N-limited Cells Research Articles

Transcriptomic analysis dissects the regulatory strategy of toxic cyanobacterium Microcystis aeruginosa under differential nitrogen forms

The critical role of nitrogen in the global proliferation of cyanobacterial blooms is arousing increasing attention. However, the mechanism underlying the algal responses to differential nitrogen forms remains unclarified. The physiological and transcriptomic changes of Microcystis aeruginosa supplied with different nitrogen forms (nitrate and ammonium) were highlighted in this study. The results indicated that ammonium behaves better in stimulating the initial growth in N-limited cells than nitrate. However, a concomitant side effect is that cellular growth and photosynthesis decreased due to photosystem II damage induced by excess absorbed light energy under 10 mg L−1 ammonium. By contrast, adequate nitrate supply favored more efficient photosynthesis, higher biomass yield and microcystin quotas than ammonium. Depending on the supplied nitrogen form, different transcriptomic patterns were observed in M. aeruginosa. Under nitrate, the upregulation of genes involved in Arg biosynthesis, ornithine-urea cycle and photosynthesis increased nitrogen storage and cellular growth, while genes involved in cyclic electron flow around photosystem I and CO2−concentrating mechanism were heightened to dissipate excess energy under high ammonium. These insights provided important clues for understanding the physiological and molecular effects of available nitrogen forms on the frequent outbreaks of cyanobacteria.

The phycobilisomes of Synechococcus sp. are constructed to minimize nitrogen use in nitrogen-limited cells and to maximize energy capture in energy-limited cells

Synechococcus sp. UTEX LB2380 is a coastal strain of a cosmopolitan cyanobacterial genus. In coastal waters, N and light availability are highly variable and their interplay may influence C allocation and photosynthetic performance. In this paper, we compared the impact of nitrogen (N) limitation and energy (light, E) limitation on phycobilisome composition and photosynthesis, in the presence of either NO3− or NH4+. Our hypothesis was that the phycobilisome composition would be influenced by the factor limiting growth. Our results show that N-limited cells adjusted their phycobilisome antenna to minimize N utilization, whereas E-limited cells had a phycobilisome composition tailored to alleviate E deficiency. The N-source was relevant for the pigment composition, under both limitations. When N limited growth, excess energy management may become important to decrease the risk of photoinhibition and oxidative stress; when the sink of electrons constituted by NO3− reduction was not present, the cells tended to decrease their phycobiliprotein content, possibly in order to minimize the size of PSII antennas and decrease excitation. When energy was limiting, the energy saved for N assimilation in NH4+-grown cells was invested in antenna pigments to allow for a higher energy input.

Differential cellular responses associated with oxidative stress and cell fate decision under nitrate and phosphate limitations in Thalassiosira pseudonana: Comparative proteomics.

Diatoms are important components of marine ecosystems and contribute greatly to the world’s primary production. Despite their important roles in ecosystems, the molecular basis of how diatoms cope with oxidative stress caused by nutrient fluctuations remains largely unknown. Here, an isobaric tags for relative and absolute quantitation (iTRAQ) proteomic method was coupled with a series of physiological and biochemical techniques to explore oxidative stress- and cell fate decision-related cellular and metabolic responses of the diatom Thalassiosira pseudonana to nitrate (N) and inorganic phosphate (P) stresses. A total of 1151 proteins were detected; 122 and 56 were significantly differentially expressed from control under N- and P-limited conditions, respectively. In N-limited cells, responsive proteins were related to reactive oxygen species (ROS) accumulation, oxidative stress responses and cell death, corresponding to a significant decrease in photosynthetic efficiency, marked intracellular ROS accumulation, and caspase-mediated programmed cell death activation. None of these responses were identified in P-limited cells; however, a significant up-regulation of alkaline phosphatase proteins was observed, which could be the major contributor for P-limited cells to cope with ambient P deficiency. These findings demonstrate that fundamentally different metabolic responses and cellular regulations are employed by the diatom in response to different nutrient stresses and to keep the cells viable.

