DEGRADATION AND FORMATION OF SULFOLIPID OCCURRING CONCURRENTLY WITH DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES

1. It has been demonstrated that when the cells of Chlorella protothecoidesare grown mixotrophically under illumination in a medium richin nitrogen source (urea) and poor in glucose, the normal greencells are obtained, while in a medium rich in glucose and poorin the nitrogen source, entirely chlorophyll-less cells withprofoundly degenerated plastids ("glucose-bleached" cells) areproduced, irrespective of whether in the light or in darkness.The "glucose-bleached" cells turn green with regeneration offully organized chloroplasts when incubated in a nitrogen-enrichedmedium in the light ("light-greening"), while in the dark theybecome pale green with formation of only partially organizedchloroplasts ("dark-greening"). When, on the other hand, thegreen cells are transferred into a medium enriched with glucose,they are bleached fairly rapidly with degeneration of chloro-plastsin the light as well as in darkness ("bleaching"). Using ³⁵Sas a tracer, investigations were made on the changes of contentsof the algal cells in sulfolipid and other sulfur compoundsduring the processes of the greening and bleaching.
2. By determiningthe radioactivities of chromatographically separatedsulfur-containingcompounds of the uniformly ³⁵S-labeled green("G") and "glucose-bleached"("W") cells, it was found thatthe concentration of a speciesof sulfolipid (discovered byBENSON et al.) as well as thoseof glutathione, sulfotriosesand most of the other sulfur-containingcompounds were at least5 times higher in the "G" cells thanin the "W" cells, whilesulfoquinovosyl glycerol was presentin approximately equalamounts in the two types of cells.
3. Phospholipidcontents and compositions in the two types of algalcells werefound to be practically identical.
4. The sulfolipid contentof algal cells increased and decreasedalmost in parallel withthe processes of greening and bleaching,respectively.
5. Studyingthe mode of incorporation of radiosulfate into varioussulfurcompounds of algal cells during the processes of "light-anddark-greening" and "bleaching" (lasting about 70 hr), itwasfound that active ³⁵S-incorporation into sulfolipid occurredthroughout the process of "light-greening," while in the "dark-greening"and "bleaching" the active incorporation abruptly ceased afterthe initial 24 hr period of experiments. It was suggested thatthe biosynthesis of the sulfolipid is closely related to theformation of photosynthetic apparatus in chloroplast.
6. Whenthe ³⁵S-labeled green cells were bleached in a medium containingno radiosulfate, the ³⁵S-sulfolipid and most of other ³⁵S-sulfurcompounds decreased markedly but the ³⁵S-sulfoquinovosyl glycerolincreased considerably. It was inferred that the deacylationof the sulfolipid, a surfactant lipid, with formation of watersoluble sulfoquinovosyl glycerol may be a cardinal event ofbleaching process, causing a disintegration of the intact architechtureof photosynthetic apparatus.
7. Based on these observations itwas concluded that the sulfolipidis an integral component ofphotosynthetic structure.

¹This work was partly reported at the Symposium on Biochemistryof Lipids, sponsored by the Agricultural Chemical Society ofJapan, Sapporo, July, 1964.     

EFFECTS OF GLUCOSE ON THE PROCESS OF CHLOROPLAST DEVELOPMENT IN CHLORELLA PROTOTHECOIDES

By growing Chlorella protothecoides in a medium rich in glucoseand poor in nitrogen source (urea), the entirely chlorophyll-lesscells showing no discernible plastid structure and containingonly little RNA and protein were obtained. These cells, whichwere called "glucosebleached" cells, turned green after a certainlag period, when they were incubated, in the light, in a mediumcontaining the nitrogen source and basal mineral nutrients butwithout glucose. As has been shown in previous studies, thisgreening process involves two consecutive steps : a light-independentphase, in which RNA plays an essential role, and a light-requiringphase, in which the chlorophyll formation and full organizationof chloroplast take place accompanied by the formation of acertain species of protein ("alkali-stable" protein). The wholeprocess of greening was found to be profoundly suppressed byaddition of glucose, the degree of suppression being determinedby the relative concentrations of glucose and the nitrogen source.The primary act of glucose was manifest in the repression ofsyntheses of RNA, and of the species of protein mentioned above,which participate in, or causally related to, the first andsecond phases, respectively, in the greening process. ¹ This paper was read at the Symposium on Extranuclear Self-reproducingSystems held by the Botanical Society of Japan in October, 1964     

