Turnover of Cell Wall Polysaccharides during the Cell Cycle in a Synchronous Culture of Catharanthus roseus

Pulse-chase experiments were done using a synchronous cultureof Catharanthus roseus in order to study cell wall turnoverduring the cell cycle. [¹⁴C]Glucose was fed for 1 h to cells35 and 49 h after the re-start of the cell cycle. Radioactivitywas then diluted with a large amount of cold glucose and chasedduring the early G1 phase after the first cell division, thetime at which an increase in the amount of cell walls mainlytook place. A pulse-chase with [¹⁴C]glucose was also made duringthe S phase when cell walls had not increased so much. Radioactivity of the EDTA-soluble (pectin) fraction decreasedduring the chase in the early G1 phase; whereas, the radioactivitiesof the other cell wall fractions, as well as extracellular polysaccharide(ECP) increased during the chase, both in the early G1 and inthe S phases. The radioactivity of uronic acid in ECP was higherin the early G1 phase than in the S phase. These results indicatethat an active turnover of pectin may take place in the earlyG1 phase after the first cell division. ¹ Present address and reprint requests: Biological Institute,Tohoku University, Sendai 980, Japan. (Received November 5, 1984; Accepted April 2, 1985)     

Short-term Products of 14C-acetate Assimilation by Chlorella pyrenoidosa in the Light

The kinetics of ¹⁴C-2-acetate assimilation by Chlorella pyrenoidosain the light were examined. Under aerobic conditions the primaryproduct of acetate assimilation was succinic acid which, afterten seconds, contained over 60 per cent of the ¹⁴C incorporatedby the cells. The percentage of the total ¹⁴C in succinate fellwith time, while that in citrate and glutamate increased. After1800 sec over 60 per cent of ¹⁴C was present in two compounds,glutamic acid and an unknown compound (X). Glucose-6-phosphate,fructose-6-phosphate, phosphoglyceric acid and phosphoenolpyruvicacid became labelled after 60 sec but together never containedmore than one per cent of the total ¹⁴C incorporated. Underanaerobic conditions succinate was still the primary productof acetate assimilation, and the absence of carbon dioxide resultedin a decrease in ¹⁴C incorporation into compound X. The patternof acetate assimilation in acetate grown and acetate adaptedChlorella was very similar to that in photo-autotrophicallygrown Chlorella. In the presence of 10^–6M DCMU, succinicacid was the primary product of acetate assimilation, but therewas an early Incorporation of ¹⁴C into glutamate, aspartate,and malate. 4 x10^–3M MFA did not effect the early incorporationof ¹⁴C into succinic acid, but resulted in accumulation of ¹⁴Cin citrate and a decreased amount in glutamate and in compound X.     

Cell Wall Metabolism in Developing Strawberry Fruits

Cell wall metabolism was studied in strawberry receptacles (Fragariaananassa, Duchesne) of known age in relation to petal fall (PF).Polysaccharide and protein composition, incorporation of [¹⁴C]glucoseand [¹⁴C]proline by excised tissue, and the fate of ¹⁴CO₂ fixedby young, attached fruits were followed in relation to celldivision, cell expansion, fine structure, and ethylene synthesis. Cell division continued for about 7 d after PF although vacuolationof cells was already beginning at PF and the subsequent cellexpansion was logarithmic. There was an associated logarithmicincrease in sugar content per cell and a decreasing rate ofethylene production per unit fresh weight. During cell expansion radioactivity from [¹⁴C]glucose was incorporatedinto fractions identified as starch and soluble polyuronideand into glucose and galactose residues in the cell wall. Radioactivityfrom [¹⁴C]proline was also incorporated into the cell wall,but only 10 per cent of this activity was found in hydroxyproline.Correspondingly wall protein contained a low proportion of hydroxyprolineresidues. The proportion of radioactivity from ¹⁴CO₂ fixed byfruitlets remained constant in most sugar residues in the cellwall. The proportion of radioactivity in galactose fell, indicatingturnover of these residues. Between 21 and 28 d after PF receptacles became red and softenedbut there was no change in the rate of ethylene production.Cell expansion continued for at least 28 d. Tubular proliferationof the tonoplast and hydration of middle lamella and wall matrixmaterial had begun 7–14 d after PF but became extremeduring ripening. Associated with the hydration of the wall,over 70 per cent of the polyuronide in the wall became freelysoluble, and arabinose and galactose residues lost from thewall appeared in soluble fractions. There was no increase intotal polysaccharide during ripening and incorporation of [¹⁴C]glucoseinto polysaccharides ceased, although protein increased andincorporation of [¹⁴C]proline into wall protein continued.     

