FLORIDOSIDE AS A CARBON PRECURSOR FOR THE SYNTHESIS OF CELL‐WALL POLYSACCHARIDE IN THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA)1

Although red algae are known to be obligatory photoautotrophs, the red microalga Porphyridium sp. was shown to assimilate and metabolize floridoside. A pulse‐chase experiment with [¹⁴C]floridoside showed that at the end of a 240‐min pulse, 70% of total ¹⁴C‐uptake by the cells remained in the floridoside fraction. To evaluate the assimilation of floridoside by Porphyridium sp. cells, we exposed Porphyridium sp. not only to [¹⁴C]floridoside but also to its constituents, [¹⁴C]glycerol and [¹⁴C]galactose, as compared with [¹⁴C]bicarbonate. The extent of incorporation of [¹⁴C] galactose by the Porphyridium sp. cells was insignificant (50–80 dpm·mL^?1), whereas uptake of ¹⁴C from [¹⁴C]glycerol into the algal cells was evident (2.4 × 10³ dpm·mL^?1) after 60 min of the pulse. The pattern of ¹⁴C distribution among the major constituent sugars, xylose, glucose and galactose, of the labeled soluble polysaccharide was dependent on the ¹⁴C source. The relative content of [¹⁴C]galactose in the soluble polysaccharide was highest (28.8%) for [¹⁴C]floridoside‐labeled culture and lowest (19.8%) for the [¹⁴C]glycerol‐labeled culture. Upon incubation of [¹⁴C]floridoside with a crude extract of a cell‐free system prepared from nonlabeled cells of Porphyridium sp., the label was indeed found to be incorporated into the sulfated polysaccharide. Our results suggested that the carbon metabolic pathway in Porphyridium sp. passes through the low molecular weight photoassimilatory product—floridoside—toward sulfated cell‐wall polysaccharide production.     

Effect of Brefeldin A on cell-wall polysaccharide production in the red microalga Porphyridium sp. (Rhodophyta) through its effect on the Golgi apparatus

The cells of the red microalga Porphyridium sp. are encapsulated within a complex sulphated polysaccharide, comprising cell-wall-bound and soluble fractions. The current study investigated the involvement of the Golgi apparatus in the production of the sulphated polysaccharide by treating the cultures with brefeldin A (BFA), a membrane-traffic inhibitor of the Golgi apparatus. Addition of BFA (10–25 μM) upon inoculation (logarithmic-phase cells) decreased the contents of both bound and soluble polysaccharides. Exposure of stationary-phase cultures to BFA (20 μM) inhibited the formation of the cell-wall bound polysaccharide to a greater extent than that of the soluble polysaccharide. Under conditions of nitrate starvation, BFA treatment had a more marked effect on soluble than on bound polysaccharide formation, as was supported by ¹⁴C pulse-chase experiments. BFA addition up to the first 10 h of the cell cycle affected cell division and bound polysaccharide and starch contents. An ultrastructural study showed that exposure of the cells to 20 μM BFA for 16 h disrupted the integrity of the Golgi apparatus. The integrated results of this study demonstrate clearly that BFA affects the architecture of the Golgi apparatus and hence polysaccharide production in algal cells.     

Antioxidant activity of the polysaccharide of the red microalga <Emphasis Type="Italic">Porphyridium</Emphasis> sp

The cells of the red microalga Porphyridium UTEX 637 are encapsulated within a sulfated polysaccharide whose external part (i.e., the soluble fraction) dissolves into the medium. It is thought that the main function of the polysaccharide is to protect the algal cells from the extreme environmental conditions, such as drought and high light, prevailing in their native sea-sand habitat. In this study, we evaluated the antioxidant properties of the water-soluble polysaccharide of Porphyridium sp. by determining the ability of a polysaccharide solution to inhibit: (1) autooxidation of linoleic acid, as determined by the standard thiobarbituric acid (TBA) and ferrous oxidation (FOX) assays; and (2) oxidative damage to 3T3 cells as determined by the dichlorofluorescein (DCFH) assay. In all three assays, the polysaccharide inhibited oxidative damage in a dose-dependent manner. Antioxidant activity was also exhibited by fractions of the polysaccharide obtained by sonication followed by separation on a reverse-phase HPLC with a C₈ semi-preparative column. It is suggested that the antioxidant activity of the sulfated polysaccharide protects the alga against reactive oxygen species produced under high solar irradiation, possibly by scavenging the free radicals produced in the cell under stress conditions and transporting them from the cell to the medium.     

