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    • LIGHT AND CHLAMYDOMONAS METABOLISM 213 1vironments where the temperature remains con REFERENCEStinuously below 20 C (4,8). Our data would sugges 1. BOYLEN, W., AND BROCK, D. 1973. Effects of thermal C. T.that no comparable adaptation to low temperatu additions from the Yellowstone geyser basins on the benthic algae of the Firehole River. Ecology 541282-91.occurs in temperate lakes where temperaturesto more than 30 C in the summer. Eucaryotic gaedo adapt to lower temperatures i n freshwater habitats d 2. BROCK, D., & BROCK,M. L. 1967. Methodology for T. measurement of chlorophyll, primary productivity, photo- phosphorylation, and macromolecules in benthic algae.where seasonal temperature flux has a summer high Limnol. Oceanogr. 12:600-5.of less than 20 C, as in cold mountain streams (I). 3. BUNT,J. S. 1963. Diatoms of antarctic sea ice as agents It would appear that water temperature is one of of primary production. Nature 199:1255-7.the most important environmental factors in the de- 4. - 1968. Some characteristics of microalgae isolated from Antarctic sea ice. Antarctic Res. Ser. 11:l-14.velopment of large diatom populations during a 5. MACAN, T. 1970. Biological Studies of the English T.seasonal period in which most other algal and macro- Lnkes. American Elsevier, N.Y., 72-83.phytic plants are either unable to grow or are dor- 6. MCCRACKEN, D., G U ~ A F S OT. , D., & ADAMS,M. S. M. Nmant. Despite its inability to grow optimally at the 1972. Productivity of Oedogoniunt in Lake Wingra. East- ern Deciduous Forest Biome Memo R e p . # 72-109 (Uni-temperature of its habitat, the diatom Cymbella versity of Wisconsin, Madison).develops high epiphytic populations. T h e environ- H., H. 7. MEGURO, ITO, K., & FUKUSHIMA, 1967. Ice floramental factors influencing this extensive algal de- (bottom type): A mechanism of primary production in polarvelopment and its role in detritus formation are seas and the growth of diatoms in sea ice. Arctic 20114-33.not known. 8. SOROKIN, I., & KONOVALOVA, Yu. I. W. 1973. Production and decomposition of organic matter in a bay of the Japan Sea during the winter diatom bloom. Limnol. Oceanogr. ACKNOWLEDGMENT 18962-7. This study was supported by a contract from the Atomic 9. WRIGHT, T. 1964. Dynamics of a phytoplankton com- R.Energy Commission (CCO-2161-10). munity in an ice-covered lake. Limnol. Oceanogr. 9:163-78.J . Phycol. 10,213-220 (1974) T H E EFFECT OF L I G H T QUALITY ON T H E CARBON METABOLISM AND EXTRACELLULAR RELEASE OF CHLAMYDOMONAS REINHARDTII DANGEARD T. J . Brown2 and G. H . Geen Department of Biological Sciences, Simon Fraser University, Burnaby 2, B.C., Canada SUMMARY T h e influence of red, blue, green, and white light while the percentage of proteins was highest in blueon growth and photosynthetic rates, carbon metab- light. Light quality also influenced the compositionolism, and rates of release of extracellular compounds of the ethanol-soluble fraction. The percentage ofin the freshwater alga Clilamydomonas reinhardtii organic acids was highest in cells grown in greenDangeard was examined. Relative growth constants and white light, while amino acids were highest inwere 0.28, 0.?2, 0.40, and 0.41 in green, white, blue, blue and green cultures. T h e percentage of ethanol-and red light, respectively. Photosynthetic rates were soluble sugars was greatest in cultures grown inhigher in white, blue, or red than in green light of blue and red light.the same intensity. T h e percentage release of dissolved organic car- More than 60% of the f C 0 , assimilated by cells bon into the medium was highest in white light andgrown under blue or green light was incorporated lowest in blue or red light. T h e nature of the extra-into the ethanol-insoluble fraction, compared with cellular products varied according t o the quality ofabout 50% i n cells grozun under white or red light. light under which the cells were cultured, but hadT h e percentage of sugars in this fraction was sig- n o consistent relation t o the nature or concentrationnificantly higher in cells grown under green or red of components in the ethanol-soluble fraction.light than in cells cultured in white OT blue light, INTRODUCTION Received August 23, 1973; revised March 28, 1974. Present address: Biological Station, Fisheries Research T h e chemical composition o planktonic algae in fBoard of Canada, Nanaimo, B.C., Canada. nature and cultures has received some attention (Id,