Impacts of nitrogen limitation on the sinking rate of the coccolithophorid Emiliania huxleyi (Prymnesiophyceae)

Pantorno A., Holland D.P., Stojkovic S. and Beardall B. 2013. Impacts of nitrogen limitation on the sinking rate of the coccolithophorid Emiliania huxleyi (Prymnesiophyceae). Phycologia 52: 288–294. DOI: 10.2216/12–064.1The biological carbon pump in the ocean plays an important role in controlling atmospheric CO2 levels. Approximately 1% of the yearly 50–60 Pg C of marine primary production settles in the deep ocean, where it is effectively sequestered for centuries to millennia. Central to the strength of the pump is the sinking velocity of phytoplankton cells and other organic debris. Stokes' Law indicates that the sinking velocity of a spherical cell will depend on its size and density, where larger, heavier cells will sink at a faster rate. Given that growth conditions can result in changes in cell size and macromolecular composition of phytoplankton, it might be expected that such changes could cause alterations in sinking velocity and carbon drawdown via the biological carbon pump. In the future, phytoplankton cells in the open ocean are predicted to be more subject to nutrient limitation. In this study we examined the effects of nitrogen limitation on the sinking velocity of Emiliania huxleyi, a coccolithophore responsible for significant phytoplankton blooms and biological drawdown of carbon. Nitrogen limitation caused changes in macromolecular composition, especially lipid content and coccosphere thickness. However, the overall density of the cells remained similar, and, as a consequence, cell size was the major determinant of sinking rate with N-limited cells in exponential phase sinking more slowly than N-replete cells. Cells in stationary phase showed the reverse trend with N-limited cells sinking faster, although not as fast as N-replete cells in exponential phase. N-limited cells produced more transparent exopolymers, suggesting an increased capacity for marine snow formation. These observations have implications for the efficiency of the biological carbon pump in areas that have increased nutrient stress caused by global climate change.

UNDERSTANDING NITROGEN LIMITATION IN AUREOCOCCUS ANOPHAGEFFERENS (PELAGOPHYCEAE) THROUGH cDNA AND qRT-PCR ANALYSIS(1).

Brown tides of the marine pelagophyte Aureococcus anophagefferens Hargraves et Sieburth have been investigated extensively for the past two decades. Its growth is fueled by a variety of nitrogen (N) compounds, with dissolved organic nitrogen (DON) being particularly important during blooms. Characterization of a cDNA library suggests that A.anophagefferens can assimilate eight different forms of N. Expression of genes related to the sensing, uptake, and assimilation of inorganic and organic N, as well as the catabolic process of autophagy, was assayed in cells grown on different N sources and in N-limited cells. Growth on nitrate elicited an increase in the relative expression of nitrate and ammonium transporters, a nutrient stress-induced transporter, and a sensory kinase. Growth on urea increased the relative expression of a urea and a formate/nitrite transporter, while growth on ammonium resulted in an increase in the relative expression of an ammonium transporter, a novel ATP-binding cassette (ABC) transporter and a putative high-affinity phosphate transporter. N limitation resulted in a 30- to 110-fold increase in the relative expression of nitrate, ammonium, urea, amino acid/polyamine, and formate/nitrite transporters. A.anophagefferens demonstrated the highest relative accumulation of a transcript encoding a novel purine transporter, which was highly expressed across all N sources. This finding suggests that purines are an important source of N for the growth of this organism and could possibly contribute to the initiation and maintenance of blooms in the natural environment.

Characterising nutrient-induced fluorescence transients (NIFTs) in nitrogen-stressed Chlorella emersonii (Chlorophyta)

