The phycocyanin to chlorophyll {alpha} ratio and other cell components as indicators of nutrient limitation in two planktonic cyanobacteria subjected to low-light exposures

The cell composition of the planktonic cyanobacteria, Oscillatoriaagardhii (Gomont) and Oscillatona redekei (van Goor), was comparedfor cultures grown under nitrogen (N) and phosphorus (P) limitation,and light climates which were energy (E) limited (photoperiods3:21 and 6:18 light:dark (LD) and irradiances 12–153 µmolm^–2s^–1). Increases in carbohydrate/protein ratio(CHO/Prot) and declines in chlorophyll a (Cha) and phycocyanin(PC) resulted from N and P limitation. N-, P- and E-limitedcultures could be distinguished on the basis of P content andthe ratio of PC/Cha. The P content range of 0.1–0.55%of ash-free dry weight (AFDW) for P-limited cultures was lowerthan that for N- and E-limited cultures (0.56–2.2 %AFDW).Cultures limited by N were distinguishable from E-limited cellsby lower PC/Cha ratios, ranging from 0 to 4.08, compared to3.9–6.9 for E-limited cells. Under the 3:21 LD cycle,the minimum PC/Cha ratio of E-limited cells was 4.5. Increasesin the CHO/Prot ratios were proportional to the difference betweenthe nutrient-limited growth rate and the non-nutrient-limitedgrowth rate. A comparison of the composition of the two speciesshowed greater accumulation of carbohydrate by O.agardhii undernutrient-limiting conditions, but that O. redekei had higherlevels of protein, chlorophyll a and phycocyanin and, in theabsence of P limitation, higher P contents than O.agardhii.     

Effects of light intensity and quality on the relative N and P requirement (the optimum N:P ratio) of marine planktonic algae

The relative requirement of N and P (the optimum N:P ratio)by Dunaliella tertiolecta, Phaeodactylum tricornutum, Prymnesiumparvum and Thalassiosira pseudonana was studied under variouslight intensities and spectra. The ratio was determined as theratio of the minimum cell N and P concentrations (q₀N and q₀pwhen either nutrient was limiting. The ratio varied widely amongspecies; under light-saturation for growth (116 µEin ^m–2s^–1 it ranged from 11.8 in ^D. tertiolecta to 36.6 in P.tricornutum. The ratio appeared to be higher at a sub-saturatingintensity (24 µEin m^–2 s^–1 in all except P.tricornutum, mainly because of higher q_oN with little changein q_oP. In T. pseudonana Q_oP also increased, resulting in aninsignificant change in the ratio. The ratio varied little withinthe range of saturation intensity. Light quality affected q_oNand q_oP as well as the ratio, and the pattern of change variedfrom species to species. The optimum ratio of individual specieswas linearly correlated to their q_oN except in P. tricornutum.q_oN for all species showed a linear correlation with cell proteinconcentrations irrespective of light conditions. The changeof optimum N:P ratios in the three species thus appears to berelated to changes in cell protein contents. The ratio of carbohydratesto protein remained constant regardless of light intensity orquality and was higher in P-limited cultures. We conclude thatchanges in light regime can strongly influence algal nutrientrequirements and species interrelationships by altering theoptimum cellular N:P ratio.     

Effect of malformin on the major constituents of Phaseolus vulgaris

Malformin inhibits wet and dry weight, nitrogen accumulation,and cell wall, RNA, DNA and protein synthesis in Phaseolus vulgaris.The relative proportion of dry matter and nitrogen in malformedtissues is increased in the ethanol soluble fraction and decreasedin the residue remaining after hydrolysis with 0.5 N HCl. Inhibitionof cell wall and protein synthesis was generally greater thaninhibition of nitrogen accumulation and RNA and DNA synthesis.The effects of malformin on the composition of P. vulgaris aresimilar to alterations in composition reported for ethylene,and opposite to those reported for gibberellic acid. ¹This research was supported by grant GB-7158 from the NationalScience Foundation and grant E-146-F from the American CancerSociety. ²Journal Paper No. 3509 of the Purdue Agricultural ExperimentStation. (Received October 23, 1968; )     