    • 214 T. J. BROWN AND G. H. GEEN green light than under white light which was inde- / *. i pendent of light intensity has also been noted (28). i ! These studies suggest that the depth-dependent shift of the spectral composition of underwater lightI" probably alters the rate and/or direction of many 5- metabolic reactions in planktonic algae. This5YI might affect the nature and quantity of extracellularb products which could, as an example, influence the- 4- growth of heterotrophic organisms which utilize dis--cv1 solved organic compounds, or influence the forma-6I- 3- tion of organic aggregates.E The primary purpose of this work was to examine+$J *- the nature of the photosynthetic and extracellular products of the freshwater alga Chlamydomonas 14 reinhardtii grown under light of the same intensity but different spectral composition, and from these data determine whether a predictable relationship exists between the nature of the photosynthate and 400 500 600 700 the extracellular products. WAVELENGTH (Nm) MATERIALS AND METHODS FIG. 1. The spectral composition of each of the light sys-tems used for culturing C . reinhardtii. An axenic culture of Chlamydomonas reinhardtii (wild type, mating strain 89, 90) was grown in previously sterilized Beijerincks medium. Stock cultures were maintained withZ8,26-28). Variations in composition are induced by continuous stirring at 18 2 0.5 C in cotton-stoppered 2800-mla number of extrinsic factors such as temperature Fernback flasks. Light was provided by a bank of cool-white fluorescent lamps producing 2.4 X 108 ergs/(cm")sec) at the(6),pH (I?, and the inorganic nutrients in the level of the bottom of the flasks. Experimental cultures weremedium (5,7). The age of the cultured cells, stage established with 10 cells/ml and placed in incubators operat-of life cycle, and previous culture history also have ing on a 16:E hr light-dark regime. The experimental culturesan effect upon the nature of the photosynthetic were illuminated from above with General Electric white,products ( 1 3 J 9 ) . Cellular composition may also blue, or green fluorescent lamps or Sylvania red fluorescent lamps. The band width of the blue, green, or red lightsdepend on the wavelength of the incident light. reaching the cultures was more narrowly defined by placingBlue light stimulated the incorporation of 14C into plexiglass filters between the lamps and cultures. T h e maxi-amino acids, especially alanine, in barley, whereas mum energy output from the blue light system at flask levelred light stimulated 14C incorporation into sugars was 2.4 X 103 ergs/(cm)(sec) and was the energy used for culturing cells under each light regime. T h e spectral distribu-(24). Blue and green light stimulated an increase in tion of each lamp-filter combination is shown in Fig. 1.proteins relative to white light in 2 species of marine Growth rates were estimated from cell count data obtainedphytoplankton (26). An increase in some amino and with a Coulter Counter. Photosynthetic rates and distribu-organic acids in cells grown under blue light inde- tion of 14C were determined when the experimental culturespendent of light intensity, photosynthetic rate, or reached the exponential phase of growth. Four 160-ml sub- cultures were taken from the experimental cultures, inoculatedcell density was also noted. Several other workers with 400 pCi of H"CO,-, and incubated under the appropriatehave reported a blue light enhancement of produc- experimental light conditions for 30 min prior to the deter-tion of proteins and/or their precursors (4,8,9,Z2). mination of the total 14C uptake and its distribution withinLight quality may also influence the rate of photo- the cells.synthesis. Blue light produced a higher rate of Separation and identification of the Photosynthetic products. After incubation with Hl4COS-the algae were filtered ontophotosynthesis in 3 species of algae than other light Millipore filters (HA 0.45 p) using a vacuum pressure of 25qualities of the same intensity (9). cm Hg and placed for 1 h r in boiling ethanol in a reflux Since light quality and perhaps quantity may in- apparatus. This separated the photosynthetic products intofluence the metabolism of algae, it seems probable the hot-ethanol soluble (HES) and insoluble fractions. Thethat either might affect the quality and quantity of HES fraction was adjusted to pH 7.2 (to reduce the expected loss of volatile organic acids under acidic conditions) andthe extracellular organic compounds released by dried under vacuum at 35 C. The dried extract was redissolvedunicellular algae. The release of glycolate by in 5 ml distilled water and an aliquot was placed in TEGChlorella pyrenoidosa increased at high light intensi- [300 ml ethylene glycol monomethyl ether, 500 ml TOL (42ties and low CO, concentration (2130). Glycolate ml spectrofluor/liter toluene)], and the activity measured with a liquid scintillation counter. The HES fraction wasrelease by C. pyrenoidosa and C . uulgaris exposed to then run on ion exchange resins (Rexyn 101 [H+] and 201high intensity white or red light, but not by cells [OH-]) to separate the free amino acids, organic acids, andgrown in blue light, has been reported (3). A smaller sugars. Each fraction was washed and evaporated 3 times with distilled water to remove the formic acid and ammoniumpercentage extracellular release by cultures and hydroxide used to elute the organic and amino acids fromnatural populations of algae grown under blue or the ion exchange resins.