DEGRADATION OF THE CELL-WALL POLYSACCHARIDE OF PORPHYRIDIUM SP. (RHODOPHYTA) BY MEANS OF ENZYMATIC ACTIVITY OF ITS PREDATOR,GYMNODINIUM SP. (PYRROPHYTA)1

The dinoflagellate Gymnodinium sp., which preys specifically on cells of the red microalga Porphyridium sp., possesses enzymes that degrade exocellular polysaccharides of the Porphyridium sp. A crude extract of Gymnodinium sp. was applied to this polysaccharide, and the degradation products were characterized by charge and size separations. Charge separation revealed the presence of a fraction that was not found in the native polysaccharide. This fraction, which was eluted from an anion-exchange resin with water alone, was composed mostly of glucose and xylose (in a 1:1 weight ratio). Size separation of the degradation products revealed three fractions; the molecular weight of the main one was 5 × 10⁶ daltons, whereas that of the native polysaccharide was 7 × 10⁶ daltons. The carbohydrate composition of these fractions was determined. Although the main product of degradation had a relatively high molecular weight, its viscosity was significantly reduced relative to the native polysaccharide. Additional enzymatic degradation is required for further exploration of the structure of the exocellular polymer of Porphyridium sp.     

Impact of the salt stress on the photosynthetic carbon flux and C-label distribution within floridoside and digeneaside in Solieria chordalis

The flux of photosynthetic carbon used in the synthesis of low-molecular weight carbohydrates (digeneaside and floridoside) was investigated by ¹³C and ¹H NMR spectroscopy in samples of the red seaweed, Solieria chordalis, incubated at different salinities (22, 34 and 50 psu). Carbohydrates were labelled, by pulse-chase, with the stable isotope ¹³C from NaH¹³CO₃. In vivo NMR analyses carried out with a cryogenic probe optimised for ¹³C detection were performed directly on the living algal tissues to evidence the labelling of the carbohydrates with neither preliminary extraction nor purification step(s). The isotopic enrichment of each compound was determined by high-resolution ¹H and ¹³C NMR spectroscopy. These analyses evidenced different orientations of the flux of the photosynthetic carbon in the algae according to the salt stress. At normal and low salinities, the photosynthetic carbon flux was responsible of 70% and 67% of the floridoside synthetized during the pulse period, respectively, whereas it was only of 30% in the thalli exposed to the high salinity, meaning a biosynthesis of high floridoside amount from endogen source leading to the osmotic regulation. Under normal and hyper-osmotic conditions, the stock of floridoside was used for cellular needs during the chase period, whereas it was not under hypo-osmotic conditions.The characterization of isotopomers composition of floridoside and digeneaside and the analysis of adjacent ¹³C-labelling gives much more details on the effects of salinity on the metabolic pathways leading to the synthesis or the degradation of these molecules. High turnover of floridoside was evidenced at normal salinity during the chase period and products issued from the degradation of floridoside would not be used for the novo biosynthesis. That suggests that synthesis and degradation of floridoside may be realized in two different cellular compartments. The presence of more numerous ¹³C-¹³C blocks in the carbon skeleton of the molecules from the up salt stressed thalli than in those from no salt stressed algae, concomitant with a slower degree of isotopic enrichment of the molecule, provided evidence that the two metabolic pathways (endogen and photosynthetic) may not share the precursor molecules involved in the floridoside synthesis and that these two routes may be totally separate until the constitution of floridoside molecule.     

Diurnal changes in allocation and partitioning of recently assimilated carbon in loblolly pine seedlings