Glycollate Formation during the Photorespiration of Acetate by Chlorella

WhenChlorella pyrenoidosa photoassimilates ³H-¹⁴C-acetate theglycollic acid formed shows a high ³H/¹⁴C ratio, the only othercompounds showing similar ratios being glycerate and serine.The ³H/¹⁴C ratio of glycollate was unaffected by the TCA cycleinhibitors MFA, diethylmalonate and arsenite showing that ³Hin glycollate does not result from the oxidation of acetatevia the TCA cycle, the resulting NADP³H₂ or NAD³H₂ being usedfor the reduction of the glycollate precursor. Although DCMUdecreased the ³H/¹⁴C ratio, complete inhibition of glycollatelabelling was not observed with 10^–6 M DCMU, at whichconcentration complete inhibition of the Hill reaction is achieved.Although the ³H/¹⁴C ratio was unaltered, total dpm of both ¹⁴Cand ³H in glycollate were increased by INH. The ³H/¹⁴C ratiosof glycerate and serine were decreased by INH, as were the totaldpm of ³H and ¹⁴C incorporated into these compounds. Thus, INHinhibits the further metabolism of glycollate to glycerate andserine. The effect of INH on incorporation of ¹⁴C-I-acetateinto various cell fractions was investigated. The incorporationof ¹⁴C into polysaccharide and lipid was decreased, while theincorporation of ¹⁴C into the water-soluble fraction of cellsand therelease of ¹⁴CO₂ were little affected. Although glycollicacid was an early product of acetate photoassimilation in Chlorellapyrenoidosa, glycollate excretion does not take place undera wide range of environmental conditions shown to favour glycollateexcretion by other algae. However, small amounts of labelledglycollate were detected in the supernatant from the cells duringthe photoassimilation of ³H-¹⁴C-acetate, but this glycollatedid not show the high ³H/¹⁴C ratio of glycollate present withinthe cell. The failure of Chlorella pyrenoidosa to excrete appreciableamounts of glycollate when photoassimilating acetate or carbondioxide was considered to result from the presence of glycollateoxidase (EC 1.1.3.1 [EC] ) which allowed the further metabolism ofglycollate. Besides glycollate oxidase, glyoxylate reductasewas also demonstrated in Chlorella pyrenoidosa so that glycollatecould function in hydrogen transfer during the photoassimilationof acetate.     

Studies on the Cell Wall of Chlorella IV. Incorporation of Glucose-Carbon into Cell Walls of Synchronously Growing Cells of Chlorella ellipsoidea

Uniformly ¹⁴C-labeIled glucose was fed to synchronously growingChlorella cells in the dark or in light. The rate of ¹⁴C-incorporationinto hemicellulose showed two maxima one in the growth phaseand one in the reproductive phase. Significant ¹⁴Cincorporationinto a "rigid wall" was found only in the reproductive phase. (Received April 14, 1983; Accepted June 15, 1983)     

The Effect of pH on the Binding of Calcium to Pea Epicotyl Cell Walls and its Implications for the Control of Cell Extension

Cell walls were prepared from the epicotyls of dark-grown pea(Pisum sativum L.) seedlings. The walls were found to bind externally-added⁴⁵Ca²⁺, with a binding constant of 4 ? 10^–4 mol dm^–3and a maximum capacity of 1.5 ? 10^–8 g-ions of Ca²⁺ perg fresh weight of epicotyl. The binding capacity decreased asthe pH of the medium was decreased below 6.0, suggesting thatthe calcium was bound by an anionic group with an apparent pKof 4.7. More than half the calcium binding was due to polygalacturonicacid in the wall, since up to 60% of the calcium binding capacitywas removed by pre-incubation of the cell walls with polygalacturonase(E.C.3.2.1.15). Only small decreases in calcium binding wereseen following pre-incubation with protease, nucleases, phospholipaseand hemicellulase. These results indicate that calcium willbe displaced from the cell wall at hydrogen ion concentrationswhich are known to occur in the wall during wall extension.They are consistent with a mechanism by which calcium inhibitswall extension by forming ionic bridges between polygalacturonicacid molecules, and also with the hypothesis that calcium andhydrogen ions exert opposing influences on cell wall extensionby competing for the same binding sites on the polygalacturonicacid. Key words: Pea epicotyl, Cell wall, Calcium, pH     