K. Shelly, T. Higgins, J. Beardall, B. Wood, D. McNaughton and P. Heraud. 2007. Characterising nutrientinduced fluorescence transients (NIFTs) in nitrogen-stressed Chlorella emersonii (Chlorophyta). Phycologia 46: 503–512. DOI: 10.2216/06-55.1Although determining the nutrient status of algae is highly desirable for water quality management, conventional methods for assessing nutrient limitation are confounded by a range of theoretical and practical limitations. The current paper examines the effectiveness of a novel rapid fluorometric technique, based on observations of nutrient-induced transients in chl a fluorescence (NIFTs), in providing accurate measurements of algal nutrient limitation. The progress of N-starvation in batch cultures of the freshwater chlorophyte Chlorella emersonii was followed and the NIFT responses of cells to N-starvation and additions of both NH4+ and NO3− were characterised using a number of techniques including NIFTs, conventional pigment analysis using UV/Visible spectrophotometry and Fourier transform infrared (FT-IR) and Raman spectroscopy. Results showed that the addition of NH4+ and NO3− elicited distinct changes in in vivo chl a fluorescence in N-limited Chlorella cells. The nature of the fluorescence change was dependent on the form of nitrogen supplied, with NH4+ additions producing a more complex effect on fluorescence than NO3− additions. Interestingly, the greatest fluorescence response following NH4+ injection occurred prior to cells becoming extremely N-starved. Despite Chlorella exhibiting many of the characteristic features of N-deficient cells such as lowered capacity for photosynthetic electron transport (rETRmax), reduced Chl a : β–carotene ratios and increased levels of carbohydrate accumulation relative to protein, values for the maximum effective quantum yield of Photosystem II (ΦPSIIe-max) remained high over the course of N-starvation.

Variations in the buoyancy response of Microcystis aeruginosa to nitrogen, phosphorus and light

Microcystis aeruginosa displays a range of variability in buoyancy in response to light which is dependent upon the previous nutrient or light history of the cell. The short-term buoyancy response to light is nested into a longer term response, over a period of days, as cyanobacteria respond to their nutrient and light climate, which may be manifested in their gas vesicle volume, photosynthetic rate and carbohydrate metabolism. Microcystis aeruginosa cultures were subjected to a range of nitrogen (N), phosphorus (P) or light pre-treatments before exposure to saturating irradiance, and these results are used to illustrate that populations can display a range of buoyancy responses dependent upon subtle changes in nutrient and light conditions. N-limited (0 µM) cells suffered a dilution in gas vesicle volume and increased carbohydrate content, which resulted in a loss of buoyancy. Cells in 10 µM N increased their gas vesicle content during light exposure; however, this was insufficient to maintain buoyancy in the majority of cells as carbohydrate increased. Cells in 100 µM N increased their gas vesicle volume, metabolized carbohydrate more efficiently than the N-limited treatments and retained positive buoyancy. During light exposure, there was minimal change in buoyancy in 0 µM P pretreated cells, although there was a dilution in gas vesicle volume and an increase in carbohydrate. In 0.5 µM P pre-treated cells, the proportion of individuals floating did not change, although gas vesicle volume and carbohydrate increased. There was a significant increase in gas vesicle volume in 10 µM P pre-treated cells during light exposure, and some buoyancy loss due to carbohydrate accumulation. Cells grown in the range of light intensities tested all increased their gas vesicle volume during the 24 h light treatment. The actual rate of gas vesicle production was equivalent for all three light pretreatments; however, the growth-dependent dilution rate was determined by the previous light history, which resulted in different relative gas vesicle volume per cell. There was minimal increase in carbohydrate per cell during the light period, yet all treatments displayed the classical buoyancy loss response.

MOBILIZATION OF beta-1,3-GLUCAN AND BIOSYNTHESIS OF AMINO ACIDS INDUCED BY NH4+ ADDITION TO N-LIMITED CELLS OF THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)

