MSX-resistant mutants of Anabaena 7120 with derepressed heterocyst development and nitrogen fixation

To investigate the role of ammonium-assimilating enzyme in heterocyst differentiation, pattern formation and nitrogen fixation, MSX-resistant and GS-impaired mutants of Anabaena 7120 were isolated using transposon (Tn5-1063) mutagenesis. Mutant Gs1 and Gs2 (impaired in GS activity) exhibited a similar rate of nitrogenase activity compared to that of the wild type under dinitrogen aerobic conditions in the presence and absence of MSX. Filaments of Gs1 and Gs2 produced heterocysts with an evenly spaced pattern in N₂-grown conditions, while addition of MSX altered the interheterocyst spacing pattern in wild type as well as in mutant strains. The wild type showed complete repression of heterocyst development and nitrogen fixation in the presence of NO₃ ^– or NH₄ ⁺, whereas the mutants Gs1 and Gs2 formed heterocysts and fixed nitrogen in the presence of NO₃ ^– and NH₄ ⁺. Addition of MSX caused complete inhibition of glutamine synthetase activity in wild type but Gs1 and Gs2 remained unaffected. These results suggest that glutamine but not ammonium is directly involved in regulation of heterocyst differentiation, interheterocyst spacing pattern and nitrogen fixation in Anabaena.     

Effects of L-methionine-DL-sulphoximine on the assimilation of newly fixed NH3, acetylene reduction and heterocyst production in Anabaena cylindrica.

The addition of exogenous L-methionine-DL-sulphoximine (MSO) to N₂-fixing cultures of the blue-green alga Anabaena cylindrica results in over half of the newly fixed NH₃ being released into the medium. MSO also inhibits glutamine synthetase (GS) activity, has negligible effect on alanine dehydrogenase activity, and glutamate dehydrogenase activity under N₂-fixing conditions is negligible. In the presence of MSO, intracellular pools of glutamate and glutamine decrease, those of aspartate and alanine + glycine show little change, and the NH₃ pool increases. MSO alleviates the inhibitory effect of exogenous NH₄⁺ on nitrogenase synthesis and heterocyst production. The results suggest that in N₂-fixing cultures of A. cylindrica the primary NH₃ assimilating pathway involves GS, and probably glutamate synthase (GOGAT), and that the repressor of nitrogenase synthesis and heterocyst production is not NH₄⁺ but is GS, GOGAT, or a product of their reactions.     

Glutamine synthetase: activity and localization in cyanobacteria of the cycadsCycas revoluta andZamia skinneri

Nitrogen-fixing cyanobacteria inhabit the zone between the inner and outer cortex of cycad coralloid roots. In the growing tip of such roots the cyanobacterial heterocyst frequency, nitrogenase activity (C₂H₂-reduction) and glutamine synthetase activity (both transferase and biosynthetic) were comparable to those found in freeliving cyanobacteria. The relative level of glutamine synthetase protein and its pattern of cellular/subcellular localization in heterocysts and vegetative cells were also similar to those of free-living cyanobacteria. However, there was a progressive decline in nitrogenase activity along the coralloid root with maximum reduction occurring in the regions farthest from the growing tip. A similar but less pronounced pattern was observed for glutamine synthetase activity. Distribution of glutamine synthetase protein in cyanobacteria in the first 2–3 mm of the root tip indicated a slight decrease in the heterocysts and vegetative cells. However, the overall level of cyanobacterial glutamine synthetase protein did not change because of a drastic increase in the numbers of heterocysts, which contain a proportionally higher level of glutamine synthetase than the vegetative cells.Abbreviation GS glutamine synthetase     

Localization of an uptake hydrogenase in anabaena

Occurrence and localization of an uptake hydrogenase were examined in three strains of the blue-green alga, Anabaena. In vivo H₂ uptake was detected (0.60-1.44 μmoles/[mg of chlorophyll a per hour]) in all three strains when grown with N₂ as the sole source of nitrogen. H₂ uptake (in vivo and in vitro) was severely suppressed in cultures grown on NH₄⁺ and lacking heterocysts. H₂ uptake in cell-free extracts could be readily measured with a methyl viologen-ferricyanide electron acceptor system. Solubilization kinetics during cavitation of aerobically grown Anabaena 7120 indicates that the uptake hydrogenase is localized solely in the heterocyst. When the same organism is grown on N₂/CO₂, vegetative cells may account for up to 21% of the total hydrogenase activity in the filaments. The results are discussed in terms of a proposed functional relationship between nitrogenase and hydrogenase.     

