Plant Physiol. (1986) 82, 136-1410032-0889/86/82/01 36/06/$0 1.00/0

Regulation of Carbon Flow by Nitrogen and Light in the RedAlga, Gelidium coulteri'

Received for publication January 29, 1986 and in revised form May 4, 1986

BRUCE A. MACLERDepartment ofBotany, University ofCalifornia, Berkeley, California 94720

ABSTRACT

The red alga Gelidium coulteri Harv. photosynthetically fixed 11'4Cbicarbonate at high rates under defined conditions in unialgal laboratoryculture. The fixation rate and flow of photosynthate into various endproducts were dependent on the nitrogen status of the tissue. Plants fedluxury levels of nitrogen (approximately 340 micromolar) showed fixationrates several-fold higher than those seen for plants starved for nitrogen.The addition of NO3 or NH4' to such starved plants further inhibitedfixation over at least the first several hours after addition. The majorityof 14C after incubations of 30 minutes to 8 hours was found in thecompounds floridoside, agar and floridean starch. In addition, aminoacids and intermediate compounds of the reductive pentose phosphatepathway, glycolytic pathway and tricarboxylic acid cycle were detected.Nitrogen affected the partitioning of labeled carbon into these compounds.Plants under luxury nitrogen conditions had higher floridoside levels andmarkedly lower amounts of agar and starch than found in plants limitedfor nitrogen. Amino acid, phycobiliprotein and chlorophyll levels werealso significantly higher in nitrogen-enriched plants. Addition of N03- tostarved plants led to an increase in floridoside, tricarboxylic acid cycleintermediates and amino acids within 1 hour and inhibited carbon flowinto agar and starch. Carbon fixation in the dark was only I to 7% ofthat seen in the light. Dark fixation of I'4Cibicarbonate yielded labelprimarily in tricarboxylic acid cycle intermediates, amino acids andpolysaccharides. Nitrogen stimulated amino acid synthesis at the expenseof agar and starch. Floridoside was only a minor component in the dark.Pulse-chase experiments, designed to show carbon turnover, indicated a2-fold increase in labeling of agar over 96 hours of chase in the light. Noincreases were seen in the dark. Low molecular weight pools, includingfloridoside, decreased 2- to 5-fold over this period under both light anddark conditions. Nitrogen status did not influence turnover. There waslittle or no organic carbon released into the culture medium over thisperiod. The results are consistent with biosynthetic pathways to florido-side and agar that share the common intermediate UDP-D-galactose. Itis hypothesized that synthesis of floridoside is regulated by nitrogen andlight at the enzymic level.

Recent work in this laboratory showed that the growth rateand the biosynthesis of the polysaccharide agar in Gelidiumcoulteri grown unialgally were affected by the specific environ-mental conditions of culture (BA Macler, unpublished data). Inparticular, N was shown to markedly affect the percent yield ofagar. When plants were starved for fixed N over several days, theyield of agar increased up to 100%. This result, called the Neisheffect, has been demonstrated by other workers in several red

'Supported by Ocean Genetics, Inc., and the National Science Foun-dation grant OCE-8360570.

algal species for both agar and the related polysaccharide, carra-geenan (2, 17). This effect appears to involve altered allocationof photosynthate as well as changes in overall growth rate.To assess the nature of N regulation of agar biosynthesis, it

became of interest to determine the pathways of carbon flowfrom primary fixation products into agar and other end products.If these pathways were known in detail, the effect of N on thephysiological levels ofparticular metabolites could be determinedand the branch points for photosynthate flow and enzymic sitesof regulation could be pinpointed (7). Previous work in this areawith a variety of red algae indicates that carbon is fixed via thereductive pentose phosphate pathway in light (10, 12). Fixationin the dark appears to be primarily via PEP2 carboxykinase (1 1).For Gelidium robustum, a significant fraction of the photosyn-thate appears in the compound floridoside (2-0-glycerol-a-D-galactopyranose) (3, 9). However, previous work utilized plantsharvested from field populations with limited control for theirnutrient levels, environmental condition or reproductive status.Plant to plant variation in field-collected material can signifi-cantly affect quantitation and subsequent conclusions. A detailedassessment of the metabolic pathways of carbon flow underdefined conditions in the red algae has not been reported.