    • LIGHT AND CHLAMYDOMONAS METABOLISM 215TABLE Total 4C/lOU cells fixed by C . reinhardtii under 1. white, blue, green, or red light and the 4C distribution between the ethanol-soluble and insoluble fractions expressed as a percent of total C fixed 4 f standard error. Total 14C Light fixed X 1o-B Ethanol Ethanol -quality DPM/lOs cells soluble, % insoluble.% Reulicates E White 59.9 f 13.6 51.8 f 0.5 48.2 f0.5 9 Y 2Blue G6.4 f 14.7 31.0 2 3.4 69.0& 3.4 4 0 YGreen 14.92 5.1 38.5 f 1.3 61.5 f 1.3 7Red * 69.1 12.2 47.1 -C 1.6 52.9-C 1.6 8 The amino acids were cochromatogrammed with standards in two dimensions using phenol:water:ammonium hydroixde (300:75:1) and butano1:acetone:diethylamine:water (120120:2450). The organic acids were run in ethano1:water:ammonium hydroxide (280:104:16) and ethyl acetate:acetic acid:water: sodium acetate (200:122:100:480). The sugars were run inpheno1:water:ammonium hydroxide (300:40:1.2) and isopropa- I 1 I 1 I 1 , 1 , I 1 I , , ,no1:ethyl acetate:water (315:45:90). The sugar chromatograms 0 4 8 12 16 20 24 28were dried and rerun in the second direction to achieve better DAYSseparation. Chromatograms were exposed to Ilford Ilfex X-ray film for FIG. 2. Growth of C . reinhardtii in red, green, blue, and4-5 weeks to locate the radioactive compounds. All radioactive white light at an intensity of 2.4 X 109 ergs/(cm?(sec).spots were excised and their activity determined. They wereidentified by their relative positions using spray reagents andknown compounds. The efficiency of the spot-counting tech- average diameter for cells grown in white, blue,nique was determined by placing a known quantity of "C- green, and red light was 5.1, 5.6, 7.2, and 6.0 p,labeled leucine, serine, glycine, and aspartic acids on chromato- respectively. The average volume of cells growngrams, running in the appropriate solvents, spraying, and under green light (198 p3) is approximately doublecounting. The ethanol-insoluble fraction was hydrolyzed in a Soxhlet that for cells grown in white, blue, or red lightapparatus. The proteins and polysaccharides were refluxed (70.4, 91.2, and 114 p3, respectively).for 24 hr in G HCI and 1N H,S04, respectively. The HCl N Cells grown under white, blue, or red light had awas removed by 3 evaporations under vacuum. The H,SO, higher photosynthetic rate in log phase cultures thanwas removed by precipitation with BaCO,. The hydrolyzates cells grown under green light of the same intensity.were concentrated under vacuum, redissolved in water, runthrough ion exchange resins, and separated paper chromato- Cells grown under green light had a rate o photo- fgraphically as previously described. synthesis about one-quarter that of cells grown in The medium in which the cells were grown was examined white light (Table 1). T h e lag phase in culturesfor extracellular products after the 30-min period of photo- grown under green or red light was longer than forsynthesis. The filtrate was brought to pH 2 and bubbled withair for 1 hr to remove the unused H14C03-. Residual bicar- cells grown under white or blue light (Fig. 2).bonate activity was further reduced during a subsequent IJC distribution in the photosynthate. Cells grownstep in which the filtrate was concentrated to 30 ml at pH in blue and green light fixed a greater percentage of7.2 under vacuum. Only 0.001% of the HWO; remained after the activity in the ethanol-insoluble fraction thanthis evaporation. A 50 aliquot was placed in TEG and did cells grown in white or red light (Table 1).counted to determine total activity in the extracellular frac-tion. The filtrate was then run through Rexyn 101 (H+) Conversely, the percentage activity in the ethanol-and 201 (OH-) to separate the amino acids, organic acids, and soluble fraction was higher in cells grown undersugars prior to chromatographic separation of the components white and red light.in each fraction. The distribution of 14C in the ethanol-insoluble fraction varied with light quality. T h e percent ac- RESULTS tivity in the protein amino acids in cells grown Growth and photosynthetic rates. The exponential under red (59.3%) and green light (68.5%) was sig-growth rate of Chlamydomonas was higher in red nificantly lower ( p < 0.01) than under white or blueand blue light than in green or white light of the light regimes (82.3 and 93.5%, respectively). Con-same intensity. Relative growth constants (K) and versely, the percent in the sugar hydrolysis fractionthe mean generation times (T) were calculated from was higher in cells grown under green or red lightthe linear portion of each growth curve on a log (31.5 and 40.7%, respectively) than under white orplot (Fig. 2). blue light (17.7 and 6.5%, respectively). The growth constants for cultures in green, white, Distribution of 14C observed in the various com-blue, and red light were 0.28, 0.32, 0.40, and 0.41, pounds of the ethanol-soluble fraction after 30 minrespectively. Corresponding mean generation times of photosynthesis is shown in Table 2. The percentwere 59.4, 52.0, 41.6, and 40.6 hr, respectively. The of 14C activity in the ethanol-soluble fraction did