KINETICS OF CELL DEATH IN THE CYANOBACTERIUM ANABAENA FLOS-AQUAE AND THE PRODUCTION OF DISSOLVED ORGANIC CARBON1

Kinetics of cell death and the production of dissolved organic carbon (DOC) were investigated in Anabaena flos-aquae (Lyngb.) Bréb grown on three different N sources (N₂nitrate, and ammonium) in a phosphorus (P)-limited chemostat. The fraction of live cells in the total population increased as growth rate increased with decreasing P limitation. Cell death was less in nitrate and ammonium media than in N₂. The specific death rate (γ), when calculated as the slope ofv^?1_x vs. D^?1, where v_xand D are live cell fraction (or cell viability) and dilution rate, respectively, was 0. 0082 day^?1 in N₂and 0.0042 day^?1 in nitrate. The slope of the plot in ammonium culture was not significant; however, the value of the live cell fraction was within the range for the NO^?₃culture. The fraction of live vegetative cells in N₂ culture was constant at all growth rates and the increase in the overall live cell fraction with growth rate was due entirely to an increase in live heterocysts. Live heterocysts comprised 3.5% of the total cells at a growth rate of 0.25 day^?1 and increased to 6.3% at 0.75 day^?1 with the ratio of live heterocysts to live vegetative cells linearly increasing with growth rate. The fraction of live vegetative cells was invariant in nitrate cultures us in N₂cultures. The live heterocysts fraction also increased with growth rate in nitrate cultures, along with the live heterocysts : live vegetative cells ratio, but the level was lower than in N₂cultures. DOC released from dead cells increased inversely with growth rate in N₂from 36.4% of the total DOC at a growth rate of 0.75 day^?1 to 54.15% at 0.25 day^?1. The contribution of cell death to the total DOC production in nitrate and ammonium media was significantly less than that under N₂DOC from dead cells consisted mainly of high-molecular-weight compounds, whereas DOC excreted from live cells was largely of low molecular weight.     

Physiological alterations and regulation of heterocyst and nitrogenase formation in Het(-) Fix(-) mutant strain of Anabaena variabilis

Physiological alterations and regulation of heterocyst and nitrogenase formation have been studied in Het⁻ Fix⁻ mutant strain of diazotrophic cyanobacterium Anabaena variabilis. Het⁻ Fix⁻ mutant strain of A. variabilis has been isolated by N-methyl-N′-nitro-N″-nitrosoguanidine (NTG) mutagenesis and was screened with the penicillin enrichment (500 μg ml⁻¹). Growth, heterocyst differentiation, nitrogenase and glutamine synthetase (biosynthetic and transferase), ¹⁴CO₂-fixation, nitrate reductase (NR), nitrite reductase (NiR), glucose-6-phosphate dehydrogenase (G6PDH), and isocitrate dehydrogenase (IDH) activities, and NO₃ ⁻, NO₂ ⁻, and NH₄ ⁺ uptake and whole cell protein profile in different metabolic conditions were studied in the Het⁻ Fix⁻ mutant strain taking wild-type A. variabilis as reference. Het⁻ Fix⁻ mutant strain was incapable of assimilating elemental nitrogen (N₂) due to its inability to form heterocysts and nitrogenase and this was the reason for its inability to grow in BG-11₀ medium (free from combined nitrogen). In contrast, wild-type strain grew reasonably well in the absence of combined nitrogen sources and also showed heterocyst differentiation (8.5%) and nitrogenase activity (10.8 ηmol C₂H₄ formed μg⁻¹ Chl a h⁻¹) in N₂-medium. Wild-type strain also exhibited higher NR, NiR, and GS activities compared to its Het⁻ Fix⁻ mutant strain, which may presumably be due to acquisition of high uptake of NO₃ ⁻, NO₂ ⁻, and NH₂ ⁺. Wild-type strain in contrast to its Het⁻ Fix⁻ mutant strain also exhibited high level of G6PDH, IDH, and ¹⁴CO₂ fixation activities. Low levels of G6PDH and IDH activities in Het⁻ Fix⁻ mutant strain further confirmed the lack of heterocyst differentiation and nitrogenase activity in the Het⁻ Fix⁻ mutant strain. NR, NiR, and GS activities in both the strains were energy-dependent and the energy required is mainly derived from photophosphorylation. Furthermore, it was found that de novo protein synthesis is necessarily required for the activities of NR, NiR, and GS in both wild-type and its Het⁻ Fix⁻ mutant strain. Received: 21 December 2001 / Accepted: 28 January 2002     