Since it has been possible to grow cloned strains of G. coulteriunder defined conditions, experiments were undertaken to char-acterize carbon fixation and subsequent partitioning of photo-synthate into various end products under a variety of environ-mental conditions. The results reported here detail some of theeffects ofN and light on carbon fixation and flow.

MATERIALS AND METHODS

Plant Material and Culture Conditions. Gelidium coulteriHarv. in unialgal culture was graciously provided by John A.West (JAW 2604). This strain originated from northern BajaCalifornia. Plants were maintained in filtered, steamed seawaterin 16-L carboys under constant aeration. Salinity was 30 g L'.Fluorescent light was provided at 250 ,uE m 2 s-' photon fluxdensity and a 16 h light period. Culture temperature was 26 ±1°C. Plants were maintained with one-eighth strength PES (15)added weekly. This was equivalent to 82 AM NO3- and 1.5 ,uMNH4+. PES also contained trace metals, vitamins and glycero-phosphate. N-limited plants were generated by culture in unen-riched seawater until phycobiliprotein levels were undetectable,which took approximately 10 d. This corresponded to a 50%decrease in total protein and was taken to indicate limiting Nconditions. N-enriched plants were generated by culture in one-half strength PES (330 sM NO;3, 6 iM NH4+) for 4 d prior to theexperiment.

1f4CjBicarbonate Labeling. N-limited and -enriched adultplants were placed in vials containing 20 ml sterile seawater.

2Abbreviations: PES, Provasoli's enriched seawater; PEP, phosphoen-olpyruvic acid.

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METABOLIC REGULATION IN RED ALGAE

Approximately 150 mg wet weight of tissue, corresponding to 4to 10 plants, were used per vial. The appropriate nutrient addi-tions were made and the cultures preincubated for 30 min at 300,gE m-2 s-' photon flux density on a rotary shaker at 100 rpm.Dark control cultures were wrapped in aluminum foil prior topreincubation. Sodium ['4C]bicarbonate (New England Nuclear)was added to each culture to an initial specific radioactivity of2.5 gCi gmol-' C. Initial bicarbonate concentration in the sea-water was 2 mM.An entire culture served as one sample. After the incubation

period, plants were separated from the medium by vacuum,washed briefly with seawater, then placed in full strength DMSOto kill the tissue and extract the soluble compounds. Aminoacids were separated from other low mol wt compounds by themethod of Larsen et al. (13). Aliquots of this crude extract weredried down under N, resuspended in distilled H20 and appliedto 6 cm cation-exchange columns (Ag 50W x 8, 200-400 mesh,H form, Bio-Rad) in Pasteur pipets. Columns were washed withglass distilled H20 to remove all low mol wt compounds but theamino acids. Amino acids were eluted with 3 N NH40H. Allfractions were acidified and taken to dryness under N2. Residueswere resuspended in 80% methanol and chromatographed onpaper in two dimensions; first in phenol:water:glacial acetic acid(42:13:5, v/v/v), then in l-butanol:water:propionic acid(74:49:36, v/v/v) as described by Pedersen et al. (19). Thecompounds were located and identified by autoradiography, co-chromatography with known compounds and use of selectivestains. Authentic floridoside and isofloridoside were graciouslyprovided by James S. Craigie. The radioactive material was elutedfrom the paper with water and quantified by liquid scintillation.Agar Extraction. Tissue residue remaining after DMSO ex-

traction was further extracted for agar following a modificationof Craigie and Leigh (4). Tissue was rinsed twice in 80% ethanolto remove any remaining DMSO, then suspended in 0.02%sodium acetate in water. Material was autoclaved for 60 min andhot filtered through Whatman GF/C paper under N2. The tissuewas reextracted as above and filtrates combined. Agar was pre-

cipitated with the addition of 3 volumes 100% ethanol. Agar wasseparated by centrifugation, washed successively in 80, 90, and100% ethanol, dried overnight and weighed. Subsamples wereresuspended in water and their radioactivity quantified by liquidscintillation. The supernatant fraction was hydrolyzed in 3 NHCO for 2 h at 100°C. Aliquots were chromatographed, identifiedand quantified as above.The remaining plant material, consisting of greater than 90%

starch and protein, was hydrolyzed in 3 N HCl for 8 h at 100°C.The supernatant fraction was separated, neutralized with NaOH,chromatographed, and quantified as above.

