to leaf expansion, fruit were more highly labeled with N derived from current-year uptake than leaves. Pistillate flower abortion was coincident with an apparent competition for N among developing vegetative and reproductive organs and preceded the period of significant N contribution from current-year uptake.     

Cyanogenic glycosides and their metabolic enzymes in barley, in relation to nitrogen levels

The possible role for cyanogenic glycosides as nitrogen storage compounds was studied in barley, Hordeum vulgare (cv. Golf), cultivated under different nitrogen regimes. Cyanogenic glycosides were absent in seeds and roots but were synthesized in seedlings where they accumulated at a level of about 150 nmol shoot⁻¹ in control plants and 110 nmol shoot⁻¹ in nitrogen-starved plants. An enzyme involved in the breakdown of cyanogenic glycosides, β-glucosidase (EC 3.2.1.-) exhibited high activity in seeds and was also detected in roots and shoots. The activity of β-cyanoalanine synthase (EC 4.4.1.9), which is involved in the metabolism of HCN, was low in seeds but very high in roots and shoots. There was no correlation between the activities of the two enzymes and the content of cyanogenic glycosides or nitrogen. The relative content of nitrogen in cyanogenic glycosides never exceeded 0.3% of total nitrogen, and the amount of cyanogenic glycosides decreased at a low rate even at a stage when nitrogen limitation inhibited growth.     

Effects of age, supra-ambient oxygen and repeated assays on acetylene reduction and root respiration in pea

An open flow-through gas system was used to investigate the effect of plant age on nitrogenase activity in relation to root respiration (measured as CO₂ release) and supra-ambient O₂ levels in 24- to 51-day-old, nodulated Pisum sativum L. cv. Bodil. The effect of assaying plants repeatedly was also studied. The respiratory efficiency of nitrogenase [mol CO₂ (mol C₂H₄)⁻¹] and the relative decline in nitrogenase (EC 1.7.99.2) activity in response to introduction of C₂H₂ in the gas stream were unaffected by plant age. In contrast, the nitrogenase-linked respiration as a proportion of total root respiration increased with time. Accordingly, the specific respiration linked-to growth and maintenace of the noduled root system decreased with time. C₂H₂ reduction and root respiration were increased by supra-ambient O₂ levels, but the tolerance to high O₂ concentrations seemed to decrease with plant age. Repeated C₂H₂ assays on the same plants decreased their rate of growth and N accumulation: in addition, nitrogenase activity and root respiration were somewhat negatively affected. The results indicate that results from experiments with plants of different ages cannot always be directly compared, and that repeated C₂H₂ assays on the same plants should be applied with caution in physiological work.     

Nitrogen regulation of erythromycin formation in Streptomyces erythreus

Abstract Erythromycin formation decreased in Streptomyces erythreus as a function of the ammonium concentration present in the medium. Total inhibition of synthesis was obtained with 100 mM NH₄Cl but medium pH and culture growth were not significantly affected. A similar effect was obtained with NH₄NO₃ or (NH₄)₂SO₄ indicating that ammonium ion probably repressed formation of antibiotic.     

Evidence for the occurrence of the alternative, vanadium-containing nitrogenase in the cyanobacterium Anabaena variabilis

Abstract Anabaena variabilis can be grown with dependence on either molybdenum (Mo) or vanadium (V) in the medium with essentially the same growth rates. Vanadium cultures reduce C₂H₂ to C₂H₄ and partly (to 2–3%) to C₂H₆. These C₂H₄ and C₂H₆ formations can be shown to be strictly light dependent, proving that the gases are formed by the cyanobacterium. C₂H₄ and C₂H₆ productions are accompanied by a H₂ formation which is much higher than in Mo cultures. Maximal C₂H₂-formation rates are 2/3 lower in V-grown cells compared to Mo control cultures. This is the first demonstration of a light-dependent ethane formation and of the occurrence of the alternative nitrogenase in any phototroph.     