Growth and acetylene reduction by Gloeotrichia echinulata (Smith) Richter in axenic culture

Gloeotrichia echinulata, isolated in axenic culture, grows in medium free of combined nitrogen but better growth occurs in the presence of low levels of combined nitrogen (5–12 mm NO^- ₃; 0·1–2·0 mm NH⁺ ₄). The addition of 5–40 mm glucose and the addition of soil extract also stimulate growth. Heterocyst frequency declines in the presence of high levels of combined nitrogen, but the effect of added glucose on heterocyst frequency is equivocal. Soil extract inhibits heterocyst production. Significant acetylene reduction activity is found only in the light with rates in the presence of NO^- ₃ and in the presence of exogenous glucose being higher than on N₂. NH⁺ ₄ inhibited nitrogenase activity.     

Effects of in vivo treatments on the activity of nitrogenase isolated from Rhodospirillum rubrum

Nitrogenase activities of partially purified extracts of Rhodospirillum rubrum grown on different nitrogen sources were examined. Most of the nitrogenase from cells grown on N₂ or glutamate was in the inactive form. This form was also predominant in extracts from cells grown on limiting N₂ or glutamate plus N₂. The enzyme from cells grown with limiting NH⁺₄ was fully active. Nitrogenase displayed varying degrees of sensitivity to in vivo inhibition by NH⁺₄, depending on the culture conditions. However, addition of NH⁺₄ to the cultures prior to harvest did not change the proportion of the active form of the enzyme in extracts from that found in control samples. Several of these observations are inconsistent with the three component model of nitrogenase regulation of Yoch and Cantu (Yoch, D.C. and Cantu, M. (1980) J. Bacteriol, 142, 899–907). A regulatory system controlled by products of NH⁺₄ assimilation is suggested.     

Akinetes of the cyanobacteriumNostoc PCC 7524: morphological changes during synchronous germination

Following dilution into fresh medium in the light, akinetes ofNostoc PCC 7524 germinated synchronously. Synchrony was maintained at a high level during the first 24 h, at which time the young filaments were composed either of three cells (with N₂ as nitrogen source) or four cells (with NO ₃ ^- or NH ₄ ⁺ ), and at a slightly lower level during the next 24 h of growth. The pattern of cell division was similar in media containing the different nitrogen sources although the timing of the major events varied. In the presence of N₂ or NO ₃ ^- , heterocysts differentiated synchronously; the first developed invariably from a terminal cell of the young filament at approximately 19 h, the second from the other terminal cell after further vegetative cell division. Heterocyst differentiation did not occur in the presence of NH ₄ ⁺ . In the absence of nitrogen (gas phase argon: CO₂) akinete germination initially followed the same pattern as that observed in N₂, this early stage probably occurring at the expense of intracellular reserve materials.During germination, a new laminated layer, similar in structure and position to that found in the heterocyst envelope, appeared in the akinete envelope. This layer was not present in the germinating akinetes of a mutant which was incapable of forming heterocysts.     