Mobilization of the reserve β-1,3-glucan (chrysolaminaran) in N-limited cells of the marine diatom Skeletonema costatum (Grev.) Cleve (Bacillariophyceae) was investigated. The diatom was grown in pH-regulated batch cultures with a 14:10-h light:dark cycle until N depletion. In a pulse-chase experiment, the cells were first incubated in high light (200 μmol photons·m−2·s−1) with 14C-bicarbonate until dissolved inorganic carbon was exhausted. Unlabeled bicarbonate (1 mM) was then added, and the cells were incubated in the dark and subsequently in low light (20 μmol photons·m−2·s−1) with additions of 40 μM NH4+. In the 14C pulse phase with high light and N depletion, β-1,3-glucan accumulated and accounted for 85% of incorporated 14C. In the subsequent 14C chase phases, added NH4+ was assimilated at an N-specific rate of 0.11 h−1 in both the dark and low light, and in both cases it caused a significant mobilization of β-1,3-glucan (dark, 26%; low light, 19%). Biochemical fractionation of organic 14C showed that free amino acids were most rapidly labeled in the early stage of NH4+ assimilation, whereas proteins and polysaccharides were labeled more rapidly after 1.2 h. Analysis of the cellular free amino acids strongly indicated that de novo biosynthesis was occurring, with a Gln:Glu ratio increasing from 0.4 to 10 within 1.2 h. After the NH4+ was exhausted, the cellular pools of glucan and amino acids became constant or slowly decreased. In another experiment, N-limited cells were first incubated in high light until dissolved inorganic carbon was exhausted and were further incubated in high light with 150 μM NH4+ under inorganic carbon limitation. Added NH4+ was assimilated at an N-specific rate of 0.023 h−1, and cellular β-1,3-glucan decreased by 15% within 6 h. Hence, β-1,3-glucan was mobilized during NH4+ assimilation, even though inorganic carbon was modifying the metabolic rates. The results provide new evidence of β-1,3-glucan supplying essential precursors for biosynthesis of amino acids and other components in S. costatum in both the dark and subsaturating light and even saturating light under inorganic carbon limitation.

EFFECT OF NUTRIENT AVAILABILITY ON THE BIOCHEMICAL AND ELEMENTAL STOICHIOMETRY IN THE FRESHWATER DIATOM STEPHANODISCUS MINUTULUS (BACILLARIOPHYCEAE).

The objective of this study was to examine the differences in the biochemical and elemental stoichiometry of a freshwater centric diatom, Stephanodiscus minutulus (Grun.), under various nutrient regimes. Stephanodiscus minutulus was grown at μmax or 22% of μmax under limitation by silicon, nitrogen, or phosphorus. Cell sizes for nutrient-limited cultures were significantly smaller than the non-limited cell sizes, with N-limited cells being significantly smaller than all other treatments. Compared with the nutrient-replete treatment, both carbohydrates and lipids increased in Si- and P-limited cells, whereas carbohydrates increased but proteins decreased in N-limited cells. All of the growth-limited cells showed an increase of carbohydrate and triglyceride, and a decrease of cell size and polar lipids as a percentage of total lipids. The non-limited cells also had a significantly higher chl a concentration and galactolipids as a percentage of total lipids than any of the limited treatments, and the low-Si and low-P cells had significantly higher values than the low-N cells. The particulate C concentrations showed significant differences between treatments, with the Si- and P-limited treatments being significantly higher than the N- and non-limited treatments. Particulate Si did not show a strong relationship with any of the parameters measured, and it was the only parameter with no differences between treatments. The low-Si cells had a significantly higher P content (about two times more) than any other treatment, presumably owing to the luxury consumption of P, and a correspondingly high phospholipid concentration. The elemental data showed that S. minutulus had a high P demand with low optimum N:P (4) and Si:P (10) ratios and a C:N:P ratio of 109:16:2.3. The particulate C showed a positive relationship with POM (r = 0.93), dry weight (r = 0.88), lipid (r = 0.87) and protein (r = 0.84, all P < 0.0001). Particulate N showed a positive relationship with galactolipids (r = 0.95), protein (r = 0.90), dry weight (r = 0.78), lipid (r = 0.75), and cell volume (r = 0.64, all P < 0.0001). It is evident that nutrient limitation in the freshwater diatom S. minutulus has pronounced effects on its biochemical and elemental stoichiometry.

The physiological significance of light and dark NH 4+ metabolism in Chlorella sorokiniana

