Characteristics of the Photosynthetic Metabolism of Carbon in Deuterated Chlorella ellipsoidea C-27

The photosynthetic metabolism of carbon in fully deuteratedcells of Chlorella ellipsoidea C-27 (D-Chlorella), obtainedby culture in medium prepared with 100 mol% D₂O, was characterizedby examining the activities of several enzymes and the levelsof metabolic regulators in a comparison with those of ordinarycells (H-Chlorella). The cellular content of starch in D-Chlorellawas more than twice that in H-Chlorella, whereas those of sucroseand glucose were significantly lower in D-Chlorella. Deuterationof Chlorella caused marked alterations in the activities ofenzymes involved in starch metabolism. There was a significantdecrease in the activity of phosphorylase, a catabolic enzyme,and a significant increase in the activity of starch synthase,an anabolic enzyme. These alterations are probably responsiblefor the increase in the amount of starch in cells. By contrast,no marked changes were observed in the activities of enzymesand the levels of metabolic inhibitors that are involved inthe synthesis of sucrose. It seems likely, therefore, that thedecrease in the amount of sucrose in D-Chlorella was causedmainly by a deficiency in sources of carbon in the cytoplasm,as a consequence of an increase in levels of starch in chloroplasts. (Received May 13, 1992; Accepted December 1, 1992)     

Enzymatic Inactivation of Extracellular Carbonic Anhydrase and its Effect on K1/2 (CO2) for Photosynthesis in Chlorella ellipsoidea C-27

The ratio of the extracellular to the intracellular activityof carbonic anhydrase (CA) in cells of Chlorella ellipsoideaC-27, adapted to low levels of CO₂ for 24 h (low-CO₂ cells),was about one to one. Treatment of intact cells with PronaseP inactivated about one-half of the extracellular CA activitywithout affecting photosynthetic activity. The CA activity incell homogenates and in cell-wall ghosts liberated during celldivision was completely inactivated by the same treatment. Pretreatmentwith Glycosidase mix, Chitosanase and Macerozyme enhanced theinactivation of the CA activity in intact cells. These resultssuggest that extracellular CA is evenly distributed throughoutthe whole cell-wall region. The apparent K_1/2 for dissolved inorganic carbon (DIC) in low-CO₂cells doubled when extracellular CA was inactivated by treatmentwith Pronase P, but the K_1/2 obtained was still one-half ofthat in high-CO₂ cells. Photosynthetic ¹⁴CO₂-fixation in low-CO₂cells was enhanced by acetazolamide, whereas H¹⁴CO₃^–-fixationwas suppressed. The results suggest that CO₂ is a dominant substrateutilized by cells and that HCO₃^– is utilized after conversionto CO₂. The present results show that both intracellular andextracellular CA contribute to the increase in affinity forDIC during photosynthesis in low-CO₂ cells of Chlorella ellipsoideaC-27. (Received May 7, 1990; Accepted July 18, 1990)     

Growth Delay and Intracellular Changes in Chlorella ellipsoidea C-27 as a Result of Deuteration

The effects of deuterium (D) on Chlorella ellipsoidea C-27 wereinvestigated. Cells grown in a medium prepared with deuteriumoxide (D₂O) showed pronounced delays in cell growth and division;the length of a cell cycle in medium with 100 mol% D₂O was morethan 5 times longer than that in medium prepared in H₂O Thedelay caused by D₂O was not overcome by either indoleaceticacid or kinetin. The biological and ultrastractural characteristicsof deuterated .Chlorella (D-Chlorella) cells were examined.The responses of D-Chlorella to cell wall-digesting enzymesdid not differ from those of normal (H-Chlorella) cells. D-Chlorellacells were enlarged, and cellular components, such as proteins,nucleic acids, lipids and ATP, were present in larger quantitiesthan those in H-cells. The chloroplast of D-Chlorella was enlarged,but the levels of component photosynthetic pigments were significantlyreduced. By contrast, mitochondria of D-Chlorella were smallerthan those of H-cells. These changes in levels of cellular componentsand in the sizes of organelles seem to be unique to deuteration. (Received May 13, 1992; Accepted July 28, 1992)     