Dark induction of nitrate reductase in the halophilic alga Dunaliella salina

The effect of nitrogen starvation on the NO₃-dependent induction of nitrate reductase (NR) and nitrite reductases (NIR) has been investigated in the halophilic alga Dunaliella salina. When D. salina cells previously grown in a medium with NH ₄ ⁺ as the only nitrogen source (NH ₄ ⁺ -cells) were transferred into NO ₃ ^? medium, NR was induced in the light. In contrast, when cells previously grown in N-free medium were transferred into a medium containing NO ₃ ^? , NR was induced in light or in darkness. Nitrate-dependent NR induction, in darkness, in D. salina cells previously grown at a photon flux density of 500 umol · m^?2 s^?1 was observed after 4 h preculture in N-free medium, whilst in cells grown at 100 umol · m^?2 s^?1 NR induction was observed after 7–8 h. An inhibitor of mRNA synthesis (6-methylpurine) did not inhibit NO ₃ ^? -induced NR synthesis when the cells, previously grown in NH ₄ ⁺ medium, were transferred into NO ₃ ^? medium (at time 0 h) after 4-h-N starvation. However, when 6-methylpurine was added simultaneously with the transfer of the cells from NH ₄ ⁺ to NO ₃ ^? medium (at time 0 h), NO ₃ ^? induced NR synthesis was completely inhibited. The activity of NIR decreased in N-starved cells and the addition of NO ₃ ^? to those cells greatly stimulated NIR activity in the light. The ability to induce NR in darkness was observed when glutamine synthetase activity reached its maximal level during N starvation. Although cells grown in NO ₃ ^? medium exhibited high NR activity, only 0.33% of the total NR was found in intact chloroplasts. We suggest that the ability, to induce NR in darkness is dependent on the level of N starvation, and that NR in D. salina is located in the cytosol. Light seems to play an indirect regulatory role on NO ₃ ^? uptake and NR induction due to the expression of NR and NO ₃ ^? -transporter mRNAs.     

Growth and Major Nutrient Concentrations in Brassica campestris Supplied with Different NH4^＋/NO3 Ratios

In the present study, we investigated whether growth and main nutrient ion concentrations of cabbage （Brassica campestris L.） could be increased when plants were subjected to different NH4^＋/NO3- ratios. Cabbage seedlings were grown in a greenhouse in nutrient solutions with five NH4^＋/NO3- ratios （1：0; 0.75：0.25; 0.5：0.5; 0.25：0.75; and 0：1）. The results showed that cabbage growth was reduced by 87% when the proportion of NH4^＋-N in the nutrient solution was more than 75% compared with a ratio NH4^＋/NO3- of 0.5：0.5 35 d after transplanting, suggesting a possible toxicity due to the accumulation of a large amount of free ammonia in the leaves. When the NH4＋/NO3- ratio was 0.5：0.5, fresh seedling weight, root length, and H2PO4- （P）, K^＋, Ca^2＋, and Mg^2＋ concentrations were all higher than those in plants grown under other NH4^＋/NO3- ratios. The nitrate concentration in the leaves was the lowest in plants grown at 0.5： 0.5 NH4^＋/NO3-. The present results indicate that an appropriate NH4^＋/NO3- ratio improves the absorption of other nutrients and maintains a suitable proportion of N assimilation and storage that should benefit plant growth and the quality of cabbage as a vegetable.     

RAPID AMMONIUM‐ AND NITRATE‐INDUCED PERTURBATIONS TO CHL a FLUORESCENCE IN NITROGEN‐STRESSED DUNALIELLA TERTIOLECTA (CHLOROPHYTA)1

When NH₄ ⁺ or NO₃ ^? was supplied to NO₃ ^? ‐stressed cells of the microalga Dunaliella tertiolecta Butcher, immediate transient changes in chl a fluorescence were observed over several minutes that were not seen in N‐replete cells. These changes were predominantly due to nonphotochemical fluorescence quenching. Fluorescence changes were accompanied by changes in photosynthetic oxygen evolution, indicating interactions between photosynthesis and N assimilation. The magnitude of the fluorescence change showed a Michaelis‐Menten relationship with half‐saturation concentration of 0.5 μM for NO₃ ^? and 10 μM for NH₄ ⁺ . Changes in fluorescence responses were characterized in D. tertiolecta both over 5 days of N starvation and in cells cultured at a range of NO₃ ^? ‐limited growth rates. Variation in responses was more marked in starved than in limited cells. During N starvation, the timing and onset of the fluorescence responses were different for NO₃ ^? versus NH₄ ⁺ and were correlated with changes in maximum N uptake rate during N starvation. In severely N‐starved cells, the major fluorescence response to NO₃ ^? disappeared, whereas the response to NH₄ ⁺ persisted. N‐starved cells previously grown with NH₄ ⁺ alone showed fluorescence responses with NH₄ ⁺ but not NO₃ ^? additions. The distinct responses to NO₃ ^? and NH₄ ⁺ may be due to the differences between regulation of the uptake mechanisms for the two N sources during N starvation. This method offers potential for assessing the importance of NO₃ ^? or NH₄ ⁺ as an N source to phytoplankton populations and as a diagnostic tool for N limitation.     