N-limited cells of Chlorella sorokiniana Shihira and Krauss 211/8k exhibited rates of respiratory oxygen consumption and photosynthetic oxygen evolution which were 70 and 17%, respectively, of those of N-sufficient cells. N-sufficient cells absorbed NH 4 + in light at a linear rate, and absorption was 100% inhibited by darkness. N-limited cells, by contrast, absorbed NH 4 + at almost similar rates in light and darkness. The rate of uptake in darkness decreased after 20 min, and decreased further after 60 min. In the light, by contrast, it decreased to a constant higher value, which appeared to approximate the sum of the rate of the dark absorption plus the rate of the light-dependent NH 4 + absorption by N-sufficient cells. NH 4 + in darkness did not affect respiration of N-sufficient cells. However, NH 4 + supply to N-limited cells in darkness resulted in a sudden increase of respiratory oxygen consumption, suggesting that short-term control mechanisms were triggered; it also resulted in time-dependent increase in photosynthetic activity and chlorophyll content, suggesting long term control of the photosynthetic apparatus. NH 4 + supply also resulted in a resumption of growth, in light but not in darkness. It is proposed that the use of NH 4 + in darkness by N-limited cells serves to recover from N-limitation, whereas in light it serves to sustain both recovery and growth. This could also explain why cells which are in an N-sufficient status and need nitrogen for growth only, exhibited exclusively a light-dependent NH 4 + uptake.

Regulation of ammonia assimilation in an obligate methylotroph Methylobacillus flagellatum under steady-state and transient growth conditions.

The obligate methylotroph Methylobacillus flagellatum was grown in the presence of different ammonium concentrations and the regulation of the enzymes associated with ammonium assimilation was investigated in steady-state and transient growth regimes. As the medium changed from C-limitation to dual C/N- and finally to N-limitation, the culture passed through three definite growth phases. The NADP(+)-dependent glutamate dehydrogenase (GDH) was present under ammonium limitation of the culture growth (at 2 mmol 1(-1) of ammonium in the growth medium) and increased in response to an increase in nitrogen availability. Glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were negligible during C- and C/N-limitation. In N-limited cells the GOGAT activity increased as the dilution rate increased up to 0.35 h-1, and then sharply dropped. In the N-sufficient cultures both NAD(+)-and NADP(+)-dependent isocitrate dehydrogenase (NAD-ICDH and NADP-ICDH) activities were up-regulated as dilution rate increased, but in the N-limited culture the NAD-ICDH activity was up-regulated whereas NADP-ICDH one was down-regulated. Pulse additions of ammonium and methanol demonstrated the coordinate regulation of the GDH and ICDHs activities. When pulses were added to the C/N-limited cultures, there was an immediate utilization of the nutrients, resulting in an increase in biomass; at the same time the GDH and ICDH activities increased and the GS and GOGAT activities decreased. When the same ammonium/methanol pulse was added into the N-limited culture, there was a 3 h delay in the culture response, after which the substrates were utilized at rates close to the ones shown by the C/N-limited culture after the analogous pulse.

Physiologic, morphologic and behavioral responses of perpetual cultures ofCaulobacter crescentus to carbon, nitrogen and phosphorus limitations

Perpetual cultures ofCaulobacter crescentus were maintained in a chemostat and provided with media that provided glucose, glutamate, ammonium chloride, and Na/K phosphate as sources, respectively, of C, of C and N, of N and of P. All cultures were maintained at the same relative flow rate, f/V, so that reproductive rate was constant. As the C∶N∶P ratio was shifted from a balanced ratio of 100 μg C∶9–10 μg N∶1 μg P, the cells modulated their rates of uptake, reducing the rates of uptake of excess element-sources and accelerating the uptake of potential sources of the limiting element. The change in N-limited cells was the greatest, resulting in a nearly 150-fold increase in rate of uptake of amino acids. Poly-β-hydroxybutyrate or polyphosphate was stored whenever C or P, respectively, was not the limiting element. Direct measurement of cell and stalk surface areas on purified peptidoglycan sacculi of P-limited cells revealed that the surface of both swarmer and stalked cells, as well as the stalk surface, participated in the accelerated rate of phosphate uptake. Swarmers from N-limited populations were the only cells that exhibited chemotactic responsiveness-to methionine, NH4Cl and glutamate, but not to glucose or phosphate-sufficient to be detectable in a microcapillary assay. In populations that were C-limited and provided with ammonium ions as the principal source of N, morphogenesis and reproduction deteriorated and a steady state could not be maintained. Generally, the responses ofC. crescentus were appropriate to manage unbalanced nutrient supplies, but this oligoheterotrophic organism did not tolerate excesses of inorganic nutrients when limited for its sources of C, of energy, and of ammonium acceptor.