Inhibiting Effect of Auxin on Glucosamine Incorporation into Cell Walls of Rice Coleoptiles

Auxin induced growth and decreased the hexosamine content ofthe cell walls of rice coleoptile sections. Indole-3-aceticacid (IAA) at 10^–5 M inhibited the incorporation of ¹⁴C-glucosamineinto the cell walls. IAA did not affect the ¹⁴C-incorporationinto the cytoplasm, while inhibitors of glycoprotein synthesis,unicamycin and monensin, suppressed the incorporation into boththe cytoplasm and the cell walls. The radioactivity due to labeledglucosamine in the cell walls increased during the chase, butthis increase was inhibited by IAA. Among the cell wall fractions,the increase in radioactivity and its inhibition by IAA wereconspicuous in the hemicellulose I fraction. The inhibitoryeffect of IAA on glucosamine incorporation into the cell wallswas observed even in the presence of 0.15 M mannitol solutionwhich completely suppressed the IAA-induced growth. These resultssuggest that auxin induces growth at least partly by inhibitingthe transport of asparagine-linked glycoproteins from the cytoplasmto the cell walls. ¹ Present address: Department of Biology, Faculty of Science,Osaka City University, Sumiyoshi-ku, Osaka 558, Japan (Received July 23, 1986; Accepted December 22, 1986)     

Regulation of CO2-fixation in Chlorella by light of varied wavelengths and intensities

1) The wavelength effects on ¹⁴CO₂-fixation by Chlorella cellswere studied, using monochromatic light of different light intensities. 2) Blue light (453 mµ) stimulated the incorporation of¹⁴C into aspartate, glutamate and malate. Red light (679 mµ),on the other hand, stimulated its incorporation into P-esters,free sugars and insoluble material. 3) The blue light effect was observed in the presence of CMUat concentrations completely suppressing ordinary photosyntheticCO₂-fixation. 4) The blue light effect in the presence of CMU was inducedat very low intensities. At 453 mµ, 300 erg cm^–2sec^–1 was sufficient for complete saturation. 5) Time courses of ¹⁴C-incorporation into individual compoundswere investigated. Irrespective of the wavelength of the illuminatinglight, the first stable CO₂-fixation product formed under weaklight (400–500 erg cm^–2 sec^–1) was citrulline.At higher light intensities (4,000–7,000 erg cm^–2sec^–1), PGA was the first stable CO₂-fixation product.The incorporation of ¹⁴C into citrulline was not inhibited byCMU. 6) Experimental results indicate that both blue light-inducedincorporation of ¹⁴C into amino and organic acids and the incorporationof ¹⁴C into citrulline induced by low intensity light are operatedby a mechanism(s) independent of ordinary photosynthetic CO₂-fixation.Possible effects of light regulating the carbon metabolism inalgal cells are discussed. (Received July 24, 1969; )     

Effect of Brefeldin A on the synthesis and transport of cell wall polysaccharides and proteins in pea root seedlings