DISTRIBUTION AND TURNOVER OF PHOSPHATE COMPOUNDS IN GROWING CHLORELLA CELLS

1. Using the Chlorella cells which had been uniformly labeled with³²P, the distribution of phosphorus in various fractions ofcell material was investigated. Uniformly ³²P-labeled Chlorellawas further grown in a P-free medium or in a standard "cold"medium, and the change of distribution of ³²P (as well as theuptake of exogenous P) in various cell fractions was followed.
2. Analysis of the ³²P-labeled algal cells showed that the highestin P-content was the fraction of RNA followed by those of polyphosphates,lipid, nucleotidic labile phosphate compounds, DNA and protein(in decreasing order). ATP and ADP were found to be only minorfractions of the total labile phosphates.
3. On incubating the³P-labeled alga in a P-free medium, the P.contentsin the fractionsof DNA, protein, lipid and ATP increased, thosein polyphosphatesand ADP decreased, and that in RNA remainedalmost unchanged.When the ³²P-labeled alga was further grownin the normal "cold"medium, DNA and protein increased withthe expenditure of endogenous³²P, but with practically no incorporationof external P. Inthe meantime the P in polyphosphates decreasedconsiderably,and the RNA fraction incorporated a large amountof externalP but only a little of endogenous³²P.
4. It was inferred that,under the experimental conditions of thepresent study, thephosphorus used in the syntheses of DNA andprotein was primarilytaken from polyphosphates, while thatused in the synthesesof RNA, phospholipid and polyphosphateswas, for the most part,taken from the extracellular P-source.

¹A part of this paper was read at the Vth International Congressof Biochemistry, Moscow, August 10–16, 1961. (Received June 4, 1961; )     

Effect of UV-Irradiation on Total Protein and Nucleic Acids in Alternaria alternata and Fusarium solani

UV-A^* irradiation caused increases in total protein in Fusariumsolani, while its effect on Alternaria alternata was variable,and not as clear-cut as in F. solani. On the other hand, UV-Birradiation stimulated protein production in both fungi. UV-Airradiation showed an inhibitory effect on total DNA in bothfungi, while the effect on RNA was stimulatory in F. solanibut had no effect on A. alternata. Short fluences of UV-B inhibitedDNA production to some extent in both fungi, however longerfluences increased DNA content especially in F. solani. Theeffect of UV-B on RNA production was inhibitory in F. solanibut not in A. alternata. A. alternata is much more resistantto UV-irradiation than is F. solani, and increases in proteinin the former after UV-irradiation suggests that protein mayplay a part in protection against the harmful effect of UV-irradiation. UV-A, UV-B, fluence, protein, nucleic acids, Alternaria alternata, Fusarium solani     

Nutrient status of phytoplankton communities in Norwegian waters (marine, brackish, and fresh) as revealed by their chemical composition

The chemical composition of nutrient-saturated cultures of Emilianiahuxleyi, Amphidinium carterae, and Staurastrum luetkemuelleriwas studied. The variation in chemical composition of naturalphytoplankton communities in the North Sea, the Trondheimsfjord,and a eutrophic lake was also studied. Nutrient status was evaluatedby measurement of the algal protein/carbohydrate, N/C, P/C,and N/P ratios. Tests for P-deficiency were carried out by measuringthe increase in ATP upon addition of phosphate. At saturationthe N/C ratio was {small tilde}0.14 in marine species and {smalltilde}0.05 in Staurastrum. Saturation P/C ratios (excludingpolyphosphates) were species-dependent, ranging from 0.017 (Skeletonema)to 0.006 (Amphidinium). Amphidinium and Staurastrum store polyphosphateswhen grown in P-rich media; true marine planktonic species donot. Natural communities tended to be close to nutrient saturationat low biomass densities and nutrient deficient at high densities.In the North Sea, nitrogen was clearly limiting. In waters offthe Møre coast and in the Trondheimsfjord, growth wasnearly balanced with respect to N and P at high salinities (>25)and clearly P-limited in brackish fjord waters. In dense communities,the N/P ratio was inversely related to salinity. Freshwatercommunities were clearly P-limited, but responses were dampenedwhen daphnia or whitefish were introduced, due to increasedexcretion of nutrients. ¹Contribution No. 212, Trondheim Biological Station, N-7001Trondheim, Norway.     