CHANGES IN CONTENTS OF CARBOHYDRATE AND FATTY ACID IN THE CELLS OF CHLORELLA PROTOTHECOIDES DURING THE PROCESSES OF DE- AND RE-GENERATION OF CHLOROPLASTS

1. Previous work has demonstrated that when cells of Chlorellaprotothecoides are grown mixotrophically under illuminationin a medium rich in nitrogen source (urea) and poor in glucose,normal green cells are obtained, while in a medium rich in glucoseand poor in the nitrogen source, strongly bleached cells containingapparently no discernible chloroplast structures — called"glucose-bleached" cells — are produced either in thelight or in darkness. When the green cells are incubated ina glucose-enriched mineral medium without added nitrogen source,they are fairly rapidly bleached with concomitant degenerationof chloroplast structures (" bleaching "). When, on the otherhand, the "glucose-bleached" cells are transferred in a nitrogen-enrichedmedium without added glucose under illumination, they turn greenwith regeneration of chloroplasts (" greening "). In the presentstudy changes in contents of carbohydrate and fatty acid inalgal cells were followed during these processes of "bleaching"and "greening.".
2. During the process of "bleaching", the quantityof glucose existingin the insoluble carbohydrate fraction ofalgal cells increasedrapidly and markedly. A considerable increasewas also observedin the contents of cells in oleic, linoleicand palmitic acids.It was noted, however, that linolenic aciddecreased in quantityduring the most active phase of cell bleaching.
3. During the process of "greening", the glucose in the insolublecarbohydrate fraction rapidly decreased, suggesting that itis utilized, as carbon and energy sources, for the chloroplastregeneration. Linolenic acid was found to be synthesized inparallel with formation of chlorophyll. A peculiar pattern ofchange in contents was observed with oleic and palmitic acids,which was interpreted as being related with the process of cellulardivision occurring incidentally during the process of greening.

(Received September 24, 1966; )     

PHOTOSYNTHESIS OF GLYCINE AND SERINE IN GREEN PLANTS

1. The effects of "carbonyl" reagents on the photosyntheticin-corporation of ¹⁴CO₂ into the assimilation products of tobaccoand spinach leaves were studied. The presence of "carbonyl"reagents causes an increase in the ratio of ¹⁴CO₂ incorporatedin glycine and a decrease in serine. The incorporation of ¹⁴Cfrom glycolate-1-¹⁴C and glycolaldehyde-2-¹⁴C into glycine andserine was also affected by "carbonyl" reagents, as in the caseof ¹⁴CO₂-experiment. 2. The feeding experiments of glycine-1-¹⁴C and serine-1-¹⁴Cin the presence and in the absence of "carbonyl" reagents revealedthat these reagents inhibit the conversion of glycine to serine. 3. The results obtained above, together with the effects ofthiols on ¹⁴CO₂ incorporation presented in this paper, supportthe assumption that glycine and serine are formed via glycolateand glyoxylate during photosynthesis in green plants. 4. Comparison of ¹⁴C incorporation in malate from ¹⁴CO₂, glycolate-1-¹⁴C,glycine-1-¹⁴C and serine-1-¹⁴C in the presence and in the absenceof "carbonyl" reagents suggested the occurrence of the pathwayof the malate formation via glycolate and glyoxylate, not passingthrough glycine and serine, during photosynthesis. ¹ A part of this paper was presented at the Symposium on "Nitrogenand Plant" by the Japanese Society of Plant Physiologists, inOctober, 1963 ² Present address: Radiation Center of Osaka Prefecture, Sakai,Osaka     