The in vivo effect of Brefeldin A (BFA) on the synthesis andtransport of cell wall polysaccharides and proteins in the rootsof pea seedlings (Pisum sativum L. cv. Alaska) was investigated.BFA (10µgml^—1 inhibited the synthesis of cell wallmatrix polysaccharides by approximately 43%. Under the sameconditions, cellulose synthesis was inhibited by approximately77%. The percentage of incorporation of L—[U—¹⁴C]leucineand L-[U-14C]proline into cytosolic, membrane and cell wallproteins was only slightly changed in the presence of BFA. Inaddition, the drug did not change the pattern of newly synthesizedproteins in the three fractions as judged by SDS—PAGEfluorography. Double labelling of proteins and cell wall polysaccharidesconfirmed the above reported data. All these results showedthat the synthesis and transport of proteins to the cell wallwas only slightly affected by BFA under similar conditions tothose which brought about a strong inhibition of the synthesisof matrix and cellulosic polysaccharides. BFA had no effecton the activity of membrane-bound and digitonin-solubilizedmannan and glucomannan synthase isolated from the third internodeof pea seedlings. This would exclude an effect of BFA at thelevel of the catalytic site of the synthases. The inhibitionof polysaccharide synthesis by the drug was rapidly eliminatedafter its removal. It is concluded that the effect of BFA onthe biosynthesis of cell wall polysaccharides could be causedby an interaction of the drug with the topological organizationof the synthase complexes in the membranes. This effect wouldprecede the action of the drug at the level of vesicle transportto the walls. Key words: Brefeldin A, cell wall polysaccharides (synthesis and transport), Pisum sativum L, polysaccharide synthases, proteins (synthesis and transport)     

Induction of Cell Division Synchrony and Variation of Cytokinin Contents through the Cell Cycle in Tobacco Cultured Cells

Cell division synchrony was induced in tobacco {Nicotiana tabacum)cultured cells by several treatments. Very high synchrony throughouttwo cell cycles was induced by aphidicolin treatment (inhibitorof DNA polymerase , 10 µg/ml) and by treatment with lowtemperature (4°C) and hydroxyurea (50 µg/ml). Themitotic index reached its maximum (52% and 40% in aphidicolinand hydroxyurea treatments, respectively) at 11 h after removalof the added chemical. During the treatments, the cells werearrested in the G₁/S phase of the cell cycle. In the aphidicolin-inducedsystem, incorporation of ¹⁴C-thymidine confirmed that DNA synthesiswas started immediately after removal of the chemical. The aphidicolin-induced synchronous cells were used to studythe contents of butanol-soluble cytokinins during the cell cycle.Cytokinin contents increased conspicuously at the G₂/M boundary. ¹Present address: Department of Biology, Otsuma Women's University,Chiyodaku, Tokyo 102, Japan. (Received May 14, 1985; Accepted November 8, 1985)     

Amino Acid-Sugar Linkages in Rice Coleoptile Cell Walls

The nature of amino acid-sugar linkages in cell walls was investigatedin a monocotyledonous tissue, rice coleoptiles. The molar ratiosof aspartic acid, threonine, and serine in cell walls were decreasedby hydrazinolysis in coleoptiles grown both on and under water.The molar ratios of threonine and serine were decreased alsoby a NaOHNaBH₄ treatment, while the alanine content was increased,and -aminobutyric acid was not formed. The cell walls were treated with NaOH in the presence of NaB³H₄,hydrolyzed, then divided into amino acid and sugar fractions.Two distinct radioactive peaks were detected in the thin-layerchromatography of the amino acid fractions. One was identifiedas alanine derived from glycosylated serine; the other was confirmedto be an oxidation product of glucosaminitol. There was justone ³H-labeled product in the sugar fractions, galactitol. Theseresults suggest the presence of serine-O-galactose and asparagine-N-N-acetylglucosamine linkages in rice coleoptile cell walls. The existence of glucosamine linked to amino acids was furthersupported by the incorporation of ¹⁴C-glucosamine into cellwalls. These linkages were also detected in the cell walls ofa dicotyledonous tissue, Vicia epicotyls. (Received April 2, 1981; Accepted June 24, 1981)     