Observation of microplasmodesmata in both heterocyst-forming and non-heterocyst forming filamentous cyanobacteria by freeze-fracture electron microscopy

Structures which may establish cytoplasmic continuity between adjacent cells of filamentous cyanobacteria have been observed by freeze-fracture electron microscopy. They are visible in the septum region of the plasma membrane as pits on the E-face (EF) and corresponding protrusions on the P-face (PF). Between 100 and 250 of these structures, termed microplasmodesmata, were present between adjacent vegetative cells in all four strains of heterocyst-forming filamentous cyanobacteria, Anabaena cylindrica Lemm, A. variabilis (IUCC B377), A. variabilis Kütz. (ATCC 29413) and Nostoc muscorum, examined. Only 30–40 microplasmodesmata were observed between adjacent cells in two species, Phormidium luridum and Plectonema boryanum, that do not form heterocysts. The results suggest that in species that form heterocysts a greater degree of cytoplasmic continuity is established, presumably to facilitate the exchange of metabolites. In species capable of forming heterocysts, the number of microplasmodesmata per septum between two adjacent vegetative cells remained constant whether the filaments were grown in the presence of NH₄ and lacked heteroxysts or under N₂-fixing conditions and contained heterocysts. When a vegetative cell differentiates into a heterocyst, about 80% of the existing microplasmodesmata are destroyed as the poles of the cell become constricted into narrow necks leaving smaller areas of contact with the adjacent vegetative cells.     

NUTRIENT CONTROLS ON NITROGEN UPTAKE AND METABOLISM BY NATURAL POPULATIONS AND CULTURES OF TRICHODESMIUM (CYANOBACTERIA)

The effects of inorganic nutrient (ammonium [NH₄ ⁺ ] and nitrate [NO₃ ⁻ ]) and amino acid (glutamate [glu] and glutamine [gln]) additions on rates of N₂ fixation, N uptake, glutamine synthetase (GS) activity, and concentrations of intracellular pools of gln and glu were examined in natural and cultured populations of Trichodesmium. Additions of 1 μM glu, gln, NO₃ ⁻ , or NH₄ ⁺ did not affect short-term rates of N₂ fixation. This may be an important factor that allows for continued N₂ fixation in oligotrophic areas where recycling processes are active. N₂ fixation rates decreased when nutrients were supplied at higher concentrations (e.g. 10 μM). Uptake of combined N (NH₄ ⁺ , NO₃ ⁻ , and amino acids) by Trichodesmium was stimulated by increased concentrations. For NO₃ ⁻ , proportional increases in NO₃ ⁻ uptake and decreases in N₂ fixation were observed when additions were made to cultures before the onset of the light period. GS activity did not change much in response to the addition of NH₄ ⁺ , NO₃ ⁻ , glu, or gln. GS is necessary for N metabolism, and the bulk of this enzyme pool may be conserved. Intracellular pools of glu and gln varied in response to 10 μM additions of NH₄ ⁺ , glu, or gln. Cells incubated with NH₄ ⁺ became depleted in intracellular glu and enriched with intracellular gln. The increase in the gln/glu ratio corresponded to a decrease in the rate of N₂ fixation. Although the gln/glu ratio decreased in cells exposed to the amino acids, there was only a corresponding decrease in N₂ fixation after the gln addition. The results presented here suggest that combined N concentrations on the order of 1 μM do not affect rates of N₂ fixation and metabolism, although higher concentrations (e.g. 10 μM) can. Moreover, these effects are exerted through products of NH₄ ⁺ assimilation rather than exogenous N, as has been suggested for other species. These results may help explain how cultures of Trichodesmium are able to simultaneously fix N₂ and take up NH₄ ⁺ and how natural populations continue to fix N₂ once combined N concentrations increase within a bloom.     