Protein was measured on dried, ground plants by the methodof Lowry et al. (14). Chl was quantified on DMSO/ methanolextracts of fresh material by the method of Duncan and Harrison(5).

RESULTS

Effects of Light and N on Total Photosynthetic I'4ClBicarbon-ate Fixation. The initial set of experiments examined carbonfixation in the light and the dark under three conditions of N.These were (a) plants previously cultured with 340 uM total Nenrichment (PES), (b) plants grown in seawater without enrich-ment until limited for N (starved), and (c) starved plants prein-cubated 30 min with I mm N source prior to the experiment torelieve the N limitation. When the experiments were initiated,exponential growth rates for the PES plants were 8 to 11% d-'.The starved plants had lower rates, 5 to 8% d-'.Growth of G. coulteri in unenriched seawater led to plants of

varying degrees of N starvation, dependent on time in such

culture and initial N status. Levels ofprotein, Chl, and phycobilin

diminished under this condition, with protein and Chl reaching50 and 20%, respectively of enriched levels and phycobilinbecoming undetectable (Fig. 1). Radiolabeling of these tissuesshowed a decrease in total fixation rate that paralleled Chl loss.When nitrogen in the form of NO3- was added to starved plants,phycobilin and Chl levels were detectably higher after 2 d.

It was found that under all conditions, total '4C fixation in thelight was linear over at least 7 h in this system. Fixation by PESplants was up to severalfold higher than that seen for starvedplants on a dry weight basis (Table I). Addition of 1 mm NO3(or 1 mm NH4', not shown) to starved plants further inhibitedcarbon fixation. When carbon fixation was calculated on a gmolC fixed mg Chl-' basis, there was no significant differencebetween starved and enriched plants. However, the inhibitionseen with NO3 addition to starved plants remained significant.

Partitioning of Fixed Carbon into Intermediate Compoundsand End Products in the Light. The resulting labeled materialfrom these experiments was separated into intermediate com-pounds and end products and quantified. The sugar phosphatesand diphosphates of the reductive pentose phosphate pathwaywere identified from the earliest time point samples (5 min) inall cases. Citrate, succinate, malate and fumarate from the tri-carboxylic acid cycle and the amino acids aspartate, glutamate,

100

90

80

70

60

0

0-0

50

40

30

20

10

0-2 3 4 5 6 7 8 9 10 11 12 13 14

starvation (days)FIG. 1. Effects of N starvation. PES enriched plants were placed in

unenriched seawater at d 0. Data are averages of 10 plants + SE.

Table I. Photosynthetic Fixation on Dry Weight and Chi BasesThe results represent photosynthesis via incorporation of ['4C]bicar-

bonate from 2-h incubations. Values are average of 4 replicates + SE.

Condition Photosynthesisgmol Cg dry wt-' gmo1 C mg Cht-'

Starved, seawater 120 ± 10 170 ± 10Starved, + I mM nitrate 89 ± 6 110 ± 10PES enriched 640 ± 30 170 ± 10

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Plant Physiol. Vol. 82, 1986

glutamine, asparagine and alanine were detected under all con-

ditions. Glycine, seine, glycolate and glycerate, associated withthe glycolate pathway, were detected. The major fractions oflabeled carbon were found in floridoside, agar, floridean starch,amino acids and protein (Table II).

Since these experiments involved relatively long labeling pe-

riods (up to 7 h), the majority of the intermediate compoundsreached steady-state levels of constant labeled pool size over

time. Other compounds accumulated label throughout an exper-

iment to become significant fractions of the total fixed carbonpool. These included floridoside, aspartate, agar, starch andprotein. This resulted in a decrease in the proportion of label inlow mol wt compounds versus polymers over time.

