ASSIMILATION OF Cl402 BY A PHOTOSYKTHESIZING RED ALGA, IRIDOPHYCUS FLACCIDUM*

BY 1%. C. BEAN A?r'D W. Z. HASSID

(Prom the Department of Plant Biochemistry, College of Agriculture, University of California, Berkeley, California)

(Received for publication, August 5, 1954)

In land plants n-glucose and n-fructose are the most abundant mono- saccharides. Sucrose, starch, cellulose, and fructosans, which are com- posed of these monosaccharides, are the main complex carbohydrates. In many marine algae these carbohydrates appear to be either completely absent or, when they occur, they do so in comparatively small amounts. The main carbohydrates in these marine algae are compounds such as mannitol, a-n-galactosylglycerol, oc-n-mannosylglycerol (l-4), and polysac- charides containing residues of mannuronic acid, L-fucose, and n-galactose (5).

In view of the difference between the predominant carbohydrates in land plants and in these marine algae, it seemed possible that the path of car- bon in photosynthesis leading to their formation also might be different. To test this possibility, an investigation was undertaken of the intermedi- ate products formed during photosynthesis by the red alga, Iridophycus $accidum.l The major part of the carbohydrate material of this alga is comprised of compounds containing chiefly n-galactose units.

Experiments were carried out by allowing pieces of Iridophycus plants to photosynthesize for periods ranging from 8 seconds to 24 hours in an at- mosphere containing C1402. The resulting radioactive components were separated and identified mainly by paper chromatographic and radioauto- graphic techniques. Analysis of the rates of fixation of Cl4 in the various compounds served to indicate their probable position in the sequence of reactions leading to the formation of carbohydrates.

EXPERIMENTAL

Plant Material-Actively growing thalli of I. jlaccidum containing no fruiting bodies were collected from the tidal rocks at Moss Reach, Cali- fornia. The plants were placed in sea water and kept at 2” for not longer than 3 days before use. A single thallus of about 15 X 10 cm. was cut

* This work was supported in part by a research contract with the United States Atomic Energy Commission.

1 The name Iridophycus jZaccidum for t,his red alga. is now preferred to that of Irideae laminarioides (28).

411

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412 PHOTOSYNTHESIS BY I. FLACCIDUM

into sections, having an area from 6 to 24 sq. cm. and weighing from 0.25 to 1.0 gm., quickly rinsed in distilled water, blotted between filter papers, and placed in the photosynthetic chamber. One thallus section was used for each experiment.

By,,, , , ,mFf?,;;,, , ,I Ll ,

FIG. 1. Photosynthesis apparatus. A, photosynthesis chamber; B, removable bot- tom; C, cooling jacket; D, cooling flask; E, by-pass U-tube and CO2 trap; F, reflector spot lamp; G, plant sample; a, gas inlet; b, exit stop-cock to vacuum line; c, wire sup- port; d, openings for circulating water; e, rubber gaskets.

Apparatus and Photosynthetic Procedure-For photosynthetic periods up to 5 minutes the apparatus illustrated in Fig. 1 was used. Since carbon dioxide uptake was more rapid in air than in water, the photosynthetic ex- periments were conducted in a gaseous rather than in an aqueous medium. This procedure seemed justifiable, inasmuch as the alga in its normal en- vironment frequently remains exposed to the sun for long periods at low tides, often becoming severely dehydrated.

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R. C. BEAN AND XV. Z. HASSID 413

The Plexiglass apparatus contained a photosynthetic chamber, A, for placement of the leaf material. The upper part of the chamber had a built in water-cooling jacket, C. The lower part consisted of a removable bottom, B. In addition, a flat flask, D, through which cold water circu- lated, was placed between the light source, F, and the photosynthetic chamber to act as an auxiliary heat absorber. The light source, consisting of a 150 watt reflector spot lamp, was situated at a distance of 1 foot above the chamber.

