Seed dormancy in Avena fatua: Interacting effects of nitrate, water and seed coat injury

In experiments conducted under controlled conditions. KNO₃ (50 or 100 m M ) promoted germination of a dormant strain (AN 474) of Avena fatua when either one or two holes were pierced in the lower (adaxial) surface of the caryopsis in contact with the nitrate solution. Germination was increased by increasing either the KNO₃ concentration or the number of holes in the seed coat. The germination response induced by the application of water to a hole pierced in the upper surface of the caryopsis was. increased by pre-treatment of the intact caryopsis with KNO₃. Treatment with either 50 or 100 m M KNO₃ caused a transient reduction in embryo water content of intact cary-opses, but increased the nitrate and amino- N content of pierced caryopses prior to germination. Supplying a 100 m M solution of KNO₃ to pierced caryopses reduced the total water potential and osmotic potential of the embryo, and increased its pressure potential by the same amount as an equimolar solution of KC1; however, while both treatments promoted germination, the KNO₃ induced more rapid germination than the KCI. Both treatments also increased the K⁺ content of the embryo, the KNO₃ again having the greater effect. These results are consistent with the hypothesis, based on our previous investigations, that KNO₃ promotes germination of dormant caryopses by accumulating in the embryo where it acts osmotically to increase water uptake. It is also postulated, that, in contrast to KCI, KNG₃ may combine an osmotic effect on water uptake with a nutritional effect on protein synthesis.     

Effects of oxygen level on metabolism and development of seedlings of Trapa natans and two ecologically related species

Seeds of the water plant Trapa natans L. (water chestnut) can germinate in strict anoxia. The seedlings show seminal roots growing upwards while shoot buds remain quiescent until O₂ becomes available. Trapa seedlings are highly tolerant to anoxia. The rate of ethanol fermentation was 21.2 μmol (g FW)⁻¹ h⁻¹, while production of lactate was negligible and lower than that of succinate. The seminal root of Trapa compares better to the rice coleoptile rather than to the rice root, both functionally and as to the metabolic response to anoxia. The anaerobic germination of Nuphar luteum L. and Scirpus mucronatus L. was also characterized by a limited developmental program.     

Effect of fusicoccin and gibberellic acid on primary dormant Avena fatua caryopses

Fusicoccin induced germination in dormant and partially afterripened dormant caryopses of Avena fatua L. The rate of caryopsis germination was slower and final percentage germination lower in the highly dormant inbred line M73 at a given concentration of fusicoccin than in the dormant caryopses of line AN265. Gibberellic acid was more effective than fusicoccin in breaking dormancy in both lines. Promotion of germination of dormant caryopses by fusicoccin was inhibited by a 6-day pretreatment with (2-chloroethyl)trimethylammonium chloride.
The basal rate of proton efflux from embryos isolated from dormant and fully afterripened line AN265 caryopses was similar. Addition of fusicoccin increased the rate of proton efflux from the isolated embryos of dormant and afterripened caryopses by nearly 400%. Gibberellic acid had no effect on the rate of proton extrusion. The uptake of ⁸⁶Rb⁺ in dormant and afterripened A. fatua embryos was similar after a 2 h uptake period. The addition of fusicoccin to the medium doubled the uptake of ⁸⁶Rb⁴ by dormant and afterripened embryos. Gibberelleic acid had no effect on the uptake of ⁸⁶Rb⁺ by isolated embryos from either dormant or afterripened caryopses. The experimental results indicate that gibberellic acid is more versatile in its action than fusicoccin, and gibberellic acid may facilitate dormant A. fatua caryopsis germination by stimulating mechanisms other than the direct H⁺ efflux and K⁺ uptake at the membrane level.     

Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor

Abstract Until now, oxidation of ammonium has only been known to proceed under aerobic conditions. Recently, we observed that NH₄⁺ was disappearing from a denitrifying fluidized bed reactor treating effluent from a methanogenic reactor. Both nitrate and ammonium consumption increased with concomitant gas production. A maximum ammonium removal rate of 0.4 kg N · m⁻³ · d⁻¹ (1.2 mM/h) was observed. The evidence for this anaerobic ammonium oxidation was based on nitrogen and redox balances in continuous-flow experiments. It was shown that for the oxidation of 5 mol ammonium, 3 mol nitrate were required, resulting in the formation of 4 mol dinitrogen gas. Subsequent batch experiments confirmed that the NH₄⁺ conversion was nitrate dependent. It was concluded that anaerobic ammonium oxidation is a new process in which ammonium is oxidized with nitrate serving as the electron acceptor under anaerobic conditions, producing dinitrogen gas. This biological process has been given the name ‘Anammox” (anaerobic ammonium oxidation), and has been patented.     