Effects of Benzyladenine on Chlorophyll, DNA, RNA and Protein Content of Attached Young Bean (Phaseolus vulgaris L.) Leaves

N⁶-Benzyladenine (BA) was applied to intact bean (Phaseolusvulgaris L.) primary leaves at 2 and 6 days after imbibition,when they were in the cell division and post-cell division stages,respectively. BA treatment at day 2 temporarily inhibited an increase in chlorophyllcontent in the following day, but stimulated it in later days.No such inhibition by BA was observed for changes with timein DNA, RNA, and protein content and f. wt. On the other hand,BA treatment at day 6 enhanced RNA and protein content, withoutsignificant influence on DNA and chlorophyll content and f.wt. The mode of cytokinin action on greening in leaves during cell-divisiongrowth seems to be different from that in etiolated cotyledons. Phaseolus vulgaris L., bean, greening, benzyladenine, DNA, RNA, protein     

The Behaviour of Nucleic Acids and Other Constituents in Protomyces inundatus Dangeard

Estimations of contents per cell of DNA and RNA were made inProtomyces inundatus. There was twice as much DNA per cell incultures derived from fusion bodies and from mycelium in thehost (Apium nodiflorum) as in those derived from unfused endospores,indicating that the former two were diploid, the latter haploid.Dry weight/cell, volume/cell, insoluble nitrogen/cell, and metaphosphate/cell,like RNA/cell, had similar values for all three types of cultures.Giant cells resulting from camphor treatment not only had twiceas much DNA/cell as normal cultures derived from unfused endospores,but all the other characteristics studied had doubled as well.In any one culture the ratio of RNA to DNA remained constantthough the absolute values decreased as the cultures aged. TheRNA to DNA ratios in P. inundatus of haploid and diploid culturesare compared to those found in yeast and other organisms.     

Appearance of Nitrate in Soybean Seedlings and Chlorella Caused by Nitrogen Starvation

Disinfected seeds of soybean and actively growing cultures ofChlorella vulgaris were grown on nitrogen-free media. The nitratecontent of both the soybean seedlings and algal cultures substantiallyincreased during nitrogen starvation. The nitrate level in soybeanseedlings was at least eight times greater than seeds aftertwo to three weeks. Nitrogen starvation also caused an increasein the nitrate content of the algal cultures. Nitrate reductaseactivity also increased, and its appearance was sensitive tocycloheximide. Tungstate, added during starvation, inhibitedthe induction of nitrate reductase with a concomitant increasein the level of nitrate. These data suggest that oxidation ofreduced nitrogen compounds can occur in higher plants and algae. ¹ A contribution of the Texas Agricultural Experiment Station (Received June 6, 1981; Accepted August 25, 1981)     

Effects of Soil Nutrients and Moisture on Root/Shoot Ratios in Lolium perenne L. and Trifolium repens L

The dry weights of foliage and roots (excluding stembases) werecompared in a 3³x2² factorial experiment with Lolium perenneL. and Trifolium repens L. grown in pots with three nitrogen(0–300 ppm N), phosphorus (0–300 ppm P), and soilmoisture (pF 3.2, pF 2.4, and waterlogged) treatments, eachduplicated. Root/shoot ratios were influenced significantly (P < 0.001)by N, P, and W (soil moisture), but these main effects werequalified by highly significant first-order interactions andsecond-order interactions which included N and P. The lowestR/S ratios occurred where N, P, and W were all non-limiting,and the highest where there was an imbalance of these factors. The explanation suggested is that a decrease in the availabilityof N, P, or W causes an increase in the relative weight of roots,but in the presence of N, P, or W deficiency an increase inany one of these factors causes a further increase in the R/Sratio by increasing the relative efficiency of the foliage.     