Significant quantitative differences in labeled pool sizes were

seen between the three N states (Tables II and IV). While underall conditions the most heavily labeled compound was florido-side, the amount varied from 62% or more of the total label forPES plants to 40% for starved plants after a 2 h incubation.Percent carbon flow into agar and floridean starch was 2 to 3times higher in starved versus PES plants. Label in amino acidsand proteins was higher in PES plants. This was particularlymarked for serine/glycine and aspartate, where levels in PESplants were 2-fold higher than in starved plants, and for gluta-mine and asparagine, which were trace in starved plants, butstrongly labeled in PES plants.

Addition of NO3- to starved plants appeared to stimulatefloridoside biosynthesis and inhibit the production of agar within1 h. Flow ofcarbon into amino acids and proteins was stimulated,as evidenced by a 2-fold increase in citrate and glutamate andan 8-fold increase in alanine. Glycine, seine and glycerate levelswere reduced to trace amounts.Carbon Fixation and Flow in the Dark. Carbon fixation in the

dark was between 1 and 7% of photosynthetic fixation (TableIII). Under these conditions, starved plants were as active asplants grown with NO3- or PES. This indicated that N limitationdid not affect dark fixation as it did photosynthetic fixation. Incontrast with G. coulteri grown in light, carbon fixation in thedark yielded very low levels of sugar phosphates and diphos-phates. Of the low mol wt compounds, the majority of label was

Table 11. Partitioning ofPhotosvntheticallv Fixed ['4C]BicarbonatePlants were incubated in 2 mm ['4C]bicarbonate in seawater for 2 h

prior to sampling, then processed as described in Materials and Methods.Nitrate was added 30 min prior to labeling. Values are averages of 5replicates ± SE. Percent values are of total fixation.

Starved Starved, PESSeawater Enriched

1 mM Nitrate

,umol C mg Chi`Total fixation 170 ± 10 120 ± 10 170 ± 10

100% 100% 100%Floridoside 67 ± 8 61 ± 7 105 ± 8

40% 50% 62%Tricarboxylic acid 4.1 ± 0.4 5.8 ± 0.8 1.9 ± 0.2

cycle2.4% 4.8% 1.1%

Amino acids 13 ± 1 13 ± 1 25 ± 27.9% 11% 15%

Nucleotide sugars 0.043 ± 0.005 0.023 ± 0.004 0.024 ± 0.040.53% 0.44% 0.27%

Agar 50 ± 3 16 ± 2 17 ± 230% 13% 10%

Floridian starch 20 ± 2 10 ± 1 9.0 ± 212% 8.6% 5.3%

Protein 7.0 ± 1.0 6.7 ± 0.8 9.2 ±0.94% 6% 5%

Table III. Partitioning ofDark Fixed['4CBicarbonate.Plants were treated as described in Table II.

Starved, Starved, PESeawaterr Seawater Enriched+ Nitrate

Mmol C mg Chl'Total fixation 8.5 ± 0.8 9.2 ± 1.0 1.6 ± 0.1

100% 100% 100%Floridoside 0.85 ± 0.07 0.74 ± 0.2 0.063 ± 0.014

10% 8% 4%Tricarboxylic acid cycle 2.0 ± 0.1 2.4 ± 0.2 0.24 + 0.03

24% 26% 16%Amino acids 1.0 ± 0.2 3.8 ± 0.6 0.98 ± 0.15

12% 38% 64%Agar 3.5±0.3 1.5±0.2 0.14±0.05

41% 16% 9%Floridean starch 0.94 ± 0.15 0.82 ± 0.06 0.092 ± 0.015

11% 9% 6%

Table IV. Amino Acid LabelingPlants were treated as described in Table II.

Starved Starved, PESSeawater Seawater + EnrichedNitrate

,umol C mg Chl'Light:Ala 1.0 ± 0.4 8.3 ± 2.0 1.7 ± 0.4Asp 7.4 ± 1.5 2.5 ± 0.8 12 ± 3Asn 0.2 ± 0.1 tra 3.4± 1.5Glt 1.3±0.3 2.4±0.3 1.1±0.4Gln tr 0.2 ± 0.1 1.3 ± 0.8Gly/Ser 2.6± 1.0 tr 5.0± 1.8