We examined diurnal fluctuations in acquisition and partitioning of recently assimilated ¹⁴CO₂, and in subsequent allocation and partitioning to roots of loblolly pine (Pinus taeda L.) seedlings. Nonmycorrhizal seedlings were grown under optimal nutrient conditions in continuously flowin solution culture. Shoots of 15-week-old loblolly pine seedlings were labeled with ¹⁴CO₂ for 30 min at four separate labeling times: 1000, 1200, 1400 and 1600 h. Six whole plant harvests were conducted during a 48 h chase period, i.e. 0, 4, 8 12, 24 and 48 h after the end of the labeling and evacuation periods. Although assimilation of ¹⁴CO₂ was constant between 1000 and 1400 h, there were significant differences in partitioning of ¹⁴C-labeled assimilate in needles of all age classes. The highest percentage of recently assimilated ¹⁴CO₂ in the ethanol-soluble fraction of photosynthesizing tissue was observed near the beginning and end of the photoperiod. Partitioning of ¹⁴C in the ethanol-soluble fraction declined between the 1000 and 1400 h labeling eriods, and was accompanied by an increase in partitioning of recently assimilated ¹⁴CO₂ toward starch and a decrease in respiratory losses. These data suggest that most of the ¹⁴CO₂ assimilated at 1000 h was used to support shoot metabolic activities and possibly restore soluble sugar reserves. Peak starch accumulation in needles during the 1400 h labeling period, concomitant with minimal respiratory loss, indicated that photosynthate production exceeded demand and export out of source leaves. A possible feedback regulation of photosynthesis by starch and/or sugar accumulation may be responsible for the observed decline in assimilation of ¹⁴CO₂ during the 1600 h labeling period. Net accumulation of recently assimilated ¹⁴CO₂ in roots was correlated with assimilation rate of ¹⁴CO₂, but independent of partitioning of recently assimilated carbon in photosynthetic tissue. However, the percentage of total seedling ¹⁴C allocated to roots was essentially the same throughout the 48 h chase, regardless of time of labeling and assimilation rate. The data suggest a strong diurnal regulation of starch and soluble sugars synthesized from recently assimilated carbon in needles of loblolly pine seedlings that was independent of assimilation rate. Allocation and transport of recently assimilated carbon to roots of loblolly pine seedlings were not subject to short-term fluctuations in supply and demand.     

Extracellular polysaccharide production in outdoor mass cultures of Porphyridium sp. in flat plate glass reactors

This work concerns an attempt to develop large scalecultivation of Porphyridium sp. outdoors. Theimpact on cell growth and production of solublesulphated polysaccharides of light-path length (LP)was studied in flat plate glass reactors outdoors. TheLP of the plate reactors ranged from 1.3–30 cm,corresponding to culture volumes of 3–72 L. The sidewalls of all reactors were covered, ensuring similarilluminated surfaces for all reactors. Maximal daytemperature was maintained at 26 ±1 ^°C.Growth conditions of pH (7.5), stirring (withcompressed air) and mineral nutrients, were optimal.Maximal volumetric concentration of the soluble sulfated polysaccharide (1.32 g L^-1) was obtained in winter with the smallest light-pathreactor (1.3 cm ) at a cell density of 1.37 ×10¹¹cells L^-1. Under these conditions, theviscosity of the culture medium was also highest,being inversely proportional to the culture'slight-path. Highest areal concentration of solublepolysaccharides (60 g m^-2) and areal cell density(3.01 × 10¹²m^-2) was recorded in the 20 cmLP reactor, progressively lower values being obtainedas the light path became shorter. A similar patternwas obtained for the areal productivity ofpolysaccharides, the highest being 4.15 g m^-2day^-1 (considering the total illuminated reactorsurface), produced in the 20-cm LP reactor.The main sugar composition (i.e. xylose, galactose andglucose) of the sulfated polysaccharides was similarin all reactors. As viscosity increased with timeduring culture growth, there was a substantial declinein bacterial population. Cultivation throughout mostof the year provided good evidence that a light pathlength of 20 cm in flat plate reactors under theseconditions is optimal for maximal areal solublepolysaccharide production of Porphyridium sp.     

CELL-WALL FORMATION DURING THE CELL CYCLE OF PORPHYRIDIUM SP. (RHODOPHYTA)