Calcium Antagonist TMB-8 Inhibits Cell Wall Formation and Growth in Pea

The effects on auxin-stimulated growth and cell wall formationof 8-(N, N-diethylamino)-octyl-3, 4, 5-trimethoxybenzoate.HCI(TMB-8), an intracellular Ca²⁺ antagonist, were investigatedin abraded stem segments from aetiolated seedlings of Pisumsativum L. cv. Alaska. Incubation of segments at pH 6.0 with200 mmol m^–3 TMB-8 resulted in a 50% inhibition of auxin-stimulatedgrowth. Added Ca²⁺ did not restore normal auxin-stimulated growth,presumably because of its well-known stiffening effect on thecell wall. In segments incubated at a pH (7–2) which preventedelongation, auxin promoted the incorporation of [³H]glucoseinto the cell wall relative to total uptake of label. TMB-8abolished about 60% of the total incorporation of label intocell walls in the presence of auxin, but was not effective inthe absence of auxin. Exogenous CaCl₂ reversed the inhibitoryeffect of TMB-8 on relative cell wall incorporation in a parabolicmanner, with a 50% reversal at about 100 mmol m^–3 andcomplete reversal at 1.0 mol m^–3 Ca²⁺. Other ions tested(Mg²⁺, Mn²⁺, Cu²⁺, Zn²⁺) were without substantial effect atconcentrations of 0.5 mol m^–3. Both apparent uptake ofCa²⁺ and consequent reversal of TMB-8 inhibition of cell wallincorporation were blocked by the Ca²⁺ channel blockers verapamiland La³⁺. The data provide further evidence that auxin-stimulatedgrowth is dependent upon continued cell wall incorporation,and suggest that a Ca²⁺ messenger system may be involved inthe promotory actions of auxin on cell wall synthesis and long-termgrowth. Key words: Auxin, calcium, cell wall synthesis     

Pyrimidine Nucleotide Biosynthesis in Vinca rosea Cells: Changes in the Activity of the de novo and Salvage Pathways during Growth in a Suspension Culture

Marked changes in the activity of the ‘de novo’and ‘salvage’ pathways of pyrimidine biosynthesisduring growth of Vinca rosea cells in a batch suspension culturewere observed. The activity of these pathways was investigated by determiningthe contribution of ¹⁴C of [2-¹⁴Cluracil, 12-¹⁴Cluridine. and[6-¹⁴Clorotate to the cell constituents and by measuring theactivity of the several enzymes of these pathways. During the lag phase of the culture, ‘uracil-’ and‘uridine-salvage’ pathways made the predominantcontribution to nucleotide biosynthesis, but, following theinitiation of cell division, the ‘de novo’ pathwayfor nucleotide biosynthesis operated appreciably. These results suggest that nucleotide synthesis during cellgrowth in a suspension culture can be divided into two stages:a ‘turnover stage’, during the lag phase of cellgrowth, and a ‘true biosynthetic stage’, which isinitiated in the cell division phase.     

The Role of Glycollic Acid in the Photoassimilation of Acetate by Chlorella pyrenoidosa

The kinetics of ³H-acetate assimilation by Chlorella pyrenoidosain the light were examined. The primary products of assimilationwere glycollate and succinate. After 10 sec glycollate contained45 per cent and succinate 25 per cent of the tritium incorporatedby the cells. The percentage of the total tritium in glycollateand succinate fell with time while that in citrate increased.Initially the specific activities (µc of ³H per µmoleof acid) of succinate and glycollate were greater than citrate.When ³H-¹⁴C-2-acetate was added to the cells, total dpm for³H and ¹⁴C in glycollate rapidly reached a steady state andgave a ³H/¹⁴C ratio of 10, compared with a ³H/¹⁴C ratio of 4in the acetate. This ³H/¹⁴C ratio in glycollate is found because³H is derived from ³H-¹⁴C-2-acetate and because the ¹⁴C is dilutedwith cold carbon from elsewhere. The addition of ¹⁴CO₂ at thesame time as ³H-¹⁴C acetate decreased the ³H/¹⁴C ratio in glycollatebut incorporation of ¹⁴C from ¹⁴CO₂ into glycollate was slowerthan incorporation from ¹⁴C-2-acetate. Although ¹⁴C from acetaterapidly appeared in glycollate, ¹⁴C-labelled glyoxylate wasnot detected. The ³H/¹⁴C ratio observed in glycollate rulesout formation of glycollate from acetate via glycoaldehyde.The available evidence did not support glycollate formationvia the Calvin cycle. ¹⁴C from ¹⁴C-Z-acetate appeared in glycollatebefore it did in phosphoglyceric acid. Total dpm for ³H, ¹⁴C,and ³H/^l4C ratio in Calvin cycle intermediates were not in equilibriumwith glycollic acid.     