Photoproduction of ammonium by immobilized mutant strains of Anabaena variabilis

Summary Ethylenediamine (EDA) is toxic to the cyanobacterium Anabaena variabilis and inhibits nitrogenase activity. The inhibition of nitrogenase was prevented by pretreatment of cells with l-methionine-d,l-sulphoximine (MSX). Mutant strains of Anabaena variabilis (ED81, ED92), resistant to EDA, had low levels of glutamine synthetase (GS) biosynthetic activity compared with the wild type strain. ED92 had a low level of GS protein whereas ED81 had a similar level to that of the parent strain as estimated using antibodies against GS. Both strains fixed N₂ and liberated NH₄ ⁺ into the media. Following immobilization of the mutant strains, sustained photoproduction of NH₄ ⁺ was obtained in air-lift reactors at rates of up to 50 mol NH₄ ⁺ mg chl a^–1 h^–1, which were comparable to the rates obtained when immobilized cyanobacteria were treated with MSX.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - MSX l-methionine-d,l-sulphoximine     

PHYSIOLOGICAL ACCLIMATION OF MARINE PHYTOPLANKTON TO DIFFERENT NITROGEN SOURCES1

We examined the energetic dependency of the biochemical and physiological responses of Thalassiosira pseudonana Hasle and Heimdal. Chaetoceros gracilis Schütt, Dunaliella tertiolecta Butcher, and Gymnodinium sanguineum Hirasaka to NH₄⁺, NO₃^?, and urea by growing them at subsaturating and saturating photon flux (PF). At subsaturating PF, when energy was limiting, NO₃^? and NH₄⁺ grown cells had similar growth rates and C and X quotas. Therefore, NO₃^? grown cells used up to 48% more energy than NH₄⁺ grown cells to assimilate carbon and nitrogen. Based on our measurements of pigments, chlorophyll-a-specific in vivo absorption cross-section, and fluorescence-chlorophyll a^?1, we suggest that NO₃^?, grown cells do not compensate for the greater energy requirements of NO₃^? reduction by trapping more light energy. At saturating PF, when energy is not limiting, the utilization of NO₃^?, compared to NH₄⁺ resulted in lower growth rates and N quotas in Thalassiosira pseudonana and lower N quotas in Chaetoceros gracilis, suggesting enzymatic rather than energetic limitations to growth. The utilization of urea compared to Nh₄⁺ resulted in lower growth rates in Chaetoceros gracilis and Gymnodinium sanguineum (saturating PF) and in lower N quotas in all species tested at both subsaturating and saturating PF. The high C:N ratios observed in all urea-grown species suggest that nitrogen assimilation may be limited by urea uptake or deamination and that symptoms of N limitation in microalgae may be induced by the nature of the N source in addition to the N supply rate. Our results provide new eridence that the maximum growth rates of microalgae may be limited by enzymatic processes associated with the assimilation of NO₃^?, or urea.     

Absorption of nitrate and ammonium ions by Lolium perenne from flowing solution cultures at low root temperatures