The cell wall of the red microalgae Porphyridium sp. (UTEX 637) comprises a complex amorphous polysaccharide (6–7 × 10⁶ Da). The polysaccharide is made up of xylose, glucose, and galactose as the main sugars, as well as some minor sugars, protein, sulfate, and glucuronic acid, the latter two conferring a negative charge on the polysaccharide. In this study, we used synchronized cultures as one of the ways of unraveling the mechanism of biosynthesis of this complex polysaccharide by following cell-wall formation during the cell cycle. Synchronization of Porphyridium sp. was achieved with an alternating light:dark regime of 12:12 h LD and dilution of the culture at the end of the cycle. Under these conditions, cell duplication occurred between the 12th and 14th hours of the cycle. The following order of building toward formation of the final polysaccharide appeared to take place: Intermediate polysaccharides with molecular masses ranging from 0.5 × 10⁶ to 2 × 10⁶ Da appeared in succession during hours 2–6 of the cycle, and the full-sized polysaccharide was detected by the 8th hour. At the beginning of the cycle, xylose was the predominant sugar. Sulfur peaked at hours 2–4; glucose, galactose, and glucuronic acid at hours 8–12; and the minor sugars at hours 12–14. Upon incubation of low molecular mass polymer (0.5 × 10⁶ Da) collected from the 4th hour with cellular crude extract from cells of the 6th hour of the cycle, two intermediates were formed (0.8 × 10⁶ Da and 2 × 10⁶ Da). We suggest that the 0.5 × 10⁶ Da polymer intermediate, which is composed mainly of xylose, is the first polymer secreted into the medium, where it is further polymerized enzymatically to produce the 2 × 10⁶ Da polymer via an intermediate 0.8 × 10⁶ Da polymer. Later, the full-size polysaccharide is produced.     

Stable isotope labelling method for studies of saccharide metabolism in Agardhiella subulata

Stable isotopes are preferable in many ways to radioactive isotopes for metabolic studies designed to elucidate biosynthetic pathways. We have developed the methodology to utilize ¹³C-labelled compounds in tracer studies of saccharide metabolism in the red algae. Cultures of Agardhiella subulata were pulse-chase labelled with ¹³C0₂ and ¹²C0₂. Gas chromatography/mass spectrometry (GC-MS) and ¹³C-NMR provided for positive identification of labelled carbohydrate metabolites. In addition, GC-MS can be used to profile the monosaccharide composition of algal species and combined GC-MS and ¹³C-NMR can disclose which carbon(s) is (are) labelled and the extent of labelling. In ¹³C0₂ incubated plants, the label is clearly detected in floridoside and floridean starch. After chasing the labelled alga with ¹²CO₂ for three days or storing the pulse-chase labelled alga in darkness for 6 days, labels disappeared from both floridoside and starch and the contents of these two carbohydrates became very low. More detailed biochemical analysis is being continued to identify labelled cell wall polysaccharides and/or their precursors.     

Synthesis and Turnover of Cell-Wall Polysaccharides and Starch in Photosynthetic Soybean Suspension Cultures

Soybean (Glycine max [L.] Merr.) suspension cultures grown under photoautotrophic and photomixotrophic (1% sucrose) culture conditions were used in 14CO2 pulse-chase experiments to follow cell-wall polysaccharide and starch biosynthesis and turnover. Following a 30-min pulse with 14CO2, about one-fourth of the 14C of the photoautotrophic cells was incorporated into the cell wall; this increased to about 80% during a 96-h chase in unlabeled CO2. Cells early in the cell culture cycle (3 d) incorporated more 14C per sample and also exhibited greater turnover of the pectin and hemicellulose fractions as shown by loss of 14C during the 96-h chase than did 10- and 16-d cells. When the chase occurred in the dark, less 14C was incorporated into the cell wall because of the cessation of growth and higher respiratory loss. The dark effect was much less pronounced with the photomixotrophic cells. Even though the cell starch levels were much lower than in leaves, high 14C incorporation was found during the pulse, especially in older cells. The label was largely lost during the chase, indicating that starch is involved in the short-term storage of photosynthate. Thus, these easily labeled and manipulated photosynthetic cells demonstrated extensive turnover of the cell-wall pectin and hemicellulose fractions and starch during the normal growth process.     

The fate and path of assimilation products in the stem of 8-year-old Scots pine (Pinus sylvestris L.) trees

Summary ¹⁴CO₂ at ambient concentration was administered to a section of an upper branch of 8-year-old Scots pines and the import of radiocarbon into the stem and roots was determined after various chase periods. ¹⁴CO₂ fixation was performed in October when export of carbon into the stems and roots was maximal. In the short-term experiments the trees were harvested 1 h, 2 days and 5 days after a 3-h ¹⁴C pulse, while chase periods of 5 or 8 months were used in the long-term experiments. Loss of ¹⁴C was initially substantial, and even after a 5-day chase had not come down to a rate which indicated decrease only by respiration. After 5 days, more than 10% of the recovered radiocarbon (53% of the ¹⁴C translocated into the stem) had entered the roots and approximately the same amount was found in the stem. Extension of the chase period beyond 5 months did not result in a further significant loss of ¹⁴C by respiration, and the bulk of the label could be localized in the cell-wall fraction. No substantial redistribution of radiocarbon prior and subsequent to the formation of the new shoots could be observed, thus indicating that the stored material was utilized for thickening the stem and roots. Radioautography of stem cross-sections revealed a narrow helical strip of ¹⁴C from the feeding branch to the root in the phloem region. In the tree harvested after bud break the utilization of the ¹⁴C-labelled material stored in the stem for the production of the first layers of earlywood and the corresponding phloem was apparent.     