The CY4-labeled carbon dioxide was generated externally and trapped in the by-pass U-tube, E. For each sample, 1.0 mg. of BaCY403, having a specific activity of 45.3 PC. per mg., was used. The U-tube was attached by a standard taper joint, a, to one end of the chamber. A vacuum line was attached to the exit stop-cock, b, at the other end. The algal sample was placed on the bottom surface of the chamber, B (wire supports kept the sample from touching either face), and a little water was spread over the bottom surface to maintain a humid atmosphere during the experi- ment. The bottom face was then tightly clamped against the rubber gas- ket with rubber bands. Air was drawn slowly through the chamber via the by-pass of the U-tube, the light turned on, and the plant allowed to photosynthesize for 10 minutes. A vacuum (about 200 mm. pressure) was momentarily drawn on the chamber by closing Stop-cock 2 of the U-tube. The vacuum line stop-cock, b, was shut completely and the radioactive carbon dioxide was admitted into the chamber by opening the 2-way Stop- cocks 1 and 2 of the U-tube to allow air to sweep through the U-tube into the chamber. After closing the stop-cocks and allowing the plant to photo- synthesize for a given period, a beaker containing boiling ethanol was placed under the chamber and the bottom face removed, letting the thallus drop into the alcohol.

Experiments involving photosynthetic periods longer than 5 minutes were carried out with a modified apparatus and procedure which has been previously described (6).

In order to determine the extent to which carbon dioxide was fixed non- photosynthetically by the plant material, experiments were conducted by the same procedure except that light was excluded from the photosynthetic chamber.

Extraction of Xample-The sample was extracted by boiling with 80 per cent ethanol for 5 minutes and decanting the extract. A small amount of water, about double the original fresh weight of the sample, was added2 to the residue and the moist material heated to rehydrate the tissue. Ap-

2 This operation facilitates the extraction of the products formed during photosyn- thesis. The highly colloidal sulfurylated galactan which constitutes the bulk of the plant tends to occlude smaller molecules.

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414 PHOTOSYNTHESIS BY I. FLACCIDUM

proximately 12 volumes of 95 per cent alcohol were added, the mixture was boiled for 5 minutes, and the extract was decanted and added to the first extract. After repeating this procedure three times, the combined ex- tracts were concentrated on a steam bath to a small volume and dried in vacua, and the residue was weighed. This material was set aside to be used for subsequent paper chromatographic analysis.

The above procedure did not remove all the labeled phosphate esters from the alcohol-insoluble residue. To complete the removal of the phos- phate esters, the following method was adopted: the residue was suspended in water (1 ml. to 0.25 gm. of original wet weight sample), the reaction adjusted with 0.5 N HCI to pH 3, and the mixture heated with stirring for less than 1 minute on a steam bath. This treatment caused complete dis- integration of the tissue and reduced the viscosity of the sulfurylated galactan sufficiently so that it became possible to use aliquots of this sus- pension for paper chromatographic analysis.

Chromatographic Analysis-The chromatographic procedures used in this work were similar to those described by other authors (7, 8). The alcohol extracts and suspensions obtained by heating the residues were chromatographed separately on sheets of 18 X 22 inch Whatman No. 4 fil- ter paper which had been washed previously with oxalic acid. For the analysis of the products from the long photosynthetic experiments (over 5 minutes) unwashed No. 1 Whatman filter papers were used. Four ali- quots of approximately 10 y (l/25 of total sample) of the alcohol extract’s and six similar aliquots (3/50 of total sample) of the residue suspensions were taken for analysis. The amount of radioactivity in each sample was determined by measuring the activity on the paper with a rate meter as described below. The papers were developed in the long dimension for 20 hours with water-saturated, redistilled phenol. After drying, they were developed in the second dimension for 16 hours in a mixture of 52 per cent n-butanol, 13 per cent acetic acid, and 35 per cent water.

Radioautograms of the chromatograms were made by leaving the papers in contact with 14 X 17 inch Eastman medical No-Screen x-ray films. The length of exposure was estimated to be inversely proportional to the amount of activity originally applied to the paper, the standard of ex- posure being 4 days for a paper containing 4000 c.p.m.

Not all of the phosphate esters could be completely resolved by two- dimensional chromatography. Further treatment of the phosphate ester fraction was therefore required in order to determine the complete activity distribution of the individual phosphate esters. For this purpose, the phosphate ester spots were eluted, hydrolyzed with phosphatase (General Biochemicals phosphatase, 1 per cent solution) for 8 to 16 hours, and re- chromatographed and the activities in the phosphate-free organic residues counted. This procedure served to establish the identity of the esters.

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R. C. BEAN AND W. 2. HASSID 415

When a spot obtained by two-dimensional chromatographic separation was suspected to contain more than one compound, the spot was eluted and rechromatographed in a mixture of 52.5 per cent n-butanol, 35 per cent ethanol, and 15.5 per cent water (8). This solvent gave an especially wide separation of the amino acids from the neutral sugars.