The physiological basis of seed dormancy in Arena fatua V. Action of ethanol and other organic compounds

Ethanol (50-200 mM )induced germination in four genetically pure dormant lines of Avena fatua L. The sensitivity to this treatment was moderate immediately after harvest and increased steadily during six months of after-ripening. This sensitivity to ethanol was detectable much earlier during after-ripening than with two other germination promoters, NaN³, and NaNO³, Because ethanol can overcome dormancy in freshly harvested caryopses, the mode of action of ethanol in these caryopses apparently differs from that of the two other promoters, azide and nitrate. Nevertheless, it is clear that induction of germination by the three promoters is fully gibberellin-dependent since in each case this response can be blocked by the administration of 2-chlorocthyl trimethylammonium chloride, an inhibitor of gibberellin biosynthesis. Short-term incubation treatments with ethanol were relatively more effective than continuous treatments. These brief treatments were most effective when presented near the beginning of seed imbibition. Among other organic compounds tested only acetaldehyde significantly promoted germination in all lines tested. Propan-1-ol, butan-l-ol, chloral hydrate, procaine, methanol and chloroform were marginally effective on the least dormant lines, while ether, formaldehyde, acetone and ethyl acetate were ineffective. The mode of action of ethanol in overcoming dormancy in both freshly harvested and partly after-ripened caryopses is discussed and the possible role as a metabolic substrate or anaesthetic is indicated.     

Utilization of Stored Nitrate during the Anaerobic Germination of Rice Seeds

The presence of nitrate in the seed and its utilization duringanaerobic germination of rice (Oryza sativa L.) were studied.The results indicate that nitrate was reduced and assimilatedby the coleoptile for 11 days during anaerobic growth aftergermination. The importance of the anoxic utilization of nitrateis discussed. (Received June 19, 1992; Accepted January 4, 1993)     

Regulation of the germination of Aleppo pine (Pinus halepensis) by nitrate, ammonium, and gibberellin, and its role in post-fire forest regeneration

Aleppo pine ( Pinus halepensis Mill.) regenerates after wildfires only by germination. This post-fire germination is controlled by temperature, light and the chemical characteristics of the ash-bed. The extremely high pH caused by the ash inhibits germination of Aleppo pine as well as of many other species. Here we report that ash extracts at neutral pH stimulate P. halepensis germination. The effects of ash extracts, nitrate, ammonium (in concentrations present in the ash) and GA₃ on the germination of P. halepensis seeds and on lipase activity at early germination were studied under controlled conditions. Sub-optimal concentrations of nitrate- and ammonium-stimulated germination and the activity of lipase in the seeds. It is proposed that nitrate increases seed sensitivity to GA₃ while ammonium directly affects lipase activity. The balance between the inhibition of germination by the high pH of ash and its stimulation by the nitrate and ammonium present in the ash determines the overall effect of the ash on post-fire germination of Aleppo pine. The pine seedlings appear in high density among the trees but because of the effects of the ash they appear in sparse stands under the projection of the burned pine canopies. These seedlings benefit from weak inter- and intra-specific competition as well as improved mineral nutrition and have good chances to grow into large trees that will form the regenerating forest; therefore, the enhancement of P. halepensis germination by nitrate and ammonium and its inhibition by high pH can be considered as ecophysiological adaptations to post-fire regeneration.     

Nonstructural carbohydrates in dormant and afterripened wild oat caryopses

Nonstructural carbohydrates were determined in both embryo and endosperm of dormant (nongerminating) and afterripened (germinating) intact caryopses of wild oat ( Avena fatua L.). No changes in endosperm starch or soluble sugar were observed at the onset of germination (18 h). No changes in glucose, fructose, sucrose or starch within dormant or afterripened embryos correlated with onset of visual germination. In afterripened embryos, depletion of raffinose (18 h), stachyose (18 h) and galactose (24 h) was correlated with germination. In contrast, raffinose-family oligosaccharide levels in dormant embryos remained constant for 7 days following imbibition. Germination of isolated dormant embryos on 88 m M galactose-containing media was accompanied by decreased endogenous levels of raffinose and stachyose. Isolated embryos from dormant caryopses incorporated ¹⁴C from ¹⁴C-fructose into both raffinose and stachyose during 24 h of imbibition. In contrast, no ¹⁴C incorporation into stachyose was observed in embryos from afterripened caryopses. No ¹⁴C incorporation into raffinose was observed at 18 and 24 h. When in vitro activities of α galactosidase were measured, no temporal differences between dormant or afterripened caryopses were detected in either embryo or endosperm tissue. Although the mechanism associated with differences in utilization of raffinose and stachyose is yet unidentified, alterations in raffinose-family oligosaccharide metabolism in the embryo appear to be a unique prerequisite for afterripening-induced germination.     