Bicarbonate stimulates non‐structural carbohydrate pools of Camptotheca acuminata

The role of root‐derived dissolved inorganic carbon (DIC) has been emphasized lately, as it can provide an alternative source of carbon for photosynthesis. The fate of newly fixed DIC and its effect on non‐structural carbohydrate (NSC) pools has not been thoroughly elucidated to date. To this end, we used ¹³C (NaHCO₃) as a substrate tracer to investigate the incorporation of newly fixed bicarbonate into the plant organs and NSC compounds of Camptotheca acuminata seedlings for 24 and 72 h. NSC levels across the organs were all markedly increased within 24 h of labeling treatment and afterward only decreased in stems at 72 h. The variation range of NSC concentrations in roots was considerably smaller than in the stem and leaves. As time passed, the δ¹³C in NSC compounds was significantly affected by ¹³C labeling and was more positive in the roots than in the stem and leaves. Starch was more ¹³C‐enriched than was soluble carbohydrate, and the δ¹³C of root starch was as high as ?4.70‰. Bicarbonate incorporation into newly formed NSC compounds contributed up to 0.24% of the root starch within 72 h. These data provided strong evidence that bicarbonate not only acted as a C source that contributed slightly to the NSC pools but also stimulated the increase in NSC pools. The present study expands our understanding of the rapid change of NSC pools across the organs in response to bicarbonate.     

Antiviral effect of red microalgal polysaccharides on Herpes simplex and Varicella zoster viruses

The cell-wall sulphated polysaccharide of the red microalga Porphyridium sp. has impressive antiviral activity against Herpessimplex viruses types 1 and 2 (HSV 1, 2) and Varicella zoster virus(VZV). Treatment of cells with 1 g mL^-1 polysaccharideresulted in 50% inhibition of HSV-infection as measured by the plaqueassay. Inhibition of the production of new virus particles was also shownwhen pre-infected cell cultures were treated with the polysaccharide. Inaddition, there was indirect evidence for a strong interaction between thepolysaccharide and HSV and a weak interaction with the cell surface.Depending on the concentration, the polysaccharide completely inhibitedor slowed down the development of the cytopathic effect in HSV or VZVpreinfected cells, but did not show any cytotoxic effects on Vero cells evenwhen a concentration as high as 250 g mL^-1 was used. Itseems therefore that the polysaccharide is able to inhibit viral infection bypreventing adsorption of virus into the host cells and/or by inhibiting theproduction of new viral particles inside the host cells. Thus, this alga seems tobe a good candidate for the development of an antiviral drug.     

NDP-sugars, floridoside and floridean starch levels in relation to activities of UDP-glucose pyrophosphorylase and UDP-glucose-4-epimerase in Solieria chorda l is (Rhodophyceae) under experimental conditions

Under limited nutrient availability (i.e. unenriched sea‐water) and under 75 mol photons m^–2 s^–1 irradiance 12:12 LD, thalli of Solieria chordalis J. Agardh accumulated floridean starch and floridoside. When they were transferred into nutrient‐enriched seawater (150 umol L^?1 NO3¹‐ and 7 umol L^?1 P0₄³i at 35 umol photons m^?2 s^?1 in irradiance 12:12 LD, starch and floridoside levels decreased. The main nucleotide diphosphate (NDP) sugars (i.e. UDP‐glucose, UDP‐galactose and ADP‐glucose) and the activities of UDP‐glucose pyrophosphorylase [Enzyme Code (EC) 2.7.7.9] and UDP‐glucose‐4‐epimerase (EC 5.1.3.2) were measured under these controlled culture conditions. Both UDP‐glucose and UDP‐galactose in the thal l i increased under conditions known to favor the accumulation of floridean starch and floridoside, whereas they decreased under conditions leading to floridean starch and floridoside breakdown. On the other hand, ADP‐glucose level only varied slightly. Although UDP‐glucose pyrophosphorylase activity rose under conditions of floridean starch synthesis, little variation was observed in UDP‐glucose‐4‐epimerase activity. These results suggest a possible enzymatic regulation of the NDP‐sugar and carbohydrate pool in which UDP‐glucose pyrophosphorylase would play a major role.     