To provide labeled material for further identification of the compounds, the remainder of each of the alcohol extracts was streaked in bands on No. 1 Whatman filter paper and separated by band chromatography (6, 8). The chromatograms were developed first in the phenol-water solvent and the bands located by radioautography. The individual bands were cut out and eluted and the eluates reapplied on paper in bands, with butanol- acetic acid-water as a developing solvent. The bands were eluted and the material was used for confirmatory chemical tests.

Counting Methode and Calculations-Two instruments were used for making radioactivity measurements. One was a Tracerlab rate meter (SU-3A) supplied with an unshielded Geiger tube with a screened end window 1 inch in diameter; the other a Berkeley decimal scaler (model 2105) with a lead-shielded, 1 inch end window Geiger tube. The Tracer- lab rate meter can be read with an accuracy of &5 per cent of the full scale deflection in ranges of 200, 2000, and 20,000 c.p.m.; the Berkeley scaler with a greater accuracy.

Counting was done with the two instruments directly on filter paper and with samples on copper disks. To determine the factors for conversion of direct counts on paper to counts on copper disks, samples of known ac- tivity were counted both ways. Thus it was found that 1 PC. corresponded to 66,700 c.p.m. when the material was absorbed on Whatman No. 1 filter paper, and to 200,000 c.p.m. when counted on a copper disk. This relation was found to apply to both instruments.

The total activities of the alcohol extracts were determined by applying 0.01 ml. aliquots on Whatman No. 1 filter paper sheets and counting with the Tracerlab instrument. After chromatographing the extracts, the indi- vidual spots were located by reference to the radioautogram, cut out, and counted with the Berkeley scaler. In cases in which the radioactive spot covered an area larger than the end window of the Geiger tube, the spot was cut into two or more pieces, each small enough to be covered by the tube, and the total activity of t,he spot obtained by summing the activities of the sections. When mixtures of known amounts of radioactivity were subjected to the methods of chromatographic separation and radioassay used in this work, the accuracy of the recoveries was found to he within f5 per cent.

Activity in the alcohol-insoluble, water-soluble fraction n-as determined by direct,ly counting weighed samples on copper disks and by applying aqueous aliquots of these samples to filter paper and counting as in the case

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416 PHOTOSYNTHESIS BY I. FLACCIDUM

of alcohol extracts. The ratio of counts on paper to those on copper disks was found to be 1:3, which is the same as that for the standards. The ac- tivity in the insoluble residue was determined by counting weighed samples on copper disks, applying the same 1:3 conversion factor as for the water- soluble fraction.

ldentijication of Labeled Products-Tentative identification of the labeled products was made from their RF values in various solvents. Further evi- dence regarding their identity was obtained from observation of the color reactions with specific spray reagents. When the concentration of the active material was too low to give an adequate color reaction, the com- pound was further identified by chromatographing the active sample after mixing it with an authentic inactive sample and determining whether the outline of the radioautogram spot coincided with that of the spot obtained upon spraying the paper.

For detection of amino acids, a 0.1 per cent alcoholic solution of nin- hydrin was used as a spray. An approximately 1 per cent solution of p- anisidine hydrochloride in butanol (9) served for locating reducing and non-reducing sugars that are easily hydrolyzed to reducing monosaccha- rides. For detection of sugar alcohols and non-reducing sugars, a 1 per cent solution of KMn04 in 2 per cent sodium carbonate was used. A neutral 0.04 per cent alcoholic solution of brom cresol green was employed to indicate organic acids (10). The reagent, ammonium molybdate in perchloric acid, used by Hanes and Isherwood (ll), served as a spray for visual detection of the phosphorylated compounds.

Some of the radioactive photosynthetic products were identified by their specific chemical or enzymatic reactions. Thus, radioactive floridoside (or-n-galactopyranosyl-2-glycerol) was hydrolyzed with an invertase prep- aration containing melibiase (or-galactosidase) (12). When the hydrolysis products were mixed with authentic samples of inactive galactose and glycerol, chromatographed, and sprayed, the radioactive spots coincided with the colored spots.