Dark:Ala 0.7±0.3 1.5±0.3 0.12±0.03Asp 0.3±0.1 1.5±0.3 0.50±0.10Asn NDb tr 0.13 ± 0.04Glt tr 0.7 ± 0.2 0.13 ± 0.03Gln ND tr 0.10 ± 0.02Gly/Ser ND ND ND

a Trace. b Not detected.

found in intermediates of the tricarboxylic acid cycle and in theamino acids (Table III). In PES plants, these compounds ac-counted for 80% of total labeled carbon. For all conditions,percent floridoside labeling was low relative to plants in the light.Compounds of the glycolate pathway, glycolate, glycerate, serineand glycine, were undetectable.As was found for plants grown in the light, N status had a

large effect on carbon partitioning (Table IV). Labeling ofaminoacids, particularly aspartate, glutamate, glutamine and aspara-gine, was 5-fold higher in PES plants. The inverse was true foragar and floridoside, which were 4-fold higher in starved plants.Addition of NO3 to starved plants led to a 3-fold increase inamino acids, with decreases in label in floridoside and agar.

Pulse-Chase Experiments. Certain low mol wt compounds,such as floridoside, citrate and aspartate, did not level off inlabeling over 7 h, but continued to accumulate throughout anexperiment. These compounds could be end products that turnover carbon slowly, or merely intermediate compounds that havelarge pool sizes that require a longer time to fill. It was of interestto see if label continued to accumulate in the individual pools inthe period following labeling, indicating an end product, orgradually became less, indicating an intermediate. In addition,since agar and floridoside are believed to have a common pre-cursor in UDP--galactose (1'20). it is possible that there is a

138 MACLER

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METABOLIC REGULATION IN RED ALGAE

direct biosynthetic relationship between them. To address thesequestions, starved and PES plants were labeled with ['4C]bicar-bonate for 1.5 h in the light, then removed to fresh, unlabeledseawater in the light and in the dark. Samples were taken atintervals for 4 d.

It was observed that 37 to 49% of the labeled carbon was lostfrom these plants after 96 h in the light (Fig. 2A). Somewhatmore label, 51 to 56% was lost under dark conditions (Fig. 2B).Analysis of the chase media for acid-stable radioactivity, indicat-ing released organic carbon, showed that no more than 3% ofthis lost label could be accounted for by this fraction. Themajority was recovered as bicarbonate, suggesting a respiratorysource.

Cultures in the light showed large decreases in label in lowmol wt compounds over time (Fig. 2A, Table V) with themajority of this decrease associated with the floridoside pool.Label in sugar phosphates decreased below detectable levelswithin 6 h under all conditions. Label in agar and insoluble cellwall material increased for 1 to 2 d, then leveled off for both PESand starved plants. Total amino acids decreased over time,although individual amino acid pools changed at markedly dif-ferent rates. Glycine, seine and alanine became undetectable by16 h. Levels of glutamate, glutamine and asparagine were con-stant over time. Aspartate rapidly decreased to a steady statelevel quantitatively similar to glutamate. Label in citrate also wasconstant throughout the chase period.

Cultures in the dark similarly showed large decreases in labelin low mol wt compounds over time (Fig. 2B, Table V). Therewas, however, little or no increase in agar or cell wall materials.Aspartate, seine, glycine and alanine decreased much moreslowly in the dark. Glutamate and asparagine maintained steadylabeling and, in PES plants, glutamine increased 6-fold.

DISCUSSION

This work was designed to be a quantitative assessment ofcarbon fixation and intermediary metabolism in the red alga, G.coulteri. The use of uniform cultured clones and defined exper-imental conditions, determined in previous investigations (BAMacler, unpublished data), was designed to minimize artifactualand conditional errors arising from using heterogenous collec-tions of wild-type material. The use of defined conditions bothfor culture and experiment allowed a quantitative comparisonof the effects of different conditions and also allowed an exami-nation of transient phenomena when culture conditions wereexperimentally modified.

Effects ofN on Carbon Fixation and Metabolism. In the light,N starvation of G. coulteri led to large losses of photosyntheticpigments and cellular protein. Rates of photosynthetic carbonfixation fell in parallel with these decreases, thus demonstratinga limitation of photosynthesis based on cellular N levels. Lightharvesting would presumably be less effective with decreasedpigment levels. Enzymes of the reductive pentose phosphatepathway might be less abundant as well. Regardless of the formof inhibition, the net result was less triose-P exported from thechloroplast into the cytoplasm for further reactions.