    • 216 T. J. BROWN AND G. H. GEEN TABLE Distribution of I * C among compounds in the 2. 4. TABLE Distribution of C in the sugar hydrolysis fraction 4 ethanol-soluble fraction of C . reinhardtii expressed of C. reinhardtii exfiressed as a percent of total activity in as a percent of total activity recovered in the the sugar hydrolysis fraction standard error. ethanol-soluble fraction 2 standard error, Light quality White Blue Green Red wl t? Blue Green Red Glucose 49.2f12.7 26.9*5.4 83.1f2.3 66.4f4.1Malic 6.4 2 0.5 * 2 0 0.2 . 3.0 -C 0.4 5.4 2 0.5 Fructose - - 5.1 f0.1 2.6 f0.5Glycolic 3.0 f0.4 2.7 f 1.0 3.3 -+ 0.9 1.8 k 0.1 Sugar origin 8.9f 3.8 4.3 f 0 . 9 0.2*0.2 0.9 *0.6Pyruvic 6.9 f 1.5 * 0.8 0.4 2.5 -C 2.0 2 3 2 0.3 . Sugar unknown 41.6 2 9.7 68.8 * 4.6 * 11.7 2.1 3.4 f3.9Oxalic 3.9 2 0.7 * 2.1 0.6 * 2.9 0.9 1.2 k 0.33-PGA 6.1 3: 1.1 4.7 * 0.9 5.6 -+ 1.2 3.6f0.6Fumaric 1.1 2 0.3 - - - but in general showed no consistent trends whichSuccinic 0.4 f 0.3 1.7 0.3 2.9 * 0.5 1.6 k 0.2a-Ketoglutaric 0.3 f0.2 0.4 2 0.2 * 0.4 0.2 could be ascribed to light quality.Citric - - 0.4 * 0.1 0.2 f0.2 Hydrolysis of the ethanol-insoluble fraction yieldedOA origin 2.4 k 0.7 * 5.7 2.5 1 0-t 0 3 . . 0.8 4 0.2 the compounds indicated in Tables 3 and 4. SomeOA unknown 5.8 f 1.O 2.6 f0.4 * 7.1 0.9 9.3 -c 1.1 differences in the protein amino acids found in cellsO A total 36.0 20.6 29.1 26.6 grown under different light qualities were observedAspartic 10.1 f 1.3 9.8 2 1.1 8.0 * 2.1 * 0.2 0.2 (Table 3). Hydrolysis of the insoluble sugar fractionGlu tamic 10.4f 1.8 11.8" 1.5 17.4 -C 2.0 0.7 f0.58-Alanine 0.8-t- 0.8 2.7 -f. 1.6 2.6 L 0.3 * 2.7 0.3 yielded glucose and fructose in varying amountsSerine - - 1.6 -C 0.2 * 1.0 0.2 (Table 4). The percent glucose present in cellsProline 0.2 f0.2 0 6-C 0.3 . 1.9 4 0.3 grown in green and red light was significantly higherLeucine 8.3 f0.8 1.7 0.4 2.2 f 0.4 than in cells grown in white and blue light. CellsValine 1.2 f 1.2 0.8 2 0.2 * 1.3 0.2 grown in green and red light had a significantly - - lower percent activity ( p < 0.01) in the unknownArginine 0.9 f 0.4Glycine - 0.4 f 0.4 -AA origin 5.0 k 0.9 5.7 -C 1.4 1.6 2 0.3 1.4 f0 9 fraction than cells grown in white and blue light.AA unknown 1.0 f 1.0 4.9 -C 0.7 8.3 f 1.8 This unidentified sugar appeared in the same areaAA total 27 5 34.5 39.6 20.6 of the chromatograms o the sugars from cells grown fGlucose 1.3 f0.8 under all 4 light conditions.Fructose 5.9 -c 1.9 - - Extracellular organic products. The nature of theGlycerol - - 0.9 -t. 0.6 compounds released by C. reinhardtii under theSugar origin 3.7 f0 6 . 9.8 f0.3 * 5.1 0.9 8.1 f 1.5 various light regimes is shown in Table 5. CellsSugar unknown 3 . 2 8 28 . * 36.1 2 6.9 26.8 -t 2.5 42.5 2 3.9 grown under white light exuded a large percent ac-Sugar total 36.5 45.8 3. 19 52.8 -Below detectable limit. ~ tivity in each fraction (ie, free amino acids, organic acids, and sugars). Cells grown under blue and red light released mainly sugars and organic acids, re-not change appreciably in cultures incubated for spectively. The composition of the material releasedvarying periods between 15 and 180 min. The by cells grown under green light was not determinedhighest percent 1% activity in organic acids was re- because of the low activity released.covered in cells grown under white light, whereascells grown under blue or green light had the highest DISCUSSIONpercent activity in the amino acid fraction. Cellsgrown in red light had the highest percent activity This study has shown that light quality influencesin the sugar fraction. The percent activity in indi- photosynthetic rate, growth, and the nature of thevidual compounds within the HES fraction varied, TABLE Distribution of +C in the extracellular products of 5.TABLE Distribution of JC in the protein amino acids of 3. I=. reinhardtii expressed as the percent of total activity C . reinhardtii expressed as a percent of the total activity recavered in the extracellular fraction * standard error. in the protein amino acid fraction f standard error. White Blue Red White Blue Green Red G1ycolate 2 7 -+ 0.8 . - 5.1 f 1.7A spart ic 12.8 f2.5 13.4 -C 2.1 14.8 f 1.7 14.2 2 0.5 3-PGA 2.8 f 1.1 - * 19.9 3.3Glutamic 14.7 f2.4 9 3 f 1.1 . * 12.9 0.9 10.8 f 0.6 Malic - - 6.6 f0.7Glycine 5.1 k 1.1 6.4 f0.5 3.8 f 0.8 2 9f0.4 . OA origin 5.0 * 1.0 - 11.722.2Serine * 1.1 0.6 - 2.9 f0.6 1,9 f02 . OA unknown 3.8 f 1.3 - 54.9 f9.7p- Alanine * 15.6 5.0 .- 17.8 2.5 * 12.2 0.9 12.8 +- 0.6 AA origin 13.8 f2.9 - -Leucine * 16.8 3.1 * 29.4 4.0 22.6 f0.6 21.6 f0.6 AA unknown 19.5 f8.8 - -Proline 14.3 2.8 6.8 k 2.5 10.0 f 1.4 11.4 C 0.8 Sorbitol * 7.6 3.9 14.0 4 1.3 -Valine 14.7 f2.1 12.4 -C 2.1 13.3 4 0 9 13.1 -t 0.5 Sugar origin 31.6 f5.0 81.5 f5.7 -A rginine 3.9 f 1.4 1.3 -C 0.8 - 0.8 f0.5 Sugar unknown 15.7 f 4.7 4.5 f4.5 -Phenyl alanine - 3.9f 1.3 0.6 f0.6 Extracellular releaseAmino acid as a percent of unknown - 3.2 + 3.2 3.5 * 2.3 9.5 -t 0.8 total incorporation 35.6 6.6 4 .O -Below detectable limit. -Below detectable limit.