Nitrogen limitation of chemostat-grown Rhizobium etli elicits higher infection-thread formation in Phaseolus vulgaris.

The symbiotic association between rhizobia and legume roots is a complex process involving many steps. An infection thread is a tubular structure of host origin formed during the infection of legume roots by rhizobia. Previous studies with batch cultures have reported that optimal attachment of rhizobia to root hairs coincides with nutrient limitation. In this study, the ability of chemostat-grown, nutrient-limited Rhizobium etli cells to form infection threads with its symbiotic partner Phaseolus vulgaris was investigated. Rhizobia were grown in a chemostat in synthetic media under C- or N-limiting conditions. Infection-thread formation was examined after inoculation of seedlings with a rhizobial cell suspension from each treatment. The number of infection threads was estimated by light microscopy after staining root sections with o-toluidine. Exopolysaccharide (EPS) production was also measured, and the cellular content and electrophoretic pattern of lipopolysaccharide (LPS) determined semiquantitatively. N-limited cells showed a markedly higher infectivity (measured as infection-thread formation) than C-limited cells. With one of the two bean cultivars used, the number of infection threads produced by N-limited cells was higher than that produced by exponentially growing cells in batch cultures. The higher infectivity of N-limited cells was correlated with higher EPS production. Electrophoretic analysis of LPS showed that C- and N-limited cells shared a common profile but the relative concentration of short LPS forms differed.

Fluorescence Transients in Response to Nutrient Enrichment of Nitrogen- and Phosphorus-limited Microcystis aeruginosa Cultures and Natural Phytoplankton Populations: a Measure of Nutrient Limitation

This study presents the results of an investigation of the application of chlorophyll fluorescence perturbations as a bioassay for nutrient limitation in microalgae. The variable chlorophyll fluorescence (ΔF) response of Microcystis aeruginosa, chemostatically cultured under different nitrogen (N) and phosphorus (P) nutrient regimes, to spiking with NO3-, NH4+ and PO43-, was monitored. Differences were observed in the short-term (&lt;10 min) fluorescence response (F) which related to the original nutrient status of the test organism. Cells growing exponentially in full nutrient media showed no transient changes in ΔF. The addition of NH4+ or NO3- to N-limited cells resulted in transient changes to ΔF, while no response was observed following the addition of PO43-. Spiking of P-limited cells with either PO43- or NH4+ resulted in transient changes to ΔF, while no response was observed for NO3-. Thus a simple, rapid, nutrient-specific assay for nutrient limitation was available for cultured cells of M. aeruginosa. When the assay was tested on field samples from the Maude Weir Pool (34�28'S,144�18'E), Murrumbidgee River, NSW, Australia, apparent nutrient limitation, either N or P, was detected. Use of microalgal fluorescence responses to nutrient spiking may allow the rapid and specific detection of nutrient limitation in both cultures and field populations of phytoplankton.

Effects of nitrogen limitation on growth and nitrite excretion rates of the dinoflagellate Prorocentrum minimum

Continuous nitrate-limited cultures of the dinoflagellate Prorocentrum minimum were grown under saturating photon flux densities to study the effects of nitrate pulses on the time variations of nitrate uptake, nitrite excretion, and cell division rate. In the first experiment, 5 chemostats were stabilized at the same dilution rate and, after stopping of the renewal supplies, received successively 1 pulse of nitrate at 24 h intervals. In the second, nitrate pulses were added about every 12 h in 1 chemostat. In the third experiment, 4 chemostats stabilized at different growth rates received 1 pulse of nitrate. Nitrate uptake process showed decreasing initial rates and lower maximum rates in cultures subjected to longer starvation times. In all cases, the amount of nitrite excreted before reabsorption represented an important proportion of the initially supplied nitrate (up to 45 %). This suggested that for nitrogen-deprived cells of L? minimum reduction of nitrite by the nitrite reductase is the more limiting step in the nitrateassimilatory pathway. The proportion of pulsed nitrate which is excreted as nitrite increased for decreasing growth rates. For 1 and 2 d of nitrate deprivation, the ratio nitrite excretion ratehitrate uptake rate integrated during each perturbation experiment increased, but decreased after longer times of starvation. This suggests that the processes of nitrate uptake and nitrite reduction are affected at different rates during nitrogen deprivation. The implications of nitrite excretion in N-limited cells on the determination of new production are discussed.