The glucose resulting from hydrolysis of the phosphate esters was iden- tified by the following criteria: Treatment with phenylhydrazine produced a crystalline phenylosazone identical with that of glucosasone (13). The product was fermentable with Torula monosa which specifically attacks glucose, fructose, or mannose (14). Oxidation with bromine (15) resulted in an acid that yielded a chromatographic pattern identical with that of a known sample of gluconic acid. When the product was epimerized with calcium hydroxide, fructose and mannose were formed (16) and were identified chromat’ographically.

The authenticity of the galactose derived from the hydrolyzed phos- ohates was confirmed by the crystalline form of its phenylosazone (13), by

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R. C. BEAN AND W. Z. HASSID 417

enzymatic oxidation with a specific enzyme (17), and by oxidation with bromine (15).

The fructose component derived from the phosphate ester was identified by its resistance to bromine oxidation, by its phenylosazone, and by its epimerization to glucose and mannose when treated with calcium hydroxide.

The identity of sedoheptulose obtained from the hydrolysis of the phos- phate ester was confirmed by the formation of its anhydro form when heated with dilute acid (18, 19) and by the crystalline characteristics of its phenylosazone.

Gluconic acid was identified as follows: The radioactive sample was mixed with inactive gluconate and the mixture oxidized with hydrogen peroxide and ferric acetate (20). The resulting radioact,ioe product and the arabinose formed from the gluconate gave a single spot when chromat- ographed together.

The glycerol was identified by its chromatographic characteristics, its relative inertness to oxidation and epimerization reactions, and by it,s tend- ency to volatilize. Its chromatographic behavior was different from that of trioses or of amino acids. When left in contact with paper or other ob- jects, the radioactivity from the glycerol spot transferred rapidly from the spot to the object. The glycerol could be differentiated from the volatile glycolic and lactic acids by difference in RF value.

The tentative identification of uridine diphosphate galactose was based upon the following evidence. When an active spot was eluted from the chromatogram of a 16 hour photosynthetic experiment, the eluate hydro- lyzed with phosphatase, and the products chromatographed, two spots were obtained, one corresponding to galactose and one to an unknown compound. The unknown compound was eluted, mixed with inactive uri- dine, and rechromatographed. The radioactive spot and the uridine were found to coincide. A chromatogram obtained from a photosynthetic ex- periment of short duration produced a band with an absorption peak at 260 rnp which is characteristic of nucleotides. Elution of this band and hydrolysis of the eluate with phosphatase yielded labeled galactose and glucose. No Cl4 activity could be found in the uridine moiety after short periods of photosynthesis, apparently because of the slow accumulation of activity in this compound. Other bands known to correspond to the monophosphates of glucose were similarly examined but failed to exhibit this absorption peak in the ultraviolet range.

A compound tentatively identified by chromatographic analysis as uri- dine diphosphate glucose (UDPGlu) was also present among the photo- synthetic products. However, this compound could not be as completely separated from others as was the galactose derivative. Upon chromato- graphic separation of the products of a long photosynthetic experiment,

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418 PHOTOSYNTHESIS BY I. FLACCIDUM

one spot was obtained that yielded on hydrolysis glucose, fructose, and sedoheptulose or mannose, and a compound that had the same RF values in the two solvents as uridine.

The oc-n-galactosylglycerol phosphate could not be separated from the other phosphate esters, but the products of hydrolysis with phosphatase consistently showed the presence of labeled floridoside.

FIG. 2. Radioautogram showing the alcohol-soluble CP4-labeled compounds formed in I. JEaccidum after 10 hours of photosynthesis in C1402. The chromatogram was first developed in phenol and then in butanol-acetic acid-water (BAW) mixture.

Results

Long Period Photosynthesis (5 Minutes to 24 Hours)-The photosyn- thetic experiments of longer duration were intended to label strongly all the metabolic products in the alga in order to facilitate their identification and to determine their relative pool sizes. The radioautogram in Fig. 2, prepared from an extract obtained after 10 hours of photosynthesis, shows the labeled compounds formed. Table I gives the percentage distribution of Cl4 activity in the various algal fractions after 2 and 10 hours of photo- synthesis, and after 2 hours in the dark. Table II shows the distribution of activity after the same periods in the various alcohol-soluble compo- nents. The data in Table II demonstrate that the greatest amount of Cl4 accumulated in the ar-n-galactosylglycerol.