Effects of propylene and oxygen on the ethylene-producing system of apples

Hypobaric conditions and treatments with ethylene and the ethylene analogue propylene were used to investigate effects of oxygen and elhylene on 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase activity and ethylene production of apples ( Malus sylveslris Mill. cv. Golden Delicious). Prcclimacteric apples were stored in air at 6.6 kPa (reduced pressure); 6.6 kPa ventilated with pure O₂; 6.6 kPa ventilated with 2600 μl 1⁻¹ C₂H₄; and in air at 101.3 kPa (atmospheric pressure) for 4 months at 4°C. No ACC synthase activity was detectable in apples stored at 6.6 kPa, whereas ACC synthase activity was induced in apples stored at 6.6 kPa and ventilated with either O₂ or C₂H₄. In a further experiment, preclimacteric apples were stored for 14 days either in air at 20 kPa or at 20 kPa ventilated with pure O₂. Both treatments were supplied with 58 500 μl 1⁻¹ propylene from day 0 to day 9 or from day 9 to day 12. Ethylene production of apples treated with propylene from day 0 to day 9 increased earlier than ethylene production of untreated apples. Propylene treatment from day 9 to day 12 did not stimulate ethylene production. Ethylene and propylene induced and stimulated extractable ACC synthase activity and ACC formation of apples. Oxygen enhanced this effect. The results also suggest inhibition of in vivo ACC synthase activity by propylene.     

Relationship between C2H2 reduction, H2 evolution and 15N2 fixation in root nodules of pea (Pisum sativum)

The quantitative relationship between C₂H₂ reduction, H₂ evolution and ¹⁵N₂ fixation was investigated in excised root nodules from pea plants ( Pisum sativum L. cv. Bodil) grown under controlled conditions. The C₂H₂/N₂ conversion factor varied from 3.31 to 5.12 between the 32nd and the 67th day after planting. After correction for H₂ evolution in air, the factor (C₂H₂-H₂)/N₂ decreased to values near the theoretical value 3, or in one case to a value significantly ( P < 0.05) below 3. The proportion of the total electron flow through nitrogenase, which is not wasted in H₂ production but used for N₂ reduction, is often stated as the relative efficiency (1-H₂/C₂H₂). This factor varied significantly ( P < 0.05) during the growth period. The actual allocation of electrons to H₂ and N₂, expressed as the H₂/N₂ ratio, was independent of plant age, however. This discrepancy and the observation that the (C₂H₂-H₂)/N₂ conversion factor tended to be lower than 3, suggests that the C₂H₂reduction assay underestimates the total electron flow through nitrogenase.     

Patterns of ammonium absorption and acetylene reduction during soybean developmental growth

Nodulated and unnodulated soybean plants ( Glycine max (L.) Merr. cv. Amsoy 71) were grown in nutrient solution either lacking or containing N. Nodulated plants, dependent on N₂ fixation, exhibited a generalized N-stress and were less vigorous than unnodulated plants dependent on inorganic N assimilation.
Starting at preflowering throughout mid pod-filling, NH₄⁺ absorption, expressed on the basis of root dry weight, was determined for intact nodulated and unnodulated plants in short-term kinetic experiments. Depletion of NH₄⁺ was measured from the liquid phase of a mist chamber. Maximum NH₄⁺ absorption occurred for both nodulated and unnodulated plants during vegetative growth. A pattern of progressive decrease in NH₄⁺ absorption was similar in nodulated and unnodulated plants, however. NH₄⁺ absorption was consistently greater in unnodulated plants. Simultaneous measurements of C₂H₂ reduction from the gas phase of the mist chamber revealed and 41-day-old plants, corresponding to late flowering and early pod-filling.     