Metabolism of C1 unit precursors in Neurospora: Effects of media supplements on labelling of nucleic acids and protein

Mycclia of Neurospora crassa wild type (FE SC no. 853), harvestedduring the exponential phase of growth on defined minimal mediaincorporated glycine-2-¹⁴C, serine-3-¹⁴C and formate-¹⁴C intoproteins, DNA and RNA. Supplementing the growth medium with1 mM glycine increased the flow of glycine and formate carboninto these products. In contrast, this supplement decreasedthe incorporation of serine-¹⁴C. When such cultures were preincubatedfor 30 min with adenine, formaldehyde, formate or L-methionine,labelling of the nucleic acids and protein fractions by glycine-2-¹⁴Cwas altered. It is concluded that glycine increases the turnoverof C₁ units in Neurospora, resulting in greater contributionsof the C-2 in nucleic acid and protein synthesis. (Received May 14, 1977; )     

A probable lack of function of c-type cytochrome in the photosynthetic system of the blue-green alga Anabaena variabilis

Quantitative study of the cytochrome c acting in the photosyntheticsystem of the blue-green alga Anabaena variabilis (M-2) wasdone with membrane fragments and intact cells. Membrane fragments highly active in the NADP⁺-Hill reaction(above 200 µmoles/mg chl.a;-hr) retained photoresponsivecytochrome c equal only one-tenth that of P700, while the plastocyanincontent was almost equal to that of P700. The cytochrome contentin intact cells was a little larger than that in membrane fragmentson the chlorophyll a basis. However, the values relative toP700 (1/9) and plastocyanin (1/10) were identical with thosein membrane fragments. The content was also far smaller thanthat of reaction center II's (1/6). If the cytochrome mediatesall electrons from reaction center II, the cytochrome oxidation-reductionshould have a rate constant of 2.4?102 sec^–1 which isone order above of the rate constant of the cytochrome reduction(2.3 to 3.5?10¹sec^–1). These quantitative relationshipsindicate that in Anabaena variabilis (M-2), c-type cytochrome,either cytochrome f or algal cytochrome c, cannot function inthe main electron flow between two reaction centers. (Received September 8, 1978; )     

Continuous Cultivation of Glucose-Limited Bean Cells (Phaseolus vulgaris L.) in a Modified Bacterial Fermentor

A commercially available bacterial fermentor was modified toallow the continuous cultivation of glucose-limited bean cells(Phaseolus vulgaris L.) at a defined pH. A steady state wasreached using a dilution rate of 0?004 h^–1 and was maintainedfor 4 months; during this period the dry weight and packed cellvolume of the culture remained constant; the yield for glucosewas 0?34 (dry wt./dry wt) and for carbon 0?27 (g atom/g atom).Glucose-limited cells had lower C/N ratios both for cytoplasmand cell walls than cells in the exponential phase of growthin batch cultures.     

Effects of nutrient and light limitation on the biochemical composition of phytoplankton

Three marine phytoplankters (Isochrysis galbana, Chaetoceros calcitrans andThalassiosira pseudonana), commonly used in the culture of bivalve larvae, were grown in batch or semi-continuous cultures. Changes in protein, carbohydrate, lipid and some fatty acids were measured as growth became limited by nitrogen, silicon, phosphorus or light. Under N starvation (2 d) the % lipid remained relatively constant, while% carbohydrate increased and% protein decreased in all 3 species compared to cells growing under no nutrient limitation. Under Si starvation (6 h) there was no change in lipid, protein or carbohydrates. The amount of two fatty acids, 20 : 53 and 22 : 63 remained relatively constant under N, P and Si starvation, exept for a sharp drop in the cells of P-starvedT. pseudonana. However, there were pronounced species differences withI. galbana containing significantly less 20 : 5 3 thanC. calcitrans orT. pseudonana. Under light limitation the amount of lipid per cell showed no consistent trend over a range of irradiances for all 3 species. The amount of N per cell (an index of protein content) as a function of irradiance, was relatively constant forI. galbana andT. pseudonana, while the amount of N per cell was lower under low irradiances forC. calcitrans. These examples of changes in protein, carbohydrate, lipid and certain fatty acids under nutrient (N, Si or P) or light limitation, emphasize the importance of knowing the phase (e.g. logarithmic vs stationary) of the growth curve in batch cultures, since the nutritional value of the phytoplankters could change as cultures become dense and growth is terminated due to nutrient or light limitation.Presented at the XIIIth International Seaweed Symposium, University of British Columbia, Vancouver, Canada, August 1989.     