Utilization Modes of Inorganic Carbon for Photosynthesis in Various Species of Chlorella

Rates of CO₂ and HCC₃^– fixation in cells of various Chlorellaspecies in suspension were compared from the amounts of ¹⁴Cfixed during the 5 s after the injection of a solution containingonly ¹⁴CO₂ or H¹⁴CO₃^–. Results indicated that irrespectiveof the CO₂ concentration during growth, Chlorella vulgaris 11h and C. miniata mainly utilized CO₂, whereas C. vulgaris C-3,C. sp. K. and C. ellipsoidea took up HCO₃^– in additionto CO₂. Cells of C. pyrenoidosa that had been grown with 1.5%CO₂ (high-CO₂ cells) mainly utilized CO₂, whereas those grownwith air (low-CO₂ cells) utilized HCO₃^– in addition toCO₂. Cells that utilized HCO₃^– had carbonic anhydrase(CA) on their surfaces. The effects of Diamox and CA on the rates of CO₂ and HCO₃^–fixation are in accord with the inference that HCO₃^– wasutilized after conversion to CO₂ via the CA located on the cellsurface. CA was found in both the soluble and insoluble fractions;the CA on the cell surface was insoluble. Independent of the modes of utilization, the apparent K_m (NaHCO₃)for photosynthesis was much lower in low-CO₂ cells than in high-CO₂ones. The fact that the CA in the soluble fraction in C. vulgarisC-3 was closely correlated with the K_m(NaHCO₃) indicates thatsoluble CA lowers the K_m. ¹ Dedicated to the late Professor Joji Ashida, one of the foundersand first president of the Japanese Society of Plant Physiologists. ⁴ On leave from Research and Production Laboratory of Algology,Bulgarian Academy of Sciences, Sofia. (Received September 14, 1982; Accepted March 1, 1983)     

Enhancing effects of CO2 on chloroplast regeneration in glucose-bleached cells of Chlorella protothecoides II. Role of carbamylphosphate in chloroplast regeneration

Levels of the activities of glutamine-dependent carbamylphosphatesynthetase, ornithine-and aspartate-transcabamylase and phosphoenolpyruvatecarboxylase were followed in greening cells of Chlorella prolothecoides.Among the enzymes examined the activity of carbamylphosphatesynthetase was extremely low, especially at the early phaseof greening. Arginine (but not ornithine or aspartate), when administeredto algal cells at the 24th hour of greening, stimulated thesyntheses of RNA, protein and chlorophyll in the subsequentperiod. It also affected the metabolic pathway of the ¹⁴CO₂supplied simultaneously with arginine in the presence of CMU.Arginine produced a decreased incorporation of ¹⁴C into proteinand an increased incorporation into nucleic acid. The mechanismof the action of CO₂ on chloroplast regeneration is discussed.We concluded that chloroplast regeneration in glucose-bleachedcells is limited by the synthesis of carbamylphosphate, especiallyin the early phase of greening. (Received August 19, 1975; )     

Cell Cycle Timing of Replication of the Nuclear Gene Encoding Chloroplastic NADP-Specific Glutamate Dehydrogenase Isoenzymes in Chlorella sorokiniana

In synchronized Chlorella sorokiniana cells, the NH₄⁺ inducibleNADP-specific glutamate dehydrogenase enzyme (NADP-GDH) accumulatedin a linear manner throughout the first cell cycle. Early inthe following second cell cycle, an increase in its rate ofaccumulation occurred that was proportional to the increasein total cellular DNA in the previous cell cycle. In synchronizedbacterial cells, increases in rate of linear accumulation ofinducible enzymes coincide with the time of replication of theirstructural genes. To determine whether the rate change in NADPGDHaccumulation resulted from a delay in replication of its nuclearstructural gene (gdhN) in fully induced C. sorokiniana cells,the cell cycle timing of replication of this gene was comparedto that of another nuclear gene, nitrate reductase (nia), andof a chloroplast gene, ribulose bisphosphate carboxylase large-subunit(rbcL), in synchronized cells cultured in NH₄⁺ or NO₃^–(uninduced) medium. The gdhN and nia genes replicated withinthe period of nDNA synthesis and rbcL within the period of ctDNAsynthesis in cells growing in either nitrogen source. Therefore,the delayed rate change in enzyme accumulation results froma process that regulates expression of the gdhN gene after itsreplication. (Received July 16, 1994; Accepted November 28, 1994)     