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R. C. BEAN .4ND W. 2. HliSSID 419

Although free glucose could not be demonstrated by chemical analysis in the red alga, paper chromatographic methods revealed the presence of

TABLE I

Distribution of Cl4 in Various Fractions of I. flaccidurn after Photosynthesis in CQ402 and in Dark

1 gm. fresh weight of Iridophycus samples was used. The 2 hour samples were exposed to 0.45 mc. and the 10 hour samples to 1.81 mc. of C402. The BaCr403 con- tained 45.3 PC. per mg.

Photosynthesis

10 hrs. 2 hrs.

Dark fixation

2 hrs. Fraction

Total activity Per cent

PC.

331 3.2 6.0

PC PC.

3.9 0.3 0.1

4.3

97.3 1260 78.0 0.9 44 2.7 1.8 316 19.5

91 7 2

Alcohol-soluble fraction ................ Water-soluble polysaccharide. ......... Insoluble residue. .....................

Total fixation. . . . . . . . 340 1620

TABLE II

Distribution of Cl4 in Alcohol-Soluble Components of I. Jlaccidum after Long Periods of Photosynth&s in Cl402 - -

I Dark fixation Photosynthesis -

2 hrs. 10 hi-s. 2 hrs. Compound

Total uztivity

PC.

14 780

1.2

1.1 61.8

0.1

31.8 2.5 40.5 3.2

6.6 0.5 29.0 2.3 76 6.0 48 3.8

Total xtivity er cm

PC.

20.2 216

1.0 0.6 0.4

29.4

6.1 65.2

0.3 0.2 0.1 8.9

36.4 11.0 15.0 4.5

5.2 1.6

Total rctivity ‘er cent

P’G.

0.03 0.78 0.30 0.69

0.05 0.02

0.03

0.8 20.1

7.7 17.7

1.3 0.5

0.8

Phosphate esters. ..... a-n-Galactosylglycerol. Aspartic acid. ......... Glutamic “ ......... Gluconic “ ......... Organic acids .......... Alanine. ................ Glycine + serine ...... Glucose ............... Unidentified compound

.

. . . .

. . .

labeled free glucose in the extracts. It is possible that this glucose is an artifact produced by hydrolysis of phosphorylated glucose; however, con- tinual accumulation of activity in gluconic acid, a compound not normally

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420 PHOTOSYNTHESIS BY I. FLACCIDUM

found in higher plants, points to the presence of a low concentration of free glucose in the plant which may escape detection by chemical analysis. This gluconate is probably derived from glucose by the action of an enzyme shown to be present in the plant which oxidizes free glucose to gluconic acid (17).

The rate of accumulation of activity in the alcohol-insoluble fraction is comparatively slow. Activity in the sulfurylated galactan, which com- prises the major bulk of the plant (5), could be detected only after about an hour of photosynthesis. This delay in detection of activity may partly be explained by the great dilution with the non-radioactive polysaccharide.

The alcohol-insoluble, water-soluble fraction consisting chiefly of galac- tan was hydrolyzed with acid and the products were chromatographed. The radioautogram obtained from this chromatogram showed the presence of a relatively large amount of activity in glucose and only a small amount in the galactose. However, when the paper was sprayed with p-anisidine hydrochloride, the color reaction showed galactose to be the chief product, while the presence of glucose could not be detected. The ratio of the ac- tivity of glucose to that of galactose, calculated on the basis of a given volume of the hydrolysate, ranged from 3 : 1 to 20: 1 in various photosyn- thetic samples. Thus it appears that the alcohol-insoluble fraction con- tained a very small amount of a glucose polymer, which had a compara- tively high specific activity.

The experiments conducted in the dark showed that a small amount of activity was also fixed in most of the compounds (Tables I and II). The highest percentage of the total activity in the dark was found in the galac- tosylglycerol, and, as might be expected, in the amino acids associated with the respiratory cycles. Comparison of the activities fixed in the compounds after 2 hours of photosynthesis with those fixed within the same period in the dark shows that all the glutamate and most of the as- partate were probably labeled as the result of dark fixation and not by photosynthesis. On the other hand, the other amino acids appear to be derived directly from photosynthetic products.

Short Period Photosynthesis (8 Seconds to 5 Minutes)-The purpose of the shorter period experiments was to identify the products formed in the early stages of photosynthesis and to determine the path leading to carbohydrate synthesis. The data in Table III summarize the results ob- tained from a photosynthetic series. In Fig. 3 these data are plotted on a logarithmic scale in order to accentuate the differences of the low activity compounds and to indicate the probable order of appearance of activity in the intermediates.