Studies on ribonucleic acids from Chlorella protothecoides with special reference to the degradation of chloroplast RNA during the process of "glucose-bleaching"

By growing Chlorella protothecoides under certain nutritionaland light conditions the following three different types ofalgal cells were obtained: (i) normal "green" cells grown ina medium rich in a nitrogen source (urea) and poor in glucoseunder illumination, (ii) "etiolated" cells cultivated in thesame medium in darkness, and (iii) "glucose-bleached" cellsgrown, in the light or in darkness, in a medium rich in glucoseand poor in the nitrogen source. The "glucose-bleached" cellscontain profoundly degenerated plastids, and the "etiolated"cells have only partially organized plastids. From these algalcells RNA was extracted by the cold phenol method, and fractionatedby MAK column chromatography and sucrose density gradient centrifugation,making use of ³²P-labelled E. coli RNA as the internal marker.It was found that in comparison with the green cells that arerich in chloroplast ribosomal RNA as well as in nonchloroplastic("cytoplasmic") ribosomal RNA, the etiolated cells possess acomparable amount of "cytoplasmic" rRNA but a significantlylesser amount of chloroplast rRNA. Both types of rRNA existat extremely low levels in the glucose-bleached cells. During the process of bleaching (chloroplast degeneration) ofthe green cells induced by the addition of a high concentrationof glucose, marked changes were observed in the patterns offractionation of RNA as followed by the above procedures. Itwas disclosed that the chloroplast rRNA is rapidly degradedduring an early phase of the bleaching process, while the quantityof "cytoplasmic" rRNA remained almost unaltered. ¹Part of this work was reported at the Symposium on Cell Differentiationsponsored by the Institute of Applied Microbiology, Universityof Tokyo, in November 1965, and at the Symposium on Biogenesisof Subcellular Particles, the 7th Internatl. Congress of Biochemistry,Tokyo, 1967. ²Present address: Faculty of Pharmaceutical Sciences, Universityof Hokkaido, Sapporo.     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: I. SYNTHESES OF NUCLEIC ACIDS AND PROTEIN IN RELATION TO THE PROCESS OF REGENERATION OF CHLOROPLAST

When Chlorella protothecoides is grown mixotrophically in thelight in a medium rich in glucose and poor in nitrogen source(urea), one obtains the cells that are entirely devoid of chlorophylland containing only little RNA and protein. When these cells—referredto as "glucose-bleached" cells—are further grown in thelight with provision of nitrogen source, but without glucose,sequential syntheses of RNA, protein and chlorophyll take place.If the glucose-bleached cells are incubated in the dark underthe same nutritional condition, RNA, protein and chlorophyllare also successively formed in relatively small amounts. Thecells obtained under such a condition are, in many respects,similar to the cells that are obtained when the alga is grownin the dark in a medium poor in glucose and rich in the nitrogensource. These cells, which are called the "etiolated cells",are faintly green in color and contain larger amounts of RNAand protein compared with the chlorophyll-less glucose-bleachedcells. The glucose-bleached cells and the etiolated cells showapproximately the same content of DNA per cell. When the etiolatedcells are incubated in the light with provision of nitrogensource, but without glucose, they become green with active synthesisof chlorophyll and additional syntheses of RNA and protein. Based on these results and those to be reported later, it wasconcluded that the greening of the glucose-bleached cells involvesa light-independent phase followed by a light-requiring phasewhich entails the greening of cells and full organization ofchloroplasts, and that the latter process is essentially thesame as that taking place when the etiolated cells are incubatedin the light with provision of nitrogen source in the absenceof glucose. (Received September 5, 1964; )     

RIBONUCLEIC ACIDS APPEARING DURING THE PROCESS OF CHLOROPLAST REGENERATION IN THE "GLUCOSE-BLEACHED" CELLS OF CHLORELLA PROTOTHECOIDES