Expression of glutamine synthetase during the anaerobic germination of Oryza sativa L.

Glutamine synthetase (GS; EC.6.3.1.2.) occurs as cytosolic (GS₁) and plastidic (GS₂) polypeptides. This paper describes the expression of GS isoenzymes in coleoptile during the anaerobic germination of rice (Oryza sativa L.) and the influence of exogenous nitrate on this. By immunoprecipitation with anti-GS serum, two polypeptides of 41- and 44-kDa were detected of which the former was predominant. After fractionation by ion-exchange chromatography, the 41 and 44 kDa bands were identified as GS₁ and GS₂, respectively. Northern blot analysis with specific probes showed the presence of mRNA for cytosolic GS but not for the plastidic form. The presence of exogenous nitrate did not alter the activity and expression of GS in the coleoptile. The role of GS during the anaerobic germination of rice seems to induce the re-assimilation of ammonia rather than the assimilation of nitrate.Abbreviations GS glutamine synthetase - GS₁ cytosolic glutamine synthetase - GS₂ platidic glutamine synthetase We are grateful to Dr. Julie V. Cullimore for providing GS anti-serum and clones. The research was supported by the National Research Council of Italy, special project RAISA, sub-project N. 2 paper N. 1586.     

Ethylene production is associated with germination but not seed dormancy in red rice

BACKGROUND AND AIMS: The relationship between ethylene production and both seed dormancy and germination was investigated using red rice (weedy rice) as a model species. METHODS: Both fully dormant and after-ripened (non-dormant) naked caryopses were incubated with or without inhibitors of ethylene synthesis [aminoethoxyvinylglycine (AVG)] and perception [silver thiosulfate (STS)], or in the presence of the natural ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). The kinetics of ethylene emissions were measured with a sensitive laser-photoacoustic system. KEY RESULTS: Dormant red rice caryopses did not produce ethylene. In non-dormant caryopses, ethylene evolution never preceded the first visible stage of germination (pericarp splitting), and ethylene inhibitors completely blocked ethylene production, but not pericarp splitting. Accordingly, endogenous ACC appeared to be lacking before pericarp splitting. However, early seedling growth (radicle or coleoptile attaining the length of 1 mm) followed ethylene evolution and was delayed by the inhibitors. Wounding the dormant caryopses induced them to germinate and produce ethylene, but their germination was slow and pericarp splitting could be speeded up by ethylene. CONCLUSIONS: The findings suggest that, in red rice, endogenous ethylene stimulates the growth of the nascent seedling, but does not affect seed dormancy or germination inception. Correspondingly, this phytohormone does not play a role in the dormancy breakage induced by wounding, but accelerates germination after such breakage has occurred.     

Production of clover broomrape seed germination stimulants by red clover root requires nitrate but is inhibited by phosphate and ammonium

The effect of nutrients (nitrate, ammonium, urea, phosphate and potassium) on the production and/or exudation of germination stimulants for clover broomrape ( Orobanche minor Sm.), a root holoparasite, by its host red clover ( Trifolium pratense L.) was examined using hydroponically grown material. Potassium (K₂SO₄) concentrations up to 100 mg l^–1 (based on K) did not affect the production of germination stimulants by red clover while, in contrast, phosphate (NaH₂PO₄) was highly inhibitory even at concentrations as low as 1 mg l^–1 (based on P). Nitrate (NaNO₃) markedly promoted stimulant production in a dose-dependent manner from 2 to 50 mg l^–1 (based on N). Ammonium [(NH₄)₂SO₄] had no effect at 2 mg l^–1 (based on N) but was inhibitory at higher concentrations. Ammonium is known to be a seed germination inhibitor of root parasites, indicating that ammonium has a dual inhibitory action. Urea had no effect at 2 mg l^–1 (based on N) but was promotive at higher concentrations. These results provide a basis for the inhibitory effects of nitrogen fertilizer on infection by root parasitic weeds, broomrapes and witchweeds, and explain why these parasites prevail in areas where there is lower phosphorus availability in soils.     