In the shortest photosynthetic exposure of 8 seconds, 69 per cent of the fixed activity resided in the phosphoglyceric acid. Subsequent exposures

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R. C. BEAN AND W. 2. HASSID 421

show a continual decrease in the activity of the phosphoglycerate relative to the other intermediates (Table III). This fact is in accord with the data obtained by Calvin (21), showing that the first stable intermediate in photosynthesis is phosphoglyceric acid.

Considerable activity was also found after 8 seconds in the sedoheptu- lose phosphate, glucose phosphate, and fructose phosphate. Although from consideration of the rates of accumulation of activity in these com- pounds it was not possible to determine precisely the order of their appear- ance, it seemed that sedoheptulose phosphate and fructose phosphate pre- ceded the glucose phosphate in the synthetic sequence. This conclusion

TABLE III

Distribution of Cl4 in Components of I. Jlaccidum in Short Periods of

Photosynthesis with Cl402

Total activities, c.p.m. X 10-Z Per cent distribution

Compound Photosynthetic period

8 sec. 15 sec. “-160 sec.1 ,‘;;, 8 sec./S sec.1 ,“,“,, ,“,” ,‘e”c9

..~~ -.

Phosphoglycerate.. 52 137 180 192 202 69 162 Sedoheptulose phosphate. 11.4 35 70.3,157 452 15 ‘16

,38.4 22 11.3 115 18.125.0

Fructose monophosphate. 2.5 5.7 18.7 46 98 3.3 2.16 4.0 5.3 5.4 Glucose “ 6.1 28 100 259 628 8 13 21.429.834.8 UDPGlu 0.77 4.7 18.1 81 0 0.35 1.0 2.1 4.5 UDPGal 2.4 6.9 22 0 0 0.5 0.8 1.3 a-Glycerol phosphate. . 4.2 14.0 44.1 126 0 1.9 3.0 5.1 7.0 Floridoside.................... 2.4 24.2 112 0 0 0.5 2.8 6.2

~_

Total in original sample 76 220 468 869 1800

is based on the observation that the glucose phosphate concentration in the plant was considerably higher than that of the other sugar phosphates, indicating that its specific activity must have been lower at that time. Ribose phosphate and ribulose phosphate were also labeled, but their ac- tivities were too low to make a kinetic analysis possible.

In the period between 8 and 15 seconds, C14-labeled cr-glycerol phosphate and a glucose derivative, presumably UDPGlu, appeared. The evidence previously present,ed indicaates that this compound is probably identical with the IJDPGlu found by Caputto rf ab. (22) in yeast.

Between 15 and 30 seconds of photosynt.hesis a radioactive phospho- rylated galactose derivative was detected which was tent,atively identified as uridine diphosphate galact’ose (UDPGal).

During the same period, labeled floridoside (galactosylglycerol) also ap-

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422 PHOTOSYI’iTHESIS BY I. FLACCIDUM

peared. However, labeled phosphorylated floridoside, presumably because of its low concentration, could not be found at this time; it could only be

4k

0 8 15 30 60 120

TIME (SECONDS)

FIG. 3. Rate of incorporation of 04 in compounds labeled during short periods of photosynthesis with Cl402. l , phosphoglyceric acid; 0, sedoheptulose phosphate; (>, glucose phosphate; 0, fructose phosphate; A, glycerol phosphate; X, UDPGlu; curve without symbols, UDPGal; crossed curve, floridoside.

TABLE IV

Comparison of 04 Activities in Glycerol and Galactose Moieties of Galactosylglycerol after Various Periods of Photosynthesis

Experiment No. Time Glycerol, Galactose, Galactose c.p.m. x 10-z c.p.m. x 10-z Glycerol

min.

1 4 640 1210 1.9 2 5 530 1130 2.1 4 120 3550 6920 1.95 5 600 1620 3430 2.12

detected after approximately 60 seconds. The accumulation of Cl4 in the nucleotides of galactose and glucose increased with time, the glucose ac-

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R. C. BEAN AND WV. Z. HASSID 423

tivity predominating over that of the galactose by a fairly constant ratio of 3 : 1. The monophosphates also showed a large increase in activity with time. The curve (Fig. 3) for the gain in activity of the free floridoside was especially steep. Other experiments showed that within 5 minutes the gain in floridoside activity accounted for 25 to 50 per cent of the total ac- tivity fixed in the plant.