Effect of H2 evolution on 15N2 fixation, C2H2 reduction and relative efficiency of leguminous symbionts

The (C₂H₄+ H_2(C2H₂))/¹⁵N₂ ratios of 15 clover- Rhizobium symbionts. soybean, and black medick symbionts were measured. Relative efficiency based on the C2H4 production and on ¹⁵N₂ incorporation were compared, and in most symbionts there was little difference between the two measures of relative efficiency. Total measurable electron flux through nitrogenase during acetylene reduction and ¹⁵N₂ incorporation were nearly equal for most symbionts studied. The relative efficiency and the (C₂H₄+ H_2(C2H₂))/¹⁵N₂ ratio showed an inverse correlation. Use of this ratio appears preferable to use of the ratio of C2H2 reduction/N₂ reduction. Some evolution of H₂ was observed in the presence of C₂H₂.     

The role of phosphorus in nitrogen fixation by young pea plants (Pisum sativum)

The influence of P on N₂ fixation and dry matter production of young pea ( Pisum sativum L. cv. Bodil) plants grown in a soil-sand mixture was investigated in growth cabinet experiments. Nodule dry weight, specific C₂H₂ reduction and P concentration in shoots responded to P addition before any growth response could be observed. The P concentration in nodules responded only slightly to P addition. A supply of P to P-deficient plants increased both the nodule dry weight, specific C₂H₂ reduction and P concentration in shoots relatively faster than it increased shoot dry weight and P concentration in nodules. Combined N applied to plants when N₂ fixation had commenced, increased shoot dry weight only at the highest P levels. This indicates that the smaller plant growth at the low P levels did not result from N deficiency. The reduced nodulation and N₂ fixation in P-deficient plants seem to be caused by impaired shoot metabolism and not by a direct effect of P deficiency of the nodules.     

Effect of pH, temperature and culture medium composition on the production of an extracellular cysteine proteinase by Micrococcus sp. INIA 528

Growth and proteinase production by Micrococcus sp. INIA 528 in a batch-operated laboratory fermentor were investigated, with trypticase soy broth as the basal medium for studies on optimum temperature, pH and medium composition. Maximum growth was recorded at 34°C and pH 715, whereas optimum temperature and pH for proteinase production were 31°C and pH 6.25. Maximum rate of enzyme production occurred during the late log and early stationary phases of growth. Addition of 5.0 g 1^-1 yeast extract, 1.0 g 1^-1 glucose, 1.0 g 1^-1 MgSO₄ or 1.0 g 1^-1 K₂HPO₄ to basal medium resulted in a lower enzyme yield, but supplementation of basal medium with 2.5 g 1^-1 (NH₄)₂SO₄ increased enzyme production by 45%. A high initial biomass added to fresh broth supplemented with 2.5 g 1^-1 (NH₄)₂SO₄ only increased enzyme activity by 19%, compared to the maximum enzyme activity achieved with the standard inoculum.     

Glycolate metabolism in cyanobacteria. III. Nitrogen controls excretion and metabolism of glycolate in Anabaena cylindrica

The effect of nitrogen on excretion and metabolism of glycolate in Anabaena cylindrica (CCAP 1403/2a) was studied. Glycidate, an inhibitor of glutamate:glyoxylate aminotransferase (EC 2.6.1.4), reduced the L-methionine-DL-sulfoximine-induced NH₄⁺ release by ca 40%, while net CO₂ fixation and C₂H₂ reduction were not lowered. This indicates that at least a part of the glyoxylate synthesized in A. cylindrica is metabolized via glycine to serine. Addition of NH₄Cl or glutamate to the medium reduced the excretion of glycolate. At pH 9, under air, NH₄Cl reduced the excretion by 10–30% and under high pO2 (0.03 kPa CO₂ in O₂) by about 80–90%. At pH 7.5, under high pO₂, NH₄Cl and glulamate reduced the excretion by about 40 and 80%, respectively. Also, the presence of NH₄Cl stimulated the animation of glyoxylate under such conditions as shown by an increased glycine pool and a decreased glutamate pool. We suggest that nitrogen regulates the capacity of A. cylindrica to retain and recycle glycolate intracellularly and that glutamate serves as an amino donor in the conversion of glyoxylate to glycine.