    • LIGHT AND CHLAMYDOMONAS METABOLISM 217photosynthetic and extracellular products of Chlamy- additional enzymes. I t has been shown that cellsdomonas reinhardtii. The data presented in this grown at low temperatures were larger and con-study and the studies of others (26-28), using cultures tained more protein (which was presumed to beand natural populations of marine planktonic algae, largely enzymes) than cells grown at higher temper-suggest that the depth-dependent shift in light atures (11). We have shown that average cell sizequality which occurs in oceans and lakes will be was largest in green light cultures, which suggests aassociated with changes in algal metabolism, nature physiological adaptation that may account for theand rate of release of extracellular products, and long lag period observed in green light cultures.rates of growth and photosynthesis. Thus the observed differences in growth and photo- Our cultures all exhibited low growth rates and synthetic rates of the various cultures could belong generation times relative to enriched cultures related to differences in enzyme or pigment concen-(doubling every 1-2 days). These rates and genera- trations or in the efficiency with which the varioustion times are not unlike those reported for natural pigments are able to absorb or transfer energy.populations (20). Whatever the basis for the differences we observed, The lag period observed in new cultures varied it seems likely that light quality will influence theaccording to light quality, but bore no obvious re- growth and photosynthetic rates of organisms dis-lationship to growth or photosynthetic rates during tributed at various depths throughout the photicthe period of log growth. Of the various explanations zone of oceans or lakes. The effects of light qualityof the lag phenomenon (see 20 for a discussion), the on growth and photosynthetic rates in C. reinhardtiifollowing seem most likely to account for the dif- that we observed in culture were generally similarferences we observed: to those observed in natural populations of plank- tonic algae in Indian Arm and Saanich Inlet and Absence in a new culture medium of a necessary in cultures of Cyclotella nana and Dun,aliella tertio- growth promoter produced by the phytoplank- lecta (26,28). ton. Differences in the length of the lag period Light quality had a distinct effect on the distribu- under the various light regimes could result tion of 14C in the photosynthetic products of C . from differences in rates of production of an reinhardtii, as it did in marine algae (26-28). T h e essential growth promoter. relatively high percent of 14Cfixed by C. reinhardtii Differential efficiency of cells in absorbing or in the ethanol-insoluble fraction in green and blue utilizing light of different qualities. This light cultures (Table 1) agrees with the results in could be a reflection of differences in pigment the work cited above, in which it was shown that cul- or enzyme concentrations or some combination tures grown in white light incorporated the greatest of the two. proportion of 14C into the ethanol-soluble fraction, Either or both of these factors may explain the whereas cultures grown in blue or green light in-observed differences in lag period. When we trans- corporated most l4C into ethanol-insoluble com-ferred a culture growing exponentially under white pounds. This relationship was not appreciablylight to green light, a lag of several days was ob- affected by light intensity. These studies suggest aserved before growth resumed. Since the culture higher percentage protein and polysaccharide pro-had been growing exponentially prior to transfer, duction in deep, relatively clear oceanic or lacustrineany necessary growth promoter was presumably environments, given the usual predominance o bluefpresent. This suggests that a period of relatively or green light at these depths in relatively clearslow cellular adaptation is required before rapid waters. Changes of this nature could be of nu-growth can be expected under the new light regime. tritional significance to herbivores.Such adaptation could require the synthesis o addi- f We observed an increased percentage incorpora-tional pigments or enzymes. We measured pigment tion of 14C into the protein fraction in cells grownconcentrations in log phase cultures grown under under blue and white light, whereas the percentageeach of the light regimes. Concentrations of the of polysaccharides was highest in green and redchlorophylls and total carotenoids were highest in light. A blue light enhancement of protein produc-cells cultured under white light and 25 to 50% tion was observed in Chlorella pyrenoidosa (12).lower in cells cultured under blue, green, or red light. Radiant energy above 500 nm resulted in a shift o fThe significance of these differences is not im- synthesis to polysaccharides. Our results supportedmediately apparent. However, there is a substantial the view that blue light enhances protein synthesisbody of data indicating shifts in pigment concen- since those cultures grown under blue or white light,tration in response to differences in light quality or both of which contained energy in the region ofquantity. For example, the total chlorophyll in C. 450-500 nm, had a higher percentage of proteinreinhardtii grown under blue light increased 20% than did cultures grown under green or red light.during the lag period, although the chlorophyll a / b Green light enhancement of protein synthesis inratio decreased (10). 2 species of marine phytoplankton has been reported Adaptation may also occur through synthesis of (26). However, this could have been attributable to