Germination of Phaseolus vulgaris III. The role of nucleic acid and protein synthesis in the initiation of axis elongation

Excised embryonic axes of Phaseolus vulgaris L. (var. WhiteMarrowfat) begin cell elongation after approximately 4 hr ofincubation at 26°C. The incorporation of ³²P into nucleicacids and phenylalanine-l-¹⁴C into protein markedly increasesduring the 4th hr of incubation, prior to initiation of cellelongation. CH, which inhibits incorporation of phenylalanine-l-¹⁴C intoprotein by 93% during the 2nd hr after its addition, completelyprevents the initiation of axis elongation if added up to 2hr after the beginning of imbibition. Actinomycin D reducesthe fresh weight increase of the axes, and inhibits both ³²Pincorporation into nucleic acids and phenylalanine-l-¹⁴C incorporationinto protein. 5-FU inhibits ³²P incorporation into nucleic acidsbut not phenylalanine-l-¹⁴C incorporation into protein or thefresh weight increase of the axes. MAK column chromatography indicates that actinomycin D inhibitsthe synthesis of all types of nucleic acids to about the sameextent, while 5-FU almost completely inhibits the accumulationof ³²P in ribosomal RNA with lesser but significant inhibitoryeffects on accumulation of ³²P in tRNA. The results suggest an absolute requirement for protein synthesisprior to initiation of cell elongation and at least a partialrequirement for synthesis of nucleic acid species other thanribosomal RNA, tRNA and DNA. The kinetic data suggest that theaxes develop a greatly increased capacity for nucleic acid andprotein synthesis prior to initiation of axis elongation. ¹This research was supported by NSF grant GB 4145 and a grantfrom the U. S. Forest Service. (Received December 16, 1968; )     

Effects of Abscisic Acid on Nucleic Acid Synthesis and the Induction of Nitrate Reductase in Lemna polyrhiza

Abscisic acid (ABA) at low concentrations brings about the formationof turions (dormant fronds) in Lemna polyrhiza within 3 to 5days after application. The incorporation of ³H-thymidine intoDNA, separated by polyacrylamide-gel electrophoresis, is inhibitedby 80 to 90 per cent within 1 to 3 h of ABA application. Theincorporation of ¹⁴C-orotate and ³²P into RNA is not inhibiteduntil 3 to 9 h after ABA application, but 70 per cent inhibitionis reached after 24 h on 10^–5 M ABA. There is little inhibitionof ¹⁴C-leucine incorporation into protein until 2 to 3 daysafter application of ABA. The capacity of nitrate to inducenitrate reductase in cultures previously grown on nitrate-freemedium is not affected by ABA even up to 3 days after application.The results are discussed in relation to the mode of actionof ABA.     

Temperature and salinity effect on growth and chemical composition of Gyrodinium aureolum Hulburt in culture

A factorial experiment shows highly significant effects of temperature(12 5–22.5°C) and salinity (17.8–34 S) on thegrowth rate of Gyrodinium aureolum, with a significant temperature-salinityinteraction. The maximum growth rate of G aureolum is measuredto 0.61 div. day^–1 at 20°C and 22.3 S. Gyrodiniumaureolum does not grow at temperatures :10 °C or 25°Cand at salinities 12 S. The cellular content of carbon (C) andnitrogen (N) and the elemental ratios N/C, P/C and N/P are significantlyaffected by the temperature The cellular content of phosphorus(P) and the elemental ratios P/C and N/P vary significantlywith salinity Significant temperature-salinity interactionsare found for the cellular content of carbon, nitrogen and phosphorus.Variations in the N/P ratio indicate that G.aureolum has a largestorage capacity for phosphorus It is suggested that temperatureis one important limiting factor in the initiation of bloomsof G.aureolum in north European waters.     