Lolium perenne L. cv. 23 (perennial ryegrass) plants were grown in flowing solution culture and acclimatized over 49 d to low root temperature (5°C) prior to treatment at root temperatures of 3, 5, 7 and 9°C for 41 d with common air temperature of 20/15°C day/night and solution pH 5·0. The effects of root temperature on growth, uptake and assimilation of N were compared with N supplied as either NH₄ or NO3 at 10 mmol m^?3. At any given temperature, the relative growth rate (RGR) of roots exceeded that of shoots, thus the root fraction (Rf) increased with time. These effects were found in plants grown with the two N sources. Plants grown at 3 and 5°C had very high dry matter contents as reflected by the fresh weight: freeze-dried weight ratio. This ratio increased sharply, especially in roots at 7 and 9°C. Expressed on a fresh weight basis, there was no major effect of root temperature on the [N] of plants receiving NHJ but at any given temperature, the [N] in plants grown with NHJ was significantly greater than in those grown with NO₃. The specific absorption rate (SAR) of NH⁺₄ was greater at all temperatures than SAR-NO₃. In plants grown with NH⁺, 3–5% of the total N was recovered as NH⁺₄, whereas in those grown with NO^?₃ the unassimilated NO^?₃ rose sharply between 7 and 9°C to become 14 and 28% of the total N in shoots and roots, respectively. The greater assimilation of NH⁺₄ lead to concentrations of insoluble reduced N (= protein) which were 125 and 20% greater, in roots and shoots, respectively, than in NO^?₃-grown plants. Plants grown with NH⁺₄ had very much greater glutamine and asparagine concentrations in both roots and shoots, although other amino acids were more similar in Concentration to those in NO^?₃ grown plants. It is concluded that slow growth at low root temperature is not caused by restriction of the absorption or assimilation of either NH⁺₄ or NO^?₃. The additional residual N (protein) in NH⁺₄ grown plants may serve as a labile store of N which could support growth when external N supply becomes deficient.     

Different Tolerance to Light Stress in NO3−- and NH4+-Grown Phaseolus vulgaris L.

Abstract: NH₄⁺‐grown plants are more sensitive to light stress than NO₃^?‐grown plants, as indicated by reduced growth and intervenal chlorosis of French bean (Phaseolus vulgaris L.). Measuring the time course of F_v/F_m ratios under photoinhibitory light regimes did not reveal any difference in PS II damage between NO₃^?‐ and NH₄⁺‐grown plants, in spite of some indications of higher energy quenching in NO₃^?‐grown plants. Also, a direct action of NH₄⁺ as an uncoupler at the thylakoid membrane could be excluded. Instead, biochemical analysis revealed enhanced lipid peroxidation and higher activity of scavenging enzymes in NH₄⁺‐grown plants indicating that these plants make use of metabolic pathways with stronger radical formation. Evidence for higher rates of photorespiration in NH₄⁺‐grown plants came from experiments showing that electron flux and O₂ evolution were decreased by SHAM in NH₄⁺‐grown plants, and by antimycin A in NO₃^?‐grown plants. Further, the comparison of electron flux and of photoacoustic measurements of O₂ evolution suggested that in NH₄⁺‐grown plants the Mehler reaction was also increased, at least in the induction phase. However, the major cause of N form‐dependent stress sensitivity is assumed to be in the coupling between photosynthesis and respiration, i.e., NO₃^?‐grown plants can utilize the TCA cycle for the generation of C skeletons for amino acid synthesis, thus improving the ATP: reductant balance, whereas NH₄⁺‐grown plants have enhanced rates of photorespiration.     

BIOENERGETIC PROCESSES ARE MODIFIED DURING NITROGEN STARVATION AND RECOVERY IN PHORMIDIUM LAMINOSUM (CYANOPHYCEAE)1

In the non-N₂-fixing cyanobacterium Phormidium laminosum (Agardh) Gomont (strain OH-I-pCl₁), N starvation induced an increase in the rate of respiration and a decrease in the rate of O₂ evolution. When NO₃^? was added to illuminated N-starved cells, O₂ evolution immediately increased to levels shown by NO₃^? grown cells, even though N-starved cells had lost most of their in vitro photosynthetic activities. Stimulation of noncyclic electron flow was maximal under light-saturating conditions and after 2–3 days of N starvation. The respiratory rate of N-starved cells was stimulated by the addition of NO₃^? or NH₄⁺ and partially inhibited at very low irradiances, even in the presence of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). Results indicate that N-starved cells obtain the energy supply for N assimilation through a process different from that used by N-sufficient cells. N-starved cells were able to take up NO₃^? in the dark and when illuminated in the presence of DCMU under anaerobiosis. Following NO₃^? addition, the photosynthetic yield of the in vivo noncyclic electron transport slightly increased, whereas it decreased after NH₄⁺ addition. Addition of NO₃^? or NH₄⁺ favored photoinhibition of photosystem II, the effect being faster after NH₄⁺ addition.