The galactosylglycerol produced after various periods of photosynthesis was hydrolyzed, the galactose and glycerol separated chromatographically, and their activities determined. The results in Table IV show that after 4 minutes of photosynthesis the ratio of the activity of galactose to glycerol is approximately 2, which is the expected value if it is assumed that all carbon atoms of these compounds are equivalently labeled.

DISCUSSION

Most of the products formed in the early stages of photosynthesis in I. flaccidurn are the same as those observed in Chlorella and in higher plants by Benson et al. and Calvin (19,21). The rates of formation of the various compounds also appear to be similar in the different organisms. As in the higher plants, phosphoglyceric acid is found to be the first stable interme- diate compound in the photosynthetic transformation of CO2 to sugars.

It has been demonstrated that Cl4 activity accumulates in the galactosyl- glycerol to the greatest extent, showing that this compound is the major reserve constituent of this red alga. Attention has therefore been directed toward gaining information regarding the mechanism of its formation. The detection of uridine diphosphate glucose and uridine diphosphate ga- lactose in the first 30 seconds of photosynthesis indicates that probably a mechanism, similar to that shown to exist in yeast (22-24), involving the enzyme galactowaldenase operates in the alga. In this connection, it should be noted that other workers have previously identified uridine di- phosphate glucose in green plants, especially in sugar beets in which galac- tose is found as a raffinose constituent (25).

The kinetic data (Fig. 3) indicate that in the photosynthetic sequence CY4-labeled glucose monophosphate precedes radioactive glucose nucleotide and is then followed by active galactose nucleotide.

The very early appearance of Cl4 label in ol-glycerol phosphate with a relatively high activity indicates that it is probably derived through the reduction of a triose phosphate. However, radioactive triose phosphate could not be demonstrated, possibly because of its very low concentration.

It can be postulated that the galactose nucleotide, presumably UDPGal, condenses with the a-glycerol phosphate, splitting off uridine diphosphate, to form galactosylglycerol phosphate. Free galactosylglycerol is then pro- duced upon hydrolysis of the phosphate group. The polysaccharide sul-

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424 PHOTOSYNTHESIS BY I. FLACCIDUM

furic acid ester of gala&an is probably synthesized through sulfurylation of galactose units and subsequent polymerization.

Analogous to UDPGlu, shown by Leloir et al. (26, 27) to serve as a glu- cose donor in the formation of trehalose phosphate by a yeast preparat,ion and sucrose by a wheat germ preparation, UDPGal may function as the galactose donor in the synthesis of the complex galactose saccharides in the red alga.

Fig. 4 is a schematic representation of possible pathways for the forma- tion of the galactose-containing carbohydrates.

co2 PGA

/-r Triose P

Regenerative cc-D-Galactosylglycerol

f CX-D-Galactosylglycerol-P

FIG. 4. Scheme indicating probable sequence of photosynthetic production of carbohydrates from CY402 in I. jiaccidum. PGA = phosphoglyceric acid; FDP = fructose diphosphate.

SUMMARY

The path of synthesis leading to the production of carbohydrates in the red alga Iridophycus jiaccidum during photosynthesis with Cl402 has been investigated.

As in green plants, phosphoglyceric acid appears to be the first stable intermediate in the transformation of CO2 to organic compounds.

Most of the phosphorylated products formed in the earlier stages of photosynthesis of the red alga appear to be identical with those produced in the green plants. In addition, a-glycerol phosphate and cu-n-galactosyl- 2-glycerol phosphate are formed.

Glycine, serine, alanine, glyceric acid, glycolic acid, malic acid, glutamic acid, and aspartic acid are also present.

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R. C. BEAN AND W. Z. HASSID 425

The data suggest that synthesis of or-D-galactosyl-2-glycerol may occur through condensation of UDPGal with cr-glycerol phosphate, elimination of uridine diphosphate, and subsequent hydrolysis of the galactosylglycerol phosphate, thus forming the free galactoside.

The oc-n-galactosyl-2-glycerol, analogous to sucrose in the higher plants, appears to be the main reserve carbohydrate in the red alga.

BIBLIOGRAPHY

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R. C. Bean and W. Z. HassidIRIDOPHYCUS FLACCIDUM

PHOTOSYNTHESIZING RED ALGA, BY A2O14ASSIMILATION OF C

1955, 212:411-425.J. Biol. Chem. 

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