The mitochondrial external NADPH dehydrogenase modulates the leaf NADPH/NADP+ ratio in transgenic Nicotiana sylvestris

Plant mitochondria contain alternative external NAD(P)H dehydrogenases,which oxidize cytosolic NADH or NADPH and reduce ubiquinonewithout inherent linkage to proton pumping and ATP production.In potato, St-NDB1 is an external Ca²⁺-dependent NADPH dehydrogenase.The physiological function of this enzyme was investigated inhomozygous Nicotiana sylvestris lines overexpressing St-ndb1and co-suppressing St-ndb1 and an N. sylvestris ndb1. In leafmitochondria isolated from the overexpressor lines, higher activityof alternative oxidase (AOX) was detected. However, the AOXinduction was substantially weaker than in the complex I-deficientCMSII mutant, previously shown to contain elevated amounts ofNAD(P)H dehydrogenases and AOX. An aox1b and an aox2 gene wereup-regulated in CMSII, but only aox1b showed a response, albeitsmaller, in the transgenic lines, indicating differences inAOX activation between the genotypes. As in CMSII, the increaseof AOX in the overexpressing lines was not due to a generaloxidative stress. The lines overexpressing St-ndb1 had consistentlylowered leaf NADPH/NADP⁺ ratios in the light and variably decreasedlevels in darkness, but unchanged NADH/NAD⁺ ratios. CMSII insteadhad similar NADPH/NADP⁺ and lower NADH/NAD⁺ ratios than thewild type. These results demonstrate that St-NDB1 is able tomodulate the cellular balance of NADPH and NADP⁺ at least inthe day and that reduction of NADP(H) and NAD(H) is independentlycontrolled. Similar growth rates, chloroplast malate dehydrogenaseactivation and xanthophyll ratios indicate that the change inreduction does not communicate to the chloroplast, and thatthe cell tolerates significant changes in NADP(H) reductionwithout deleterious effects.     

Changes in toxins, intracellular and dissolved free amino acids of the toxic dinoflagellate Gymnodinium catenatum in response to changes in inorganic nutrients and salinity

The paralytic shellfish poison prducing dinoflagellate Gymnodiniuncatemrum was subjected to changes in salinity, phosphate, ammoniumand nitrate using continuous culture and batch culture methods.In contrast with other algae, this species showed very slowchanges in the concentration of intracellular amino acids, inthe Gln:Glu ratio, and, in contrast with Alrsandnum spp., onlyslow changes in toxin content, during such events as N-feedingof Ndeprived cells or during nutrient deprivation. This organismwas found to be very susceptible to disturbance; maximum growthrates around 0.25–0.3 day^–1 with a minimum C:N massratio of 5.5, were attained when cultures were only disturbedby sampling once a day. P-deprived cells were larger (twicethe usual C content of 4 ng C cell^–1 and volume of 20pl). The content of free amino acids was always low (5% of cell-N),with low contributions made by arginine (the precursor for paralyticshellfish toxins). Cells growing using ammonium had the lowestC:N ratios and the highest proportion of intracellular aminoacids as arginine. The toxin profile (equal mole ratios of dcSTX,GTX₅, dcGT_2/3 C₁ and C₂, and half those values for C₃ and C₄)was stable and the toxin concentration varied between 0.2 and1 mM STX equivalents (highest when ammonium was not limiting,lowest in P-deprived cells, though as the latter were largertoxin per cell was not so variable). Decreased salinity didnot result in increases in toxin content. Significant amountsof amino acids (mainly serine and glycine, with a total oftenexceeding 4 µM) accumulated in the growth medium duringbatch growth even though the cultures were not bacteria free. ⁴Present address: Instituto Español de Oceanografia,Apdo 1552, 36280, Vigo, Spain