Amino acid pools in nutrient limited Cyanidium caldarium and responses to resupply

Amino acid pools in Cyanidium caldarium (Tilden) Geitler, growing under conditions of sufficiency of nutrients (sufficient cells), or under conditions of nitrogen, potassium or phosphorus limitation (N-, K- or P-limited cells), and responses upon nutrient resupply were investigated. Glutamate was the dominant amino acid in sufficient cells [11.2 μmol ml −1 packed cell volume (pcv)], in N-limited cells (7.71 μmol ml −1 pcv) and in P-limited cells (7.46 μmol ml −1 pcv), whereas in K-limited cells the dominant amino acid was alanine (8.83 μmol ml −1 pcv). Other amino acids detected occurring at significantly high concentrations were: citrulline, alanine, aspartate, glutamine and δ-aminolevulinic acid in sufficient cells, alanine in N-limited cells; alanine, citrulline, serine, arginine and asparagine in P-limited cells; glutamate, arginine, glutamine, citrulline, asparagine, serine, aspartate and putrescine in K-limited cells. On adding NH 4 + to N-limited cells the level of glutamate decreased and then increased to the starting value; there occurred an increase in the level of glutamine, aspartate, citrulline, arginine, alanine, serine, as well as of δ-ALA and putrescine. Resupply of Pi to P-limited cells caused a significant decrease of glutamate and a concomitant increase of alanine, but was without efrect on the concentration of other amino acids. On adding K + to K-limited cells there was an increase of glutamate and citrulline, and a decrease of putrescine, whereas arginine and alanine remained unvaried at the very high level occurring before addition.

Activation of Respiration to Support Dark NO3− and NH4+ Assimilation in the Green Alga Selenastrum minutum

Short-term changes in pyridine nucleotides and other key metabolites were measured during the onset of NO(3) (-) or NH(4) (+) assimilation in the dark by the N-limited green alga Selenastrum minutum. When NH(4) (+) was added to N-limited cells, the NADH/NAD ratio rose immediately and the NADPH/NADP ratio followed more slowly. An immediate decrease in glutamate and 2-oxoglutarate indicates an increased flux through the glutamine synthase/glutamate oxoglutarate aminotransferase. Pyruvate kinase and phosphoenolpyruvate carboxylase are rapidly activated to supply carbon skeletons to the tricarboxylic acid cycle for amino acid synthesis. In contrast, NO(3) (-) addition caused an immediate decrease in the NADPH/NADP ratio that was accompanied by an increase in 6-phosphogluconate and decrease in the glucose-6-phosphate/6-phosphogluconate ratio. These changes show increased glucose-6-phosphate dehydrogenase activity, indicating that the oxidative pentose phosphate pathway supplies some reductant for NO(3) (-) assimilation in the dark. A lag of 30 to 60 seconds in the increase of the NADH/NAD ratio during NO(3) (-) assimilation correlates with a slow activation of pyruvate kinase and phosphoenolpyruvate carboxylase. Together, these results indicate that during NH(4) (+) assimilation, the demand for ATP and carbon skeletons to synthesize amino acid signals activation of respiratory carbon flow. In contrast, during NO(3) (-) assimilation, the initial demand on carbon respiration is for reductant and there is a lag before tricarboxylic acid cycle carbon flow is activated in response to the carbon demands of amino acid synthesis.

Effect of Nitrogen Supply on the Kinetics and Regulation of Nitrate Assimilation in Chlamydomonas reinhardtii Dangeard