1. Investigations were made on the modes of synthesis of differentspecies of RNA which appear during the greening (chloroplastregeneration) of the "glucose-bleached" cells of Chlorella protothecoidescontaining profoundly degenerated plastids.
2. RNAs were extractedfrom the algal cells which had been labelledwith ³²P for 1hr before harvesting at different stages of thegreening inthe light and in darkness, and subjected to columnchromatographywith methylated albumin-coated kieselguhr. Itwas found that,during the greening process, the elution profilesof RNAs, interms of the optical density at 260 mµ and³²P-radioactivity,changed profoundly.
3. Based on these and other results, it wasconcluded that duringan early phase of the chloroplast regenerationin the glucosebleachedalgal cells, there occurs an active formationof both ribosomalRNAs (rRNAs) and the RNAs corresponding tosoluble RNA (sRNA),the formation coming, however, later toa standstill when thesynthesis of chlorophyll has proceededto a certain level. Thequantity ratio of sRNA to rRNA was foundto be constant (30:70)at different stages of the greening (bothin the light and indarkness), with a few exceptions. The synthesisof the chloroplastribosomal RNA is markedly accelerated bylight, and its maximumrate is observed sometime later thanthat of the non-chloroplast("cytoplasmic") ribosomal RNA. Itwas suggested that there areat least two different sites ofsynthesis of ribosomal RNAs,one in the plastid and the otheroutside of it (most probablyin the nucleus).

¹A part of this work was reported at the Symposium on Cell Differentiationsponsored by the Institute of Applied Microbiology, Universityof Tokyo, in November 1965. ² Present address: Institute for Plant Virus Research, Ministryof Agriculture and Forestry, Aoba-cho, Chiba.     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: V. DEGENERATION OF CHLOROPLASTS INDUCED BY DIFFERENT CARBON SOURCES, AND EFFECTS OF SOME ANTIMETABOLITES UPON THE PROCESS INDUCED BY GLUCOSE

1. The green cells of Chlorella protothecoides were bleached todifferent extents when incubated (in the dark) in the nitrogen-freemedia containing, besides basal mineral nutrients, glucose,fructose, galactose, glycerol or acetate. Glucose and fructosewere found to have the strongest bleaching effect. Additionof a nitrogen source (urea) caused a considerable reductionof the bleaching. It was assumed that from the different carbonsources a certain common intermediate(s) causing the bleachingis formed, and that in the presence of the nitrogen source thesubstance is removed by reacting with it.
2. Using glucose asbleach-inducing agent, the effects of someantimetabolites uponthe processes of bleaching, division andgrowth of green algalcells were investigated, and it was demonstratedthat the processof bleaching occurs without being accompaniedby growth anddivision of the algal cells.
3. It was found that during theprocess of bleaching no net increasesin RNA and protein tookplace.

(Received March 11, 1965; )     

Further studies on the metabolism of glucose in the process of "glucose-bleaching" of Chlorella protothecoides

Previous studies have demonstrated that when cells of Chlorellaprotothecoides are incubated in a medium containing glucosebut no nitrogen source, they are profoundly bleached with degenerationof chloroplast structure and photosynthetic activity. When anitrogen source (urea) is added to the glucose medium, bleachingof algal cells is greatly suppressed. In this work the metabolismof glucose in the process of glucose-induced bleaching was studiedusing ¹⁴C-glucose as tracer. Changes in algal cell activityfor ¹⁴CO₂-evolution and ¹⁴C-incorporation into various cellularsubstances from ¹⁴C-glucose were followed. Most conspicuouswere increases in cellular activities for assimilating ¹⁴C-glucoseinto lipids (fatty acids) and glucose polymer. When urea wasadded to the glucose medium, the incorporation of ¹⁴C by algalcells into fatty acids was greatly reduced, while the assimilationof ¹⁴C into glucose polymer was increased. These and previous observations suggest that the formation oflarge amounts of lipids (fatty acids) probably is causally relatedto the induction of algal cell bleaching. (Received March 5, 1969; )     