Effect of Temperature on Starch Degradation in Chlorella vulgaris 11h Cells

Analysis of products formed in Chlorella vulgaris 11 h cellsduring photosynthesis in air containing 3,000 ppm ¹⁴CO₂ at varioustemperatures revealed that the level of ¹⁴C-starch was maximumaround 20–24?C and decreased with further rise in temperatureuntil 40?C, while ¹⁴C-sucrose greatly increased at temperaturesabove about 28?C. Elevating the temperature from 20 to 38?Cduring photosynthetic ¹⁴CO₂ fixation resulted in a remarkabledecrease in ¹⁴C in starch and a concomitant increase in ¹⁴Cin sucrose. This conversion of starch to sucrose when shiftingthe temperature from 20 to 38?C proceeded even in the dark.Hydrolysis of sucrose by rß-fructosidase showed that,irrespective of the experimental conditions, the radioactivitiesin sucrose were equally distributed between glucose and fructose.The enhancement of starch degradation with temperature risewas more remarkable than that of the activity of ribulose bisphosphatecarboxylase from the same cells. When Chlorella cells whichhad been preloaded with ¹⁴C-starch after photosynthesis for30 min at 20?C were incubated in the dark for an additional30 min at 20?C, ¹⁴C-starch was degraded by only about 4%. However,the values after 30-min dark incubation at 28, 32, 36 and 40?Cwere increased by about 10, 19, 36 and 50%, respectively. Duringthe temperature-dependent conversion of starch to sucrose, nosignificant amount of radioactivity accumulated in free glucoseand maltose. (Received October 27, 1981; Accepted January 9, 1982)     

Synthetic rates of chloroplast and cytoplasmic ribosomal ribonucleic acids during the cell cycle of Chlorella

By feeding radioisotopic precursors of RNA ([5-³H]uracil and[5-³H]uridine) to cells of Chlorella ellipsoidea at variousstages in the cell cycle effected by autotrophic synchronousculture, we examined synthetic rates of the chloroplast andthe cytoplasmic ribosomal ribonucleic acids (chl-rRNA and cyt-rRNA,respectively). The net incorporation of the precursors intochl-rRNA was higher than that into cyt-rRNA in the early stagesof the cell cycle, and vice versa in the late stages. The specificactivity of chl-rRNA was extremely high, and this phenomenonwas likely to be intrinsic to small cells at the start of thecell cycle under autotrophic conditions, namely, cell-cyclestagespecific. We conclude that algal cells grown autotrophicallysynthesize chl-rRNA at a distinctly higher rate than cyt-rRNAin the early stages of the cell cycle. (Received July 21, 1978; )     

Effects of disalicylidenepropandiamine and near far-red light on 14CO2-fixation in Chlorella cells

1) With Chlorella ellipsoidea cells, in the presence of 5x10^–6M DSPD, or in its absence, the amounts of ¹⁴CO₂ incorporatedin P-esters, serine-plus-glycine and alanine were larger underred light than under blue light, whereas blue light specificallyincreased ¹⁴CO₂-incorporation in aspartate, glutamate, malateand fumarate (blue light effect). The amount of total ¹⁴C fixedunder blue or red light was greatly decreased by the additionof DSPD. When the concentration of DSPD was raised to 5x10^–4M, practically no radioactivity was found, under blue or redlight, in aspartate, glutamate and fumarate. Radioactivity inalanine was greatly increased. Effects of higher concentrationof DSPD are explained as due to the inhibition of PEP carboxylaseactivity in Chlorella cells. 2) The percentage incorporation of ¹⁴C into aspartate and theother compounds mentioned above, under near infra-red illuminationwas significantly smaller than that under blue light and wasalmost equal to that under red light. These results along withthe effect of 5x10^–6 M DSPD, exclude the possibility thatcyclic photophosphorylation is involved in the "blue light effect"mechanism. (Received December 12, 1969; )