    • 218 T. J. BROWN AND G . H. GEENa blue light effect, since a small portion of theenergy in the green light system used in those studieswas below 500 nm. It has been shown that the addi-tion of only 4% blue light as incident energy to red -1light has essentially the same effect on the distribu-tion of 14C as blue light alone (9). There were some significant differences in the per-centage of some components of the protein hydrolysisfraction in cells cultured under different light +qualities (Table 3). Cells grown under blue light +had more leucine than cells grown in white light,while glycine was significantly higher in white andblue light than in green and red light (Table 3).Serine, on the other hand, was below detectablelimits only in cells grown under blue light. Thissuggests that light quality influences the type ofproteins synthesized although other workers havereported that light quality changed the rate but notthe direction of protein synthesis (23,26). E! Glucose was the most significant identified radio-active sugar obtained on carbohydrate hydrolysis, al-though small quantities of fructose occurred in cells FIG. 3. Percent extracellular release by C. reinhardtii cul-cultured under green and red light. These sugars tures grown in white light as a function of cell numbers.may have been derived from a reserve polysaccharideor from cell-wall polysaccharides. The cells grownunder blue and white light incorporated a smaller release of compounds by the cells and not a loss duepercent 1% into glucose and fructose than those to cell breakage or passive leaching, since the rangegrown under green or red light (Table 4). of compounds in the photosynthate (Table 2) was Within the ethanol-soluble fraction cells grown much greater than that appearing in the extracellularunder blue and green light had the highest percent fraction (Table 5). Differences in membrane perme-activity in free amino acids, while cells grown in ability related to light quality are suggested. Nitellawhite and red light contained the highest percentage shows light-induced changes in membrane potentialin the organic acids and sugar fractions, respectively which are associated with changes in cytoplasmic(Table 2). The main constituent of the sugar frac- ion concentrations (25).tion was an unknown sugar which appears not only The percentage of newly incorporated 14C exudedin algae but also in a number of vascular plants, and by C . reinhardtii was similar to the range of valueshas thus far not been identified (G. R. Lister, published in the literature, although percentagepersonal communication). extracellular release in white light was relatively Our results, and those of other workers, suggest high (Table 5). The magnitude of the release by that light quality alters the rates o metabolic f cells cultured in white light was in part a functionreactions and/or activates different metabolic path- of cell density in the cultures (5 x 104 cells/ml inways. For example, it has been suggested that blue white light and 1-3 x 106 cells/ml in blue and redlight may activate phosphoenol pyruvate carboxylase light). Our data (Fig. 3) indicate that the lower cellin Chlorella (15) or increase the rate of secondary density in the white light cultures would be associ-carboxylation of certain organic acids, thereby pro- ated with a high rate of extracellular release. Atmoting the accumulation of amino acid precursors a density of 1-3 x 106 cells/ml the release rate under(22). Changes of this type which are dependent on white light would probably have been less than 10%.light quality might influence the rate or nature of Our results did not show the positive relationshipany extracellular release. between the percentage of the newly formed photo- The nature of the dissolved organic compounds synthate in the ethanol-soluble fraction and the per-released from cells grown in white, blue, and red centage extracellular release reported earlier (28).light is shown in Table 5. Insufficient labeled nor do our data on C. reinhardtii support the sug-material was exuded by cells grown in green light to gestion that a depth-dependent change in percentagepermit identification of constituent compounds. The extracellular release could be expected in naturalextracellular products of cells grown in white light waters as a result of the change in the size o the fincluded amino acids, organic acids, and sugars, ethanol-soluble fraction. Species differences may ex-whereas sugars and organic acids were the only plain these results. I n addition, the light intensityproducts released by cells grown under blue and red under which the cells are cultured may affect thelight, respectively. These data indicate a selective extracellular release by C h Zamy domonas.