Detection of a variable intracellular acid‐labile carbon pool in Thalassiosira weissflogii (Heterokontophyta) and Emiliania huxleyi (Haptophyta) in response to changes in the seawater carbon system

Accumulation of an intracellular pool of carbon (C_i pool) is one strategy by which marine algae overcome the low abundance of dissolved CO₂ (CO_2(aq)) in modern seawater. To identify the environmental conditions under which algae accumulate an acid‐labile C_i pool, we applied a ¹⁴C pulse‐chase method, used originally in dinoflagellates, to two new classes of algae, coccolithophorids and diatoms. This method measures the carbon accumulation inside the cells without altering the medium carbon chemistry or culture cell density. We found that the diatom Thalassiosira weissflogii [(Grunow) G. Fryxell & Hasle] and a calcifying strain of the coccolithophorid Emiliania huxleyi [(Lohmann) W. W. Hay & H. P. Mohler] develop significant acid‐labile C_i pools. Ci pools are measureable in cells cultured in media with 2–30 µmol l^?1 CO_2(aq), corresponding to a medium pH of 8.6–7.9. The absolute C_i pool was greater for the larger celled diatoms. For both algal classes, the C_i pool became a negligible contributor to photosynthesis once CO_2(aq) exceeded 30 µmol l^?1. Combining the ¹⁴C pulse‐chase method and ¹⁴C disequilibrium method enabled us to assess whether E. huxleyi and T. weissflogii exhibited thresholds for foregoing accumulation of DIC or reduced the reliance on bicarbonate uptake with increasing CO_2(aq). We showed that the C_i pool decreases with higher CO₂:HCO₃^? uptake rates.     

THE PRODUCTION OF EXTRACELLULAR POLYSACCHARIDE BY THE UNICELLULAR RED ALGA PORPHYRIDIUM AERUGINEUM1,2

The unicellular red alga Porphyridium aerugineum was shown to be encapsulated by an amorphous, water-soluble, polyanionic polysaccharide of high molecular weight. The encapsulating polysaccharide is qualitatively identical with polysaccharide found dissolved in large quantity in the culture medium. The kinetics of extracellular polysaccharide production as a function of cell age was studied. Rates of production (on a per cell basis) of both encapsulating and dissolved polysaccharides are greatest in stationary phase light-grown cultures. Dissolved polysaccharide was quantitatively isolated by precipitation with cetyl pyridinium chloride, conversion to the calcium salt, and reprecipitation with ethanol. The procedure yields a spectrally pure product, which is composed of glucose, galactose, xylose, and 2 undetermined, sugar components, and has a sulfate content of 7.6% by weight. Electron microscopy of Porphyridium revealed that Golgi vesicles transport, polymerized polysaccharides to and through the cell membrane. Similar vesicles were observed in the multicellular Pseudogloiophloea, indicating that the Golgi complex plays a crucial role in the production of extracellular polysaccharides by the red algae. H¹⁴CO₃^- pulse-label experiments resulted in labeled extracellular polysaccharide in which all the constituent components contained ¹⁴C. Rates of excretion of polysaccharide were found, to follow a cyclic pattern, correlated generally with the division cycle, of the cell.     

Relationship between Photosynthesis and Protein Synthesis in Maize: I. Kinetics of Translocation of the Photoassimilated Carbon from the Ear Leaf to the Seed

To gain a better understanding of the biochemical basis for partitioning of photosynthetically fixed carbon between leaf and grain, a ¹⁴CO₂ labeling study was conducted with field-grown maize plants 4 weeks after flowering. The carbon flow was monitored by separation and identification of ¹⁴C assimilates and ¹⁴C storage components within each tissue during the chase period (from 4 to 96 hours) following a 5 minute ¹⁴CO₂ pulse. In the labeled ear leaf, the radioactivity strongly decreased to reach, at the end of the experiment, about 12% of the total incorporated radioactivity, mostly associated with sucrose and proteins. Nevertheless, an unexpected reincorporation of radioactivity was observed either in leaf starch or proteins, the day following the pulse. Conversely, the radioactivity in the grain increased to attain 66% of the total incorporated ¹⁴C after a 96 hour chase. The photosynthates, mostly sucrose, organic and free amino acids, rapidly translocated towards the developing seeds, served as precursors for the synthesis of seed storage compounds, starch, and proteins. They accumulate in free form for 24 hours before being incorporated within polymerized storage components. This delay is interpreted as a necessary prerequisite for interconversions prior to the polycondensations. In the grain, the labeling of the storage molecules, either in starch or in storage protein groups (salt-soluble proteins, zein, and glutelin subgroups), was independent of their chemical nature but dependent on their pool size.     