Besides affecting the availability of photosynthate in the cyto-plasm, N levels had large effects on particular pathways ofcarbonmetabolism: (a) Nitrogen appeared to stimulate the synthesis ofamino acids and protein via anapleurotic movement of carbonthrough tricarboxylic acid cycle intermediates to the amino acidsalanine, glutamate, aspartate, glutamine and asparagine. Thiswas consistent with work by others on spinach (13) and poppy(18) that demonstrates N stimulation of pyruvate kinase andPEP carboxylase. While evidence suggests that PEP carboxylaseactivity is minimal relative to PEP carboxykinase in red algae

25

20

15

starved PES

5IO \ - ~ ~ -t

O .* *

1 2 3 4

0

E

0C1

chase (days) chase (days)FIG. 2. "C label remaining after pulse labeling with ['4C]bicarbonate. Plants were labeled for 1.5-h in 2 mm ['4C]bicarbonate in seawater in the

light, then placed in unlabeled seawater for the indicated periods. A, Chase period in the light; B, chase period in the dark. All values are averages

of 4 replicates + SE.

co

E

-Q0

1

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Plant Physiol. Vol. 82, 1986

Table V. Partitioning of 4C during Chase Periodfollowing Labeling with ['4C]BicarbonateAll plants were incubated in 2 mM ['4C]bicarbonate in seawater in light for 2-h, then placed in label-free

seawater. Values are averages of 4 replicates ± SE. Percent values are of total fixation

Condition Fixation Floridoside Amino Acids Protein Agar Starch

Mmol C mg Chl'Starved,Nochase period 220 ± 10 120 ± 10 32 ± 3 8.4 ± 1.0 28 ± 2 22 ± 2

100% 54% 14% 4% 12% 10%96hchase,light 130± 10 21 ±2 7.2±0.6 7.6±0.9 51 ±4 30±2

100% 16% 6% 6% 38% 23%96 h chase, dark 94 ± 6 25 ± 2 8.9 ± 0.9 12 ± 2 29 ± 3 16 ± 2

100% 27% 9% 13% 30% 17%PES enriched,Nochase period 200 ± 10 120 ± 10 38 ± 5 25 ± 3 12 ± 1 8.5 ± 0.7

100% 58% 20% 12% 6% 4%96 h chase, light 100 ± 10 26 ± 4 13 ± 2 19 ± 2 28 ± 2 12 ± 1

100% 25% 12% 18% 26% 11%96 h chase, dark 95 ± 6 33 ± 3 21 ± 3 15 ± 2 14 ± 1 10 ± 1

100% 34% 22% 15% 14% 11%

(I 1), the stimulation of f3-carboxylation by N may be similar. and III). In the dark, carbon fixation would be most likely viaIncreased labeling ofalanine, which requires pyruvate, supported PEP carboxykinase (11), although the work reported here dida stimulation by N of pyruvate kinase as well; and (b) coinciden- not rule out a reductive tricarboxylic acid cycle (6). This wastal with stimulation of amino acid biosynthesis, N enhanced supported by the large percentages of labeled tricarboxylic acidfloridoside biosynthesis and relatively inhibited formation ofagar cycle components. Some form of gluconeogenesis would beand starch. An explanation of this would likewise involve a required to yield label in starch, agar and floridoside. This wouldquantitative change in the routing of photosynthate. Floridean explain the strong inhibition by N of these three compounds. Asstarch is synthesized in the cytoplasm in the red algae, unlike the in the light, N stimulated amino acid biosynthesis. The increasedstarches of higher plants (16). A simplified scheme metabolically use of carbon skeletons in amino acids would decrease theirrelating starch, agar, floridoside and the amino acids is shown in availability to form hexose phosphates.Figure 3. It can be seen that agar, starch and floridoside all In the dark, floridoside labeling was inhibited relative to agarrequire nucleotide sugars from a common metabolic pool. Flor- and starch compared to patterns seen in the light. This suggestsidoside uniquely requires a-glycerol phosphate derived directly that the floridoside biosynthetic reactions are additionally regu-from triose-P (8). In the experiments reported here, the addition lated by light. This may be indirectly via the availability ofof N to starved plants yielded a rapid increase in floridoside reductant to produce glycerol phosphate or directly on the con-coupled to a decrease in nucleotide sugars, agar and starch. While densation reaction.definitive enzymological tests remain to be done, this data could Carbon Turnover. The results of the pulse-chase experimentsbest be explained by a direct stimulation by N of the condensa- showed that labeled carbon continued to accumulate in agartion of UDP-D-galactose and glycerol phosphate to floridoside over a period of 1 to 2 d in the light when grown in unlabeledphosphate. This would strongly route carbon away from starch media. It is most likely that floridoside provided some carbonand agar biosynthesis without directly affecting their biosynthetic for this and for the continued synthesis of amino acids, as itsenzymes. label decreased over time. Floridoside is probably broken down