Photosynthetic Apparatus Formation during the Cell Cycle of Chlorella

Synchronous cell division in cultures of Chlorella vulgaris Beijerinck was induced by intermittent illumination: 9 hours light, 6 hours darkness. The rate of photosynthetic O₂ evolution per cell increases 4-fold in a one-step manner at the beginning of the light period, to the same extent as the increase in cell number. Over the division cycle, the following accumulation times during the light period were found: chlorophyll a, between 2 and 8 hours, chlorophyll b, between 5 and 8 hours, reaction centers of photosystems I and II, between 2 and 6 hours; and cytochrome f, between 2.5 and 5 hours. Cytochrome f accumulation is closely followed by an increase in amplitude of the rapid phase in light-induced absorption increase at 520 nanometers and in intensity of the delayed light emission. Enhancement of the delayed fluorescence yield per flash under continuous illumination (caused by the establishment of the pH difference across the thylakoid membrane) is maximal by the first hour of the light period.     

土壤干旱对水稻叶片光合速率和碳酸酐酶活性的影响

以适应性广的水稻品种“汕优 6 3”为材料 ,具有较高的光合速率但适应性较差的品系“NAU30 3”为对照 ,比较了不同阶段的叶片光合机构对于干旱处理的适应性。结果表明“汕优 6 3”具有较广泛的适应性 ,在逆境条件下仍能维持较高的光合速率 ,这可能和叶片中碳酸酐酶活性对逆境的响应程度高有关。这种对干旱逆境的适应性反应只在叶片光合功能的可逆衰退阶段发生     

Detection and first characterization of a cell-wall lytic activity in Chlorella ellipsoidea C-27

Cell wall lytic activity was detected in Chlorella ellipsoidea Gernick IAM C-27 using the [¹⁴C]-labeled cell wall as substrate. The highest activity was obtained at pH 8. and the solubilized product was a polysaccharide of high molecular weight. The lytic activity appeared to be a protease and did not hydrolyse glycosidic bonds of the cell wall polysaccharides. The activity probably solubilizes the cell wall by cleaving the peptide bonds that interconnect the polysaccharides of the cell wall.     

Correlation between the Starch Level and the Rate of Starch Synthesis during the Developmental Cycle of Chlorella ellipsoidea

The starch content as well as the rate of photosynthetic starchformation in Chlorella ellipsoidea was studied throughout thecell cycle. The starch level in Chlorella cells rose markedlyduring the growing phase in the light, but it started to decreaseafter about 14 to 16 hr regardless of illumination. The rateof starch synthesis, measured by the level of ¹⁴C-incorporationinto starch, increased rapidly in the growing phase until 10hr, and decreased promptly thereafter, even in the light. From these results, it was concluded that both the cellularlevel of starch and the rate of starch synthesis were a functionnot only of the light regime, but also of the stage of celldevelopment. ³ Present address: Yamada High School, Yamada-machi, Iwate Pref.028-13, Japan. (Received October 12, 1981; Accepted May 12, 1982)     

Intracellular Carbonic Anhydrase Activity Sensitizes Cancer Cell pH Signaling to Dynamic Changes in CO2 Partial Pressure

Carbonic anhydrase (CA) enzymes catalyze the chemical equilibration among CO₂, HCO₃⁻ and H⁺. Intracellular CA (CA_i) isoforms are present in certain types of cancer, and growing evidence suggests that low levels correlate with disease severity. However, their physiological role remains unclear. Cancer cell CA_i activity, measured as cytoplasmic CO₂ hydration rate (k_f), ranged from high in colorectal HCT116 (∼2 s⁻¹), bladder RT112 and colorectal HT29, moderate in fibrosarcoma HT1080 to negligible (i.e. spontaneous k_f = 0.18 s⁻¹) in cervical HeLa and breast MDA-MB-468 cells. CA_i activity in cells correlated with CAII immunoreactivity and enzymatic activity in membrane-free lysates, suggesting that soluble CAII is an important intracellular isoform. CA_i catalysis was not obligatory for supporting acid extrusion by H⁺ efflux or HCO₃⁻ influx, nor for maintaining intracellular pH (pH_i) uniformity. However, in the absence of CA_i activity, acid loading from a highly alkaline pH_i was rate-limited by HCO₃⁻ supply from spontaneous CO₂ hydration. In solid tumors, time-dependence of blood flow can result in fluctuations of CO₂ partial pressure (pCO₂) that disturb cytoplasmic CO₂-HCO₃⁻-H⁺ equilibrium. In cancer cells with high CA_i activity, extracellular pCO₂ fluctuations evoked faster and larger pH_i oscillations. Functionally, these resulted in larger pH-dependent intracellular [Ca²⁺] oscillations and stronger inhibition of the mTORC1 pathway reported by S6 kinase phosphorylation. In contrast, the pH_i of cells with low CA_i activity was less responsive to pCO₂ fluctuations. Such low pass filtering would “buffer” cancer cell pH_i from non-steady-state extracellular pCO₂. Thus, CA_i activity determines the coupling between pCO₂ (a function of tumor perfusion) and pH_i (a potent modulator of cancer cell physiology).     