Effect of CO2 and phosphate deprivation on the control of nitrate, nitrite and ammonium metabolism in Chlorella

Chlorella vulgaris Beijerinck, strain 211/12, uses nitrate, nitrite and ammonium at pH 8.2 but not at pH 6.4 when kept under conditions of CO₂-deprivation, as observed in cell suspensions aerated with CO₂-free air during a 20–30. h period Most of the nitrate absorbed at pH 8.2, however, was not assimilated but was released into the external medium as nitrite and ammonium. Cells of Chlorella previously grown in phosphate-limited continuous cultures were unable to absorb nitrate, nitrite or ammonium under conditions of phosphate starvation at either pH 6.4 or 8.2 in cell suspensions flushed with air containing 5% CO₂, However, in cell suspensions flushed with CO₂-free air, the capacity of the alga to absorb and reduce nitrate and to excrete nitrite and ammonium at pH 8.2 was restored.
It is hypothesized that in Chlorella the metabolism of nitrate, nitrite and ammonium is influenced by the availability of other nutrients and controlled by the cell's carbon status at the level of ion entry into the cell. With respect to nitrate this carbon-dependent control is distinct and works independently of that triggered by the cell's nitrogen status.     

Anaerobic nitrate reduction to ammonium in two strains isolated from coastal marine sediment: A dissimilatory pathway

Abstract: A total of 28 nitrate-reducing bacteria were isolated from marine sediment (Mediterranean coast of France) in which dissimilatory reduction of nitrate to ammonium (DRNA) was estimated as 80% of the overall nitrate consumption. Thirteen isolates were considered as denitrifiers and ten as dissimilatory ammonium producers. ¹⁵N ammonium production from ¹⁵N nitrate by an Enterobacter sp. and a Vibrio sp., the predominant bacteria involved in nitrate ammonification in marine sediment, was characterized in pure culture studies. For both strains studied, nitrate-limited culture (1 mM) produced ammonium as the main product of nitrate reduction (> 90%) while in the presence of 10 mM nitrate, nitrite was accumulated in the spent media and ammonia production was less efficient. Concomitantly with the dissimilation of nitrate to nitrite and ammonium the molar yield of growth on glucose increased. Metabolic products of glucose were investigated under different growth conditions. Under anaerobic conditions without nitrate, ethanol was formed as the main product; in the presence of nitrate, ethanol disappeared and acetate increased concomitantly with an increased amount of ammonium. These results indicate that nitrite reduction to ammonium allows NAD regeneration and ATP synthesis through acetate formation, instead of ethanol formation which was favoured in the absence of nitrate.     

The physiological basis of seed dormancy in Avena fatua. IX. Characterization of two dormancy states

The dormancy-breaking effect of several known germination promoters was studied in 9 genetically pure lines of Avena fatua L. during a period of controlled after-ripening. Changes in the germination response show at least two dormancy states in the caryopses of these lines. The first state is overcome by a short period of after-ripening and is insensitive to nitrate and azide, while the second state is more persistent and is sensitive to nitrate and azide. Both states are sensitive to gibberellic acid (OA,) and ethanol. In the most dormant lines a third ethanol-insensitive dormancy state is present. The duration of both major dormancy states was related to several environmental factors influencing plant growth and seed storage. Duration was increased in caryopses produced from plants matured under low temperatures (15°C) and decreased in caryopses produced from plants matured under high temperatures (25°C). Duration was increased in caryopses after-ripened under low temperatures (4°C) and decreased in caryopses after-ripened under high temperatures (45°C). Dehulling the seeds prior to after-ripening reduced the duration of both major dormancy states. The multiple state dormancy system and its environmentally induced plasticity are discussed with reference to previous explanations of the dormancy mechanism in wild oats.     

Effects of oxygen, pH and nitrate concentration on denitrification by Pseudomonas species

Abstract The production of nitrogen-containing gases by denitrification in three organisms was examined using membrane inlet mass spectrometry. The effects of O₂ (during both growth and maintenance) and of pH, nitrate concentration and carbon source were tested in non-proliferating cell suspensions. Two strains of Pseudomonas aeruginosa were capable of co-respiration of NO₃⁻ and O₂ and, under controlled O₂ supply, gave oscillatory denitrification. Variations in culture and assay conditions affected both the rate of denitrification and the ratio of end products (N₂O:N₂). Higher rates were seen following anaerobic growth. Optimum values of pH and nitrate concentration for denitrification are given. Generally, the optimum pH was 7.0–7.5, approximately that of the growth medium. Optimum nitrate concentration was generally 20 mM.     