Effects of Light and Dark. A different pattern of carbon flow first to free sugars (16), so that recycling into agar would beappeared in the dark. Tricarboxylic acid cycle intermediates and indirect and subject to competing reactions. The majority ofamino acids were a more significant fraction of the total fixed respired carbon was recovered in the medium as ['4C]bicarbon-carbon, while floridoside labeling was much reduced (Tables II ate, but refixation of label would have been negligible due to the

Phosphoglyceric acid

Triose phosphate Phosphoenolpyruvate TCAcycle - IAminoac ids

Fructose bisphosphate

Glucose phosphate _

a-Glycerol phosphate (Zlcephosphate FIdh FIG. 3. Metabolic paths from phosphoglyceric

_-Florephosphte- loridoside acid. N, indicates possible site of nitrogen regula-tion. TCA = tricarboxylic acid.

UDP-glucose ' DUDP- D-ga lactose

Mannose phosphate F snFlorsdeon starch]co

GDP-mannose - GDP l-alctose

140 MACLER

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METABOLIC REGULA

low specific activity.It is important to remember that labeling data only indicate

rate of synthesis and not total cellular carbon in any compound.Only when quantitative data are available for total pool sizes forindividual compounds may rates be discussed in terms ofpercentof total carbon or carbon turnover.

Total carbon fixation rates for G. coulteri, when calculated interms of percent cellular carbon fixed per day, gave values of 5.2for starved plants and 8.4 for PES plants, which were essentiallythe same as the actual exponential growth rates determined forthese plants via weight measurement prior to the experiments.Similar calculations for the floridoside pools indicated that about30% of the starved plant pool and 50% of the PES plant poolwere labeled in one day. For agar, the values were respectivelyonly 1.4 and 1.0% d-'. This is consistent with the data from thepulse-chase experiments. Label in floridoside turned over with ahalf-life of about I d (Fig. 2), but no agar label was apparentlylost from the plants over at least 4 d.These observations support a role for floridoside similar to

that of sucrose in higher plants, as a low mol wt, stable inter-mediate that serves as a short-term carbon reservoir ( 16). Whilecompeting with agar for precursors, floridoside is ultimatelyrecycled to provide carbon for cellular needs. Its synthesis appearsto be highly regulated and positively correlated with the availa-bility of N. Agar appears to be a stable end product that doesnot serve a carbon storage role in addition to its role in the redalgal cell wall. Its synthesis is stimulated under N-limiting con-ditions due to the increased availability of carbon skeletons andthe cellular need for cell wall components to maintain growth.

Presentation of Photosynthetic Data. In previous work, growthrates of these plants under N limitation were observed to bemaintained at control levels for 5 to 8 d before gradually decreas-ing (BA Macler, unpublished data). This growth pattern resultedin a dilution of N-containing compounds by carbohydrates, aresult consistent with the changes observed in the routing offixed carbon shown here. As a consequence of this, photosyn-thetic rates based on dry weights of tissue varied substantiallybetween genetically identical plants of different N status. Sinceit is typical for studies of this type involving red algae to usefield-collected material of undetermined genetic variability andundefined nutrient status, this suggests that red algal photosyn-thetic data be presented in a form that includes a N-basedcomponent, such as Chl or protein. This would allow a clearerseparation ofelements ofgenetic variability between populationsof field collected plants where nutrient levels are uncontrolledfrom the nutrient effects themselves.