STOICHIOMETRIC RESPONSES OF PHYTOPLANKTON SPECIES TO THE INTERACTIVE EFFECT OF NUTRIENT SUPPLY RATIOS AND GROWTH RATES1

Three species of phytoplankton, Rhodomonas sp., Phaeodactylum tricornutum Bohlin, and Isochrysis galbana Parke, were cultivated in semicontinuous culture to analyze the response of carbon (C):nitrogen (N):phosphorus (P) stoichiometry to the interactive effect of five N:P supply ratios and four growth rates (dilution rates). The relationship between cellular N and P quotas and growth rates fits well to both the Droop and Ågren’s functions for all species. We observed excess uptake of both N and P in the three species. N:P biomass ratios showed a significant positive relationship with N:P supply ratios across the entire range of growth rates, and N:P biomass ratios converged to an intermediate value independent of N:P supply ratios at higher growth rates. The effect of growth rates on N:P biomass ratios was positive at lower N:P supply ratios, but negative at higher N:P supply ratios for both Rhodomonas sp. and I. galbana, while for P. tricornutum this effect was negative at all N:P supply ratios. A significant interactive effect of N:P supply ratios and growth rates on N:P biomass ratios was found in both Rhodomonas sp. and P. tricornutum, but not in I. galbana. Our results suggest that Ågren’s functions may explain the underlying biochemical principle for the Droop model. The parameters in the Droop and Ågren’s functions can be useful indications of algal succession in the phytoplankton community in changing oceans.     

Studies on chlorophyll formation in Chlorella protothecoides I. Enhancing effects of light and added {delta}-aminoIevulinic acid, and suppressive effect of glucose on chlorophyll formation

Light-induced formation of chlorophyll in "etiolated" cellsof Chlorella protothecoides was studied under various experimentalconditions, (i) Two different types of enhancing effect of lightwere demonstrated: a "long-term" effect lasting for many hoursafter a relatively short illumination of etiolated cells anda "short-term" effect disappearing in a few hours after illumination,(ii) Addition of ALA caused enhancement of chlorophyll synthesisin etiolated cells in darkness as well as in light; the ALA-enhancedrate of dark chlorophyll synthesis, however, was much lowerthan the rate in light without added ALA. ALA was replaceablewith succinic acid plus glycine in light, but not in the dark,for enhancement of chlorophyll formation, (iii) Adding glucose,fructose, galactose, glycerol or acetate—at concentrationsmuch lower than those previously shown to induce "bleaching"of green algal cells-caused a more or less marked suppressionof light-induced greening in etiolated cells, (iv) Added glucosealmost instantaneously and completely stopped chlorophyll synthesisin light as well as in darkness either with or without addedALA. On the basis of these and other results, a tentative schemeis presented for the enhancing effects of light and the suppressiveeffects of glucose on chlorophyll formation in algal cells. (Received April 1, 1970; )     

FURTHER NOTES ON THE GROWTH AND CHLOROPHYLL FORMATION OF CHLORELLA PROTOTHECOIDES

1. Using area of a fixed concentration as a nitrogen source,cells of Chlorella protothecoides were grown in the presenceof various carbon compounds. Magnitudes of growth of the cellswere widely different depending on the carbon sources used;glucose and fructose being most favourable substrates and galactose,glycerol and acetate coming next. But the amounts of chlorophyllformed in the cells during the experimental period were almost,the same irrespective of the different carbon sources, withsome exceptions. The similarity of the chlorophyll level observedin these experiments seemed to indicate that the formation ofchlorophyll was limited largely by the nitrogen source but notby the carbon source. 2. Strong bleaching effect was recognized with glucose and fructoseat their high concentration, which produced totally chlorophyll-lesscells. On the other hand, a stimulating effect on chlorophyllformation was observed with galactose at the different concentrationsexamined. 3. Effects of glycine and ammonium carbonate as the nitrogensource on the algal growth and pigmentation were studied insome details. The results were similar to those previously obtainedwith urea, confirming our previous conclusion that the algalpigmentation is profoundly affected by the concentration balancebetween glucose and nitrogen source. ¹Present address: Tokyo Research Laboratories, Tanabe SeiyakuCo., Toda-machi, Saitama.     