    • LIGHT AND CHLAMYDOMONAS METABOLISM 219 T h e extracellular compounds released by C . rein- on the distribution of carbon-14 in the early productshardtii in our studies were somewhat different than of photosynthesis. Nature 17989-90. 5. CONSTANTOPOULOS,1970. Lipid metabolism of Mg- G.those reported by other workers (2,21). For example, deficient algae. Plant Physiol. 45:76-80.23% of the organic material formed by C . reinhardtii G. FEDOROV, D., MAKSIMOV. N., & KHROMOV, M. V. V. V.appeared in the culture medium as glycolic and 1968. Effect of light and temperature on primary pro-oxalic acids and sugars (2). No amino acids were duction of certain unicellular green algae and diatoms.exuded under white light during an 8-day growth Soviet Plant Physiol. 15:635-43. 7. Focc, G. E. 1952. T h e production of extracellular ni-period. We detected amino acid release by this trogenous substances by a blue-green alga. Proc. Roy. SOC.species in white light cultures, although specific B . 139~372-97.compounds were not identified. Release of soluble 8. A. HAUSCHILD, H. W., NELSON, D., & KROTROV, C. G.polysaccharides by C . reinhardtii has also been re- 1962. T h e effect of light quality on the products ofported (13), although these may have been leached photosynthesis in Chlorella vulgaris. Can. J. Bot. 40: 179-89.from the mucilaginous sheath. We detected no 9. 1965, On the mode of action of blue light onlabeled polysaccharides i n the medium after 30 the products of photosynthesis in Chlorella vulgaris.min of photosynthesis, although some were observed Naturwissenschaften 52:435.after 40 min. Glycolic acid, a compound frequently 10. HESS, L. & TOLBERT, E. 1967. Changes in chloro- J. N. phyll a/b ratio and products of “CO, fixation by algaereleased by algae as a n extracellular product, was grown in blue or red light. Plant Physiol. 42:1123-30.detected in the medium of cultures grown under 11. E. JORGENSEN, G. 1968. The adaptation of planktonwhite and red, but not blue light. Similar results algae. 11. Aspects of temperature adaptation in Skeleto-were reported for Chlorella pyrenoidosa and C . vul- nema costatum. Physiol. Plant. 21:423-7.garis (3). Other work with C . pyrenoidosa and 12. W. KOWALLIK, 1965. Protein production of Chlorella in light of different wavelengths. Planta 64:191-200.Euglena gracilis has shown that under blue light 13. LEWIN, R. A. 1956. Extracellular polysaccharides ofthat glycolate neither accumulated intracellularly nor gren algae. Can. J. Microbiol. 2:665-72.was released extracellularly (14). This suggests that 14. LORD, M., CODD, A., & MERRETT, J. 1970 . The J. G. M.any glycolate formed under blue light may be effect of light quality on glycolate formation and excre- tion in algae. Plant Physiol. 46:855-6.further metabolized via glyoxylate to glycine and 15. N. OCASAWARA, & MIYACHI, 1970. Regulation of CO, S.other amino acids which are utilized during protein fixation in Chlorella by light of varied wavelengths andsynthesis. T h e absence of organic acids in the extra- intensities. Plant Cell Physiol. 11:l-14.cellular products of cells grown under blue light 16. J. OLIVE, H. & MORRISON, H. 1967. Variation in dis- J. tribution of 14C in cell extracts of phytoplankton livingmight be the result of a greater utilization of organic under natural conditions. Limnol. Oceanogr. 12:383-91.acids in blue light for protein or amino acid syn- 17. ORTH,G . M., TOLBERT, E., & JIMENU,E. 1966. Rate N.thesis. T h e explanation for the absence of amino of glycolate formation during photosynthesis at high pH. Plant Physiol. 41:143-7.acids and sugars in the exudate of cells grown under 18. T. PARSONS, R., STEPHENS, & STRICKLAND, H. 1961. K., J. D.red light is not at hand. On the chemical composition of eleven species of marine T h e chemical composition of the algae and their phytoplankton. J . Fish. Res. B d . Canada 18:lOOl-16.extracellular products may also be altered by light 19. STEWART, D. P. 1963. Liberation of extracellular ni- W. trogen by two nitrogen fixing blue-green algae. Natureintensity (29,30) or by an interaction of light quality 200:1020-1.and quantity. Species differences are probably of 20. STRICKLAND, H. 1960. Measuring the production of J. D.importance as well. We have not considered these marine phytoplankton. Bull. Fish. Res. Bd. Canada 122: 1-172.latter possibilities although it is clear that further TOLBERT, E. & ZILL,L. P. 1956. Excretion of glycolate N. 21.work will be required before many generalizations by algae during photosynthesis. J. Biol. Chem. 222:895-are possible. 906. 22. TRUKHIN, V. 1968. Effect o the carbon supply on N. f ACKNOWLEDGMENT the response of Chlorella pyrenoidosa to the effect of blue and red light. Soviet Plant Physiol. 