TION IN RED ALGAE 141

Acknowledgments-It is a pleasure to thank George Matsumoto for his capableassistance and John A. West and James A. Bassham for their advice and support.

LITERATURE CITED

1. BEAN RC, WZ HASSID 1955 Assimilation of 14CO2 by a photosynthesizing redalga, Iridophycusflacidum. J Biol Chem 212: 411-425

2. BIRD KT, MD HANISAK, J RYTHER 1981 Chemical quality and production ofagars extracted from Gracilaria tikvahiae grown in different nitrogen enrich-ment conditions. Bot Mar 24: 441-444

3. CRAIGIE JS, J McLACHLAN, RD TOCHER 1968 Some neutral constituents ofthe Rhodophyceae with special reference to the occurrence ofthe floridosides.Can J Bot 46: 605-611

4. CRAIGIE JS, C LEIGH 1978 Carrageenans and agars. In JA Hellebust, JS Craigie,eds, Handbook of Phycological Methods. Physiological and BiochemicalMethods. Cambridge University Press, Cambridge, pp 109-132

5. DUNCAN MJ, PJ HARRISON 1982 Comparison of solvents for extracting chlo-rophylls from marine macrophytes. Bot Mar 25: 445-447

6. EVANS MCW, BB BUCHANAN, DI ARNON 1966 A new ferredoxin-dependentcarbon reduction cycle in a photosynthetic bacterium. Proc Natl Acad SciUSA 55: 928-934

7. KANAZAWA T, MR KIRK, JA BASSHAM 1970 Regulatory effects of ammoniaon carbon metabolism in photosynthesizing Chlorella pyrenoidosa. BiochimBiophys Acta 205: 401-408

8. KAUss H, B SCHOBERT 1971 First demonstration of UDP-gal: sn-glycero-3-phosphoric acid 1 -a-galactosyl-transferase and its possible role in osmo-regulation. FEBS Lett 19: 131-135

9. KREMER BP 1978 Patterns of photosassimilatory products in Pacific Rhodo-phyceae. Can J Bot 56: 1655-1659

10. KREMER BP 1978 Studies on "CO2 assimilation in marine Rhodophyceae.Mar Biol 48: 47-54

11. KREMER BP 1979 Light independent carbon fixation by marine macroalgae. JPhycol 15: 244-247

12. KREMER BP, U KUPPERS 1977 Carboxylating enzymes and pathway of pho-tosynthetic carbon assimilation in different marine algae-evidence for theC4 pathway? Planta 133: 191-196

13. LARSEN PO, KL CORNWELL, SL GEE, JA BASSHAM 1981 Amino acid synthesisin photosynthesizing spinach cells. Plant Physiol 68: 292-299

14. LOWRY OH, NJ ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measure-ment with the Folin phenol reagent. J Biol Chem 193: 265-275

15. McLAUCHLAN J 1973 Growth media-marine. In JR Stein, ed, Handbook ofPhycological Methods. Culture Methods and Growth Measurements. Cam-bridge University Press, Cambridge, pp 25-52

16. MEEUSE BJD 1962 Storage products. In RA Lewin, ed, Physiology and Bio-chemistry of Algae. Academic Press, New York, pp 289-311

17. NEISH AC, PF SHACKLOCK, CH Fox, FJ SIMPSON 1977 The cultivation ofChondruc crispus. Factors affecting growth under greenhouse conditions.Can J Bot 55: 2263-2271

18. PAUL J, KL CORNWELL, JA BASSHAM 1978 Effects of ammonia on carbonmetabolism in photosynthesizing isolated mesophyll cells from Papaversomniferum. Planta 14: 49-54

19. PEDERSON TA, MR KIRK, JA BASSHAM 1966 Light-dark transients in levels ofintermediate compounds during photosynthesis in air-adapted Chlorella.Physiol Plant 19: 219-231

20. Su JC, WZ HASSID 1962 Carbohydrates and nucleotides in the red algaPorphyra perforata. II. Separation and identification of nucleotides. Bio-chemistry 1: 474-480

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