EFFECTS OF LIGHT ON THE DEOXYRIBONUCLEIC ACID FORMATION AND CELLULAR DIVISION IN CHLORELLA PROTOTHECOIDES

1. It has been demonstrated previously that when Chlorella protothecoidesis grown in a medium rich in glucose and poor in nitrogen source(urea), chlorophyll-less cells with markedly degenerated plastids—called "glucose-bleached" cells—are produced eitherin the light or in darkness. When the glucose-bleached cellsare incubated in a medium enriched with the nitrogen sourcebut without added glucose, normal green cells with fully organizedchloroplasts are obtained in the light, and pale green cellswith partially organized chloroplasts in darkness. During theseprocesses of chloroplast development in the glucose-bleachedcells, there occurs, after a certain lag period, an active DNAformation followed by a more or less synchronous cellular division.In the present study the effects of light on the DNA formationand cellular division were investigated in the presence of CMUor under aeration of CO²-free air to exclude the interveninginfluence of photosynthetic process.
2. It was revealed thatlight severely suppresses the DNA formationand cellular divisionof the glucose-bleached cells while enhancingremarkably theirgreening. The suppression was saturated atthe light intensityof about 1,000 lux. Blue light was mosteffective, being followedby green, yellow and red light inthe order of decreasing effectiveness.
3. Further experiments unveiled that light exerts two apparentlyopposing effects on the DNA formation depending upon the timeof application during the incubation of algal cells. When thealgal cells were illuminated only during the lag period beforethe active DNA synthesis, there occurred an enhancement of theDNA synthesis occurring during the subsequent dark incubation.When, on the other hand, the cells were transferred to the lightfrom darkness at or after the start of the DNA synthesis, itcaused an almost complete abolition of the subsequent synthesisof DNA in the algal cells. No such effects of light were observedwith RNA and protein (total)
4. These findings were discussedin relation to the process ofchlorophyll formation occurringconcurrently in the algal cells.

(Received August 10, 1967; )     

Growth of Phaseolus vulgaris on Various Nitrogen Sources: The Importance of Urease

Rhizobium-inoculatcd plants of Phaseolus vulgaris L. were grownwith different N-sources (nitrate, ammonium, urea) and differentconcentrations of urea. The distribution of growth between plantparts varied with N-sources. Nitrate and ammonium were moreinhibitory to nodulation than urea, which at 40 mol m^–3N had no effect. Urease activity varied in amount and locationover a range of urea concentrations. At higher concentrations,more urea was transported to and increased urease activity wasfound in the shoot Lower levels of activity in plants relianton N₂-fixation were consistent with a ureide-degradation pathwaynot involving urea. Moderate doses of urea could be assimilatedconcomitantly with N₂-fixation. At higher levels of appliedurea, nodulation and ureide transport to the shoots were reduced,although increased growth could not be maintained at concentrationsof applied urea greater than 6.0 mol m^–3 urea N. Key words: Phaseolus vulgaris, growth, nitrogen source, urease     