Incorporation of [14C]Methanol and [14C]Bicarbonate by Hyphomicrobium sp. 53-49 under Various Growth Conditions: Aerobic,Denitrifying and Autotrophic

A study was made of the incorporation of methanol and bicarbonate into the cell constituents of denitrifying or aerobic methanol grown and autotrophic H₂–O₂–CO₂ grown Hyphomicrobium sp. 53-49. Cells were incubated with [¹⁴C]methanol or [¹⁴C]bicarbonate, and the distribution of the radioactivity in the nonvolatile constituents of ethanol extracts of cells was examined. When denitrifying grown cells were incubated with [¹⁴C]methanol, the major part of the radioactivity was fixed to serine as the first stable compound. Aerobic methanol grown cells also fixed [¹⁴C]methanol mainly to serine. These results suggest that methanol grown cells assimilate methanol by the serine pathway. When denitrifying or aerobic methanol grown cells were incubated with [¹⁴C]bicarbonate, malate was mainly observed as a nonvolatile compound in the initial period of the incubation. Autotrophic grown cells also fixed the major part of [¹⁴C]bicarbonate to malate. In this case, phosphoglyceric acid was found in the phosphorylated compounds area.     

DARK CARBON FIXATION STUDIES ON THE INTERTIDAL MACROALGA ASCOPHYLLUM NODOSUM (PHAEOPHYTA) 1

The characteristics of dark carbon fixation by Ascophyllum nodosum were investigated. In longitudinal profile the maximum rates of dark and light dependent fixation are found at the apex. The use of Michaelis-Menten kinetics did not suitably describe the relationship between the uptake rate in the dark and the total inorganic carbon concentration. Dark fixation was saturated at a total inorganic carbon concentration [TIC] of 2.5 mM. The use of the Hill-Whittingham equation to describe the uptake curve indicates that the process is diffusion limited. Comparisons of dark fixation at high (8.0) and low (5.2) pH suggest that bicarbonate ions are used as a source of inorganic carbon. The transfer of ¹⁴C, fixed in the dark, from the ethanol soluble to the insoluble fraction was relatively slow irrespective of the light treatment during the chase period. Ascophyllum nodosum displays a small diel fluctuation in the pH of aqueous extracts and titratable acidity similar to that displayed by CAM plants. The significance of dark fixation to the overall carbon budget is discussed.     

RADIOLABELING STUDIES OF LIPIDS AND FATTY ACIDS IN NANNOCHLOROPSIS (EUSTIGMATOPHYCEAE), AN OLEAGINOUS MARINE ALGA1

The synthesis of fatty acids and lipids in Nannochloropsis sp. was investigated by labeling cells in vivo with [¹⁴C]-bicarbonate or [¹⁴C]-acetate. [¹⁴C]-bicarbonate was incorporated to the greatest extent into 16:0, 16:1, and 14:0 fatty acids, which are the predominant fatty acids of triacylglycerols. However, more than half of the [¹⁴C]-acetate was incorporated into longer and more desaturated fatty acids, which are constituents of membrane lipids. [¹⁴C]-acetate was incorporated most strongly into phosphatidylcholine, which rapidly lost label during a 5-h chase period. The label associated with phosphatidylethanolamine also decreased during the chase period, whereas label in other membrane lipids and triacylglycerol increased. The dynamics of labeling, along with information regarding the acyl compositions of various lipids, suggests that 1) the primary products of chloroplast fatty acid synthesis are 14:0, 16:0, and 16:1; 2) C₂₀ fatty acids are formed by an elongation reaction that can utilize externally supplied acetate; 3) phosphatidylcholine is a site for desaturation of C₁₈ fatty acids; and 4) phosphatidylethanolamine may be a site for desaturation of C₂₀ fatty acids.