15:544-8. Thanks are due to John Nelson for his skillful technical 23. VOSKRESENSKAYA, 1956. Synthesis of organic and N. P.assistance and helpful suggestions. This research was sup- amino acids during photosynthesis under various con-ported by a National Research Council of Canada grant. ditions of illumination. Fiziol. Rast. 3:49-57. REFERENCES 24. -& NECHAEVA,P. 1967. Effect of blue, red, and I. green light on the content of protein, nucleic acids, and AHMED, M. M. & RIES, E. 1969. The pattern of WO, A. chlorophyll in young barley plants. Soviet Plant Physiol. fixation in different phases of the life cycle and under 1425441. different wavelengths in Chlorella pyrenoidosa. Prog. 25. VREDENBERC, J. 1971. Changes in membrane po- W. Ph 0 tosyn. R es. 3: 1662-8. tential associated with cyclic and non-cyclic electron trans- ALLEN, R. 1956. Excretion of organic compounds by M. port in photochemical system I in Nitella translucens. Chlamydomonas. Arch. Mikrobiol. 24163-8. Biochem. Biophys. Res. Comm. 42:lll-8. BECKER, D., DOHLER, & ECLE,K. 1968. The effect J. G., 26. WALLEN, G. & GEEN,G. H. 1971. Light quality in D. of monochromatic light on the extracellular excretion of relation to growth, photosynthetic rates, and carbon glycolate during photosynthesis of Chlorella. Pflanzen- metabolism in two species of marine plankton algae. physiol. 58:212-21. Marine Biol. 10:34-43. R. CAYLE, & EMERSON, 1957. Effect of wavelength T. 27. - 1971. Light quality and concentration of pro-
    • 220 EDWARD G. DURBIN teins, RNA, DNA, and photosynthetic pigments in two 29. WAIT. W. D. 1969. Extracellular release of organic species of marine plankton algae. Marine Biol. 1044-51. matter from two freshwater diatoms. Ann. Bot. 33:427-37.28. - 1971. The nature of the photosynthate in natural 30. -& Focc, G. E. 1966. The kinetics of extracellular phytoplankton populations in relation to light quality. glycolate production by Chlorella pyrenoidosa. J . Exp. Marine Biol. 10157-68. B o t . 17:117-34.J . Phycol. 10,220-225 (1974) STUDIES ON T H E AUTECOLOGY OF T H E MARINE DIATOM THALASSIOSIRA NORDENSKIOLDII CLEVE. 1. T H E INFLUENCE OF DAYLENGTH, L I G H T INTENSITY, AND TEMPERATURE ON GROWTH’ Edward G. Durbin Graduate School o f Oceanography, University of Rhode Island Kingston, Rhodc Island 02881 SUMMARY The marine diatom Thalassiosira nordenskioldii INTRODUCTIONCleve was grown at 48 different combinations of day- The marine diatom Thalassiosira nordenskioldiilength (9:15,12:12, 15:9 LD), light intensity (0.011, Cleve has been described (16)as a neritic arctic and0.027, 0.066, 0.100 lylmin [g cal/cmz/min]), and boreal species. Extensive studies of its distributiontemperature (0,5,10, 15 C). Growth occurred at all in the North Sea and adjacent waters showed it tocombinations of light and temperature except at 15 be one of the main species of the arctic neritic plank-C at the highest light level. Maximum growth ton during the spring bloom. Because of the temper-( K = 1.8 doublings/day) occurred at 10 C under the ature range over which it was found (0.8-10.6 C),15:9 L D cycle. A t 15 C the maximum rate was 1 7 . it was described as being “rather eurythermic with adoublings/day but occurred at the shortest day- low optimum” (23). Subsequent studies have shownlength (9:15 LD). The maxima at 5 and 0 C were it to be widely distributed and an early dominant of1.32 and 0.67 doublings/day, respectively. the spring diatom bloom in arctic, boreal and even At 0 C growth was similar over a wide range of some temperate neritic waters of the northern At-light intensities ( K = 0.6-0.65), with maximum lantic and Pacific oceans (1-3,6,7,9,22,22,25,27,29,growth being attained at a much lower light in- 25,2630).tensity than at 5 C. Above 5 C there was a decrease In Narragansett Bay it is one o the major forms fin the light intensity at which maximum growth in the winter-spring bloom which it generallyoccurred and excessive light became inhibitory to initiates along with Skeletonema costatum andgrowth. At 15 C the light intensity at which maxi- Detonula conferuacea (25,26). Weekly records ofmum growth occurred was greater with shorter day- phytoplankton abundance since 1959 show that thelengths. The temperature optimum was 10 C at mean temperature at which Thalassiosira begins15:9 and 15 C at 9:15 LD. rapid growth is 2.45 C. I n Long Island Sound The chlorophyll a content of the cells was greatest Thalassiosira and Skeletonema are the 2 dominant species in the spring bloom (21). Both experimentalunder low light intensities and short daylengths, and field observations showed that Thalassiosira hadwhile temperature had a variable effect. a competitive advantage over Skeletonema at low The response of Thalassiosira in the laboratory light intensities and temperatures, becoming domi-contrasts with its apparent preference for low tem- nant under such conditions. Thalassiosira has alsoperatures in nature (0-5 C). T h e experiments sug- been found to be the early dominant in the springgest that the termination of the bloom of Thalas- bloom in other areas such as the Gulf of Mainesiosira in Narragansett Bay and elsewhere is not (2,27), off Umiak Island, Alaska (12), and the Oslosolely temperature dependent. Fjord (29). The arctic nature of this species is emphasized by its occurrence at Igloolik, near Baffin Received August 23, 1973; revised April 18, 1974. Island, where it was found under ice in May ( T =