Oxygen--A Key Regulatory Metabolite in Metabolic Defense Against Hypoxia

Two main defense strategies against hypoxia tolerant animalshave been identified in earlier studies: (i) reduction in energyturnover and (ii) improved energetic efficiency of those metabolicprocesses that remain. Two model systems were developed fromthe highly anoxia tolerant aquatic turtle—(i) tissue slicesof brain cortex (to probe cell level electrophysiological responsesto oxygen limitation) and (ii) isolated liver hepatocytes (toprobe signalling and defense). In the latter, a series of mechanismsunderpinning hypoxia defense is initiated with an oxygen sensor(probably a heme protein) and a message transduction pathwayleading to the specific activation of some genes (increasedexpression of several proteins) and to specific down regulationof other genes (decreased expression of several other proteins).The pathway seems similar to oxygen regulated schemes in othercells. The main roles for the oxygen sensing and signal transductionsystem appear to include coordinate down regulation of energydemand and energy supply pathways in metabolism. By this means,hypoxia tolerant cells stay in energy balance as they down regulateto extremely low levels of ATP turnover. The main ATP demandpathways in normoxia (protein synthesis, protein degradation,glucose synthesis, urea synthesis, and maintenance of electrochemicalgradients) are all depressed to variable degree during anoxiaor extreme hypoxia. However, Na⁺ K⁺ ATPase is the main energysink in anoxia—despite significant reductions in cellmembrane permeability ("channel arrest"). Turtle brain corticalcells also show lower permeability than do homologous hypoxiasensitive cells, but in this case under acute anoxia, thereis no further change in cell membrane conductivity. These twomodels may supply guidelines for further studies of estuarineanimals on how normoxic maintenance ATP turnover rates can bedown regulated by an order of magnitude or more—to newhypometabolic steady states prerequisite for surviving prolongedhypoxia or anoxia     

EFFECT OF LIGHT ON THE CHLOROPHYLL FORMATION IN THE "GLUCOSE-BLEACHED" CELLS OF CHLORELLA PROTOTHECOIDES

1. Previous studies have shown that when Chlorella protothecoidesis grown in a medium rich in glucose and poor in nitrogen source(urea), apparently chlorophyll-less cells with profoundly degeneratedplastids—referred to as "glucose-bleached cells—areproduced either in the light or in darkness. When the glucose-bleachedcells are incubated in a medium enriched with the nitrogen sourcebut without added glucose, an active formation of chlorophylloccurs after a certain lag period under illumination, whilein darkness a very small amount of chlorophyll is formed atabout the same time as in the light. The stimulating effectof light on the chlorophyll formation is not appreciably affectedwhen the photosynthetic CO₂-fixation of greening algal cellsis blocked by the addition of CMU. In the present study, itwas further found that the light-enhanced chlorophyll formationproceeds, although at a somewhat lower rate, under aerationof CO₂-free air. All the experiments in this work were doneunder these non-photosynthetic conditions to exclude any influenceof photosynthates.
2. The effect of light (from daylight fluorescentlamps) on thechlorophyll formation in the glucose-bleachedalgal cells wassaturating at about 1,000 lux. Blue light wasfound to be mosteffective; yellow, green and red light followingin the orderof decreasing effectiveness.
3. When the bleachedalgal cells were illuminated for a short periodin the lag phaseof chlorophyll formation and subsequently incubatedin darkness,there occurred an appreciable enhancement of chlorophyllformationin the dark. When the short illumination was appliedat differenttimes of the lag phase, the enhancement was inducedto almostthe same extent. But the longer the duration of theilluminationduring the lag phase, the greater was the enhancementof chlorophyllformation in the subsequent dark incubation.In such experimentsblue light was most effective and red lightleast, as it wasthe case in the experiments of continuous illumination.An intervenientillumination of the bleached cells at lowertemperatures orunder the atmosphere of N2 produced little orno enhancementof the chlorophyll formation in the subsequentdark incubation.
4. Based on these results, it was concluded that the light enhancementof chlorophyll formation in the glucose-bleached algal cellsis mediated by a non-chlorophyllous photoreceptor(s), absorbingmaximally blue and yellow light, and that a light-induced changeof the photoreceptor is immediately followed by a certain dark(temperaturedependent and aerobic) process(es) which is connected,directly or indirectly, to the chlorophyll synthesis.