Concentration-dependent NO3 uptake by C. reinhardtii was studied in cells cultured under three NO3-growth regimes: 10 mM NO3 (NOJ -grown), 0-25 mM NO3 (N-limited) and an 18 h period of N-deprivation following growth on 10 mM NO J (N-starved). Two NO3 uptake isotherms, with an apparent phase transition in uptake at IT mM NO3, were resolved in all three cell types over the concentration range tested (0025 to 1-8 mM N03). The apparent Kmax for uptake by N-limited cells (0-72 x 10-8 /.?mol cell-1 h-1) exceeded that for both NO3-grown (0-42 x 10-8 /??mol cell-1 h-1) and N-starved (0-49 x 10-8 /??mol cell-1 h-1) cells at NO3 concentrations less than IT mM (isotherm 0). When supplied with NOj concentrations in excess of IT mM (isotherm 1) the apparent Kmax values for uptake by N-limited (0-64 x 10-8 /?mol cell-1 h-1) and N-starved (0-41 x 10-8 /?mol cell-1 h-1) cells were depressed relative to those for isotherm 0. In contrast, NO3-grown cells exhibited a greater apparent Vmax for uptake (0-63 x 10-8/?mol cell-1 h-1) at the higher NO3 concentrations. When N-limited and N-starved cells were supplied with 1-6 mM NO J (isotherm 1), the pattern of uptake showed a rapid linear increase after 60 min. This enhanced pattern of uptake did not occur in NO J-grown cells supplied with either 0-6 or 1-6 mM NO3 or in N-limited and N-starved cells supplied with 0-6 mM NO J. Cycloheximide (10 /?g cm-3), but not actinomycin D (50 /?g cm-3), blocked the apparent 'induction' of NO3 uptake in N-starved cells supplied with l-6mM NO J. These results suggest that mechanisms controlling the observed 'inducible' aspect of NO3 uptake in N-starved cells are dependent on a translational or post translational event. It is possible that the NO3 -concentration dependent 'inducible' portion of uptake may provide the means by which C. reinhardtii cells may regulate uptake in accordance with their capacity for assimilation.

The increase of KCN-sensitive electron flow in the branched respiratory chain of Micrococcus luteus grown slowly in carbon-limited culture

Summary: The strictly aerobic bacterium Micrococcus luteus was grown in the presence of lactate as sole carbon source under conditions of excess substrates (in batch culture) or under strict lactate (C) or ammonium (N) limitation (in a chemostat, D = 0·02 h−1). KCN (2mm) stimulated the respiration of batch-grown and N-limited cells, and inhibited the oxidase activity of C-limited cells by 40–60%. The content and composition of cytochromes and the KCN-dependences of the oxidase activities were found to be the same for the membranes of C-limited and batchgrown cells. The KCN sensitivity of the oxidase activities of isolated membranes decreased in the order NADH &gt; malate &gt; lactate. NAD-dependent lactate and malate dehydrogenase activities were observed in the cytoplasm of C-limited cells but not in that of batch-grown cells. The stimulation of cell respiration by lactate, maiate, pyruvate or ethanol caused a marked increase of the steady-state level of NAD+ reduction only in C-limited cells. It is assumed that the slow growth of C-limited cells is accompanied by an increase in the formation of NADH, which is oxidized by the KCN-sensitive, tightly-coupled, main branch of the M. luteus respiratory chain. Under non-limited conditions of growth the respiration is due mainly to the direct oxidation of lactate via the weakly coupled alternative branch. The role of this switching of the electron flow from one pathway to the other during the adaptation of the bateria to the slow C- and energy-limited growth is discussed.

Chlororespiration: an adaptation to nitrogen deficiency in Chlamydomonas reinhardtii.

When grown under nitrogen limitation, pronounced chlororespiratory activity develops together with an altered composition of thylakoid membranes in Chlamydomonas reinhardtii. Relative to control cultures, the flash-inhibited, chlororespiration-dependent O2 consumption signal increases 10-fold. Also augmented is the light-sensitive respiratory activity responsible for the "Kok effect," reflecting competitive inhibition of chlororespiratory electron transport by photosystem I. Fluorescence measurements show that the thylakoid plastoquinone pool is extensively reduced in dark-adapted, N-limited cells. Thylakoids of N-limited cells have reduced amounts of cytochrome b6, cytochrome f, and light-harvesting complexes. However, thylakoid-bound NADH-PQ oxidoreductase, with major subunits of 51 kDa and 17 kDa, is increased 7-fold and two novel cytochromes of 34 and 12.5 kDa are highly abundant. Thus, components of photosynthetic and chlororespiratory electron transport pathways are differentially regulated by N availability.