Amylolytic activity and carbohydrate levels in relation to coleoptile anoxic elongation in Oryza sativa genotypes

Among starchy seeds, rice has the unique capacity to germinate successfully under complete anaerobiosis. In this conditions, starch degradation is supported by a complete set of starch-degrading enzymes that are absent or inactive in cereals except rice. A characterization of carbohydrate metabolism and starch-degrading enzyme activity across twenty-nine genotypes of Oryza sativa L. is presented here. The zymogram of amylolytic activities present in rice embryos and endosperms under anaerobic conditions seven days after sowing (DAS) revealed marked differences among cultivars. Coleoptile elongation was positively correlated with total amylolytic activities and α-amylase activity in embryos, and negatively correlated with α-amylase activity in endosperm. Moreover, carbohydrate content in embryos was found to be positively correlated with total amylolytic activities under anaerobic conditions, while a negative relationship was recorded in the endosperm. Carbohydrate status in rice seedlings has a primary importance in sustaining coleoptile elongation towards the surface. The relationship between carbohydrate level in embryo and anoxic germination, as well as with total amylolytic activities present in rice embryo under anaerobic condition 7 DAS, is consistent with the role of sugar metabolism to support rice germination under oxygen-deprived environment.     

Effects of inorganic nitrogen sources on spore germination and gametophyte growth in Botrychium dissectum

Abstract. The effects of variations in nitrogen source upon spore germination and gametophyte growth of the eusporangiate fern Botrychium dissectum forma obliquum were investigated. Spore germination and early gametophyte growth were directly related to the oxidation level of the supplied nitrogen source. Nitrate and nitrite inhibited spore germination and at concentrations above 0.035 mol m^-3 prevented it entirely. Ammonium promoted germination well above the levels attained on media without nitrogen. Concentrations of ammonium greater than 0.035 mol m^-3 often resulted in germination above 90%. The growth of young gametophytes from spores was reduced on media without nitrogen or on media with high concentrations of nitrate. Ammonium stimulated the growth of both young and older gametophytes. However, older gametophytes were able to grow on media containing nitrate as the sole nitrogen source. We conclude that a reduced nitrogen source is necessary for spore germination and early growth of Botrychium gametophytes. This requirement has several ecological implications which may be related to the distribution of these ferns and the establishment of mycorrhizal associations.     

Nutrient translocation pattern and accumulation of free amino acids in rice coleoptile elongation under anoxia

Comparing nutrient translocation to the rice ( Oryza sativa L. var. Arborio ) shoot during anoxia with the aerobic situation, it was found that anoxia reduced the translocation of K⁺, phosphorus, Mg²⁺ and Ca²⁺ with progressive intensity; Ca²⁺ translocation was practically zero in the absence of oxygen. The translocation of K⁺ and phosphorus under anoxia was still considerable and contributed to the maintenance of a high osmotic potential while the blocking of Ca²⁺ translocation caused a decrease in its concentration in the anoxic coleoptile, possibly favouring high cell wall plasticity in that organ. As anoxia proceeded, amino acids, no longer employed in protein synthesis, accumulated in the coleoptile, reaching spectacular levels [51 mmol kg of tissue-water)⁻¹] and, after 48 h of anoxia, their contribution to the osmotic potential was 80% of that of K⁺, as against less than 20% in all aerobic treatments. Anoxia caused a reduction in soluble hexose concentrations which, however, were more than compensated osmotically by the accumulation of amino acids.     

Effects of inorganic nitrogen compounds on the activity and synthesis of nitrogenase in Gloeothece (Nägeli) sp. ATCC 27152

Addition of 2 mM nitrite or ammonium to aerobically incubated cultures of Gloeothece rapidly inhibited N₂ fixation (measured as acetylene reduction). In contrast, 2 mM nitrate inhibited N₂ fixation less rapidly and less extensively, and often temporarily stimulated nitrogenase activity. The inhibitory effects of both nitrate and ammonium could be prevented by addition of 3 mM L-methionine-DL-sulphoximine, suggesting that the true inhibitor of N₂ fixation was an assimilatory product of ammonium rather than either ammonium or nitrate itself. The inhibition of N₂ fixation by nitrite could not, however, be prevented by addition of L-methionine-DL- sulphoximine. On the other hand, nitrite (unlike nitrate and ammonium) did not inhibit N₂ fixation in cultures incubated under a gas phase lacking oxygen. These findings suggest that the mechanism whereby nitrite inhibits N₂ fixation in Gloeothece differs from that of either nitrate or ammonium. The inhibitory effect of nitrite on N₂ fixation did not involve reduction of nitrite to nitric oxide, though nitric oxide was a potent inhibitor of nitrogenase activity in Gloeothece . Nitrate and nitrite inhibited the synthesis of nitrogenase in Gloeothece , while ammonium not only inhibited nitrogenase synthesis but also stimulated degradation of the enzyme. In addition, all three compounds favoured the appearance of the Fe-protein of nitrogenase in its larger, presumed inactive, form.