(Received August 10, 1967; )     

Effect of Exogenous Glycinebetaine on Na+ Transport in Barley Roots

Ahmad, N., Wyn Jones, R. G. and Jeschke, W. D. 1987. Effectof exogenous glycinebetaine on Na⁺ transport in barley roots.—J.exp. Bot. 38: 913–921. A comparison has been made of the kinetics of 22Na⁺ uptake intoexcised barley roots and roots pre-loaded with glycinebetaine.The elevated intracellular glycinebetaine or a metabolic consequencethereof increased the Na⁺ influx, and the effect was relatedto the level of internal glycinebetaine and or Na⁺ [Cl^–].The quasi-steady-state Na⁺ influx at the tonoplast rather thanthe plasmalemma influx was apparently influenced by glycinebetaineloading. The tonoplast fluxes and vacuolar Na⁺ content wereconsistently higher in glycinebetaine-loaded roots than unloadedroots. A membrane-modifying role of glycinebetaine in relationto ion compartmentation is discussed. Key words: Excised roots, glycinebetaine, Na⁺, ion fluxes, barley     

Metabolism of urea in Chlorella ellipsoidea

The effect of cyanide on ammonia and urea metabolism was studiedwith intact cells of Chlorella ellipsoidea Gerneck, a greenalga which apparently lacks urease. Ammonia uptake was inhibited more readily by cyanide than wasurea uptake. Urea uptake was stimulated by lower concentrationsof cyanide. The addition of cyanide caused the formation ofammonia from some cellular nitrogenous compounds. In the presenceof exogenously added urea, the molar ratio of ammonia accumulatedin the medium to urea taken up exceeded 2.0 as the cyanide concentrationincreased. However, the molar ratio of ammonia actually producedfrom urea nitrogen to urea taken up was less than 1.35 at anyconcentration of cyanide tested. In the presence of higher concentrationsof cyanide, the rate of incorporation of ¹⁵N into amino acidsfrom ¹⁵N-urea was higher than that from ¹⁵N-ammonium sulfate. The results suggest that Chlorella ellipsoidea possesses a pathwaythrough which urea nitrogen is assimilated directly withouta preliminary breakdown to ammonia. (Received October 18, 1976; )     

INCORPORATION OF RADIOACTIVE CARBON FROM H14CO3-INTO SUGAR CONSTITUENTS BY A BROWN ALGA, EISENIA BICYCLIS, DURING PHOTOSYNTHESIS AND ITS FATE IN THE DARK

When the fronds of Eisenia bicyclis were exposed to H¹⁴CO₃^–in the light, the radioactive carbon was rapidly incorporatedinto mannitol. Even after illumination of such a short periodas 5 min, about seventy percent of the total radioactivity incorporatedwas found in this compound, and the specific radioactivity ofthis alcohol decreased very rapidly during the subsequent darkincubation. Among various cellular polysaccharides examined,only laminaran showed a similar quick response with respectto the specific radioactivity change. On the basis of thesefindings it was concluded that mannitol and laminaran form storagesubstances in the brown alga, and they are possibly interchangeableas sucrose and starch do in higher plants. ¹This work was partly reported at the 27th Annual Meeting ofthe Botanical Society of Japan, Nagoya, 1962, and at the 28thAnnual Meeting of the Botanical Society of Japan, Okayama, 1963. ²Contribution from the Shimoda Marine Biological Station, TokyoKyoiku University, No. 149.     

Quantum yield and conformational changes during greening and bleaching of Chlorella protothecoides

Measurements of quantum requirement of oxygen evolution in greeningand bleaching cultures of Chlorella proiothecoides reveal aconstant low-quantum requirement during greening and the firsthours of bleaching. Thereafter the values increase drastically. The light-induced "conformational change," measured as straylight-dependent absorbance change, is biphasic; the second partof die signal is due to the absorbance changes caused by theshrinking of the chloroplast. Its value was used as a measureof photophosphorylation, which follows, after a certain delay,the photosynthetic oxygen evolution during greening and bleachingofthe cells. ¹ Present address: Institute of Applied Microbiology, Universityof Tokyo, 113 Tokyo, Japan. (Received January 27, 1976; )