Factors Controlling Induction of Carbonic Anhydrase in Chlorella vulgaris 11h: Effects of CO2 and O2

Increase of carbonic anhydrase activity was enhanced by decreasingthe O₂ concentration when Chlorella vulgaris 11h cells grownunder 3% CO₂2 in ordinary air were transferred to low CO₂ conditions.The carbonic anhydrase activity finally attained under the steadystate was dependent on the CO₂ concentration, irrespective ofthe O₂ concentration used. (Received April 24, 1988; Accepted February 23, 1988)     

Studies on frost hardiness in Chlorella ellipsoidea III. Changes in O2 uptake and evolution during hardening and after freeze-thawing

Chlorella ellipsoidea cells at an intermediate stage in theripening phase of the cell cycle were hardened at 3?C. Oligomycin(OGM) and 3-(3,4-dichiorophenyl)-1,1-dimethylurea (DCMU) addedduring hardening in the light inhibited the development of frosthardiness, suggesting that mitochondria and chloroplasts wereinvolved in the hardening process. The O₂-uptake activity in unhardened cells increased duringhardening in the light while the O²-evolution activity decreased,when these activities were measured at 25?C. The increase inO₂ uptake was suppressed by OGM and DCMU and the decrease inO₂ evolution was stimulated by OGM. While the algal hardinessin the dark was very limited, the addition of glucose duringhardening in the dark caused a remarkable development of frosthardiness. These results suggest that mitochondria and chloroplastsclosely interact at low temperature, and the former plays aprincipal role in the hardening process and the latter servesas substrate-donor in the light. The O₂ evolution in cells which survived freezing was remarkablydecreased by freeze-thawing while the O₂ uptake was hardly affected.The freeze-injured chloroplasts were repaired during the followingincubation. OGM inhibited the repair of freeze-injured chloroplasts.From the results, mitochondria seem to change their membranesinto a structure hardier than chloroplasts, and ATP synthesizedby mitochondria seems to be essential for the repair of freeze-injuredchloroplasts. ¹ Present address: Department of Public Health, Faculty of Medicine,Kyushu University, Maidashi 3-1-1, Higashiku, Fukuoka 812, Japan. (Received November 9, 1977; )     

Alkalinity-induced Aggregation in Chlorella vulgaris II. Changes in the Cell Wall during the Cell Cycle

In a Chlorella culture growing synchronously at pH 6.3 undera 12 hr light-12 hr dark regime, autospores are released duringthe dark period of the cycle by mechanical rupture of the cellwall. When the culture is made in an alkaline medium (pH 9.5)during the first 8 hr of the light period, the release of autosporesis prevented, and at the end of the cycle, the cells are enlarged.It was found that the diurnal fluctuations in cellulase specificactivity and in polysaccharide content, which normally takeplace at pH 6.3, do not occur at alkaline pH. The concentrationof polysaccharides, as measured by the periodic acid-Schiffreagent, and cellulase activity are almost doubled. Peak levelsare reached at the 8th hour of the cell cycle rather than theusual 4th hour and persist during the second part of the cellcycle, whereas they normally decrease. It is suggested thatunder alkaline conditions, the diurnal increase in cellulaseactivity is amplified and prolonged, resulting in increasedflexibility of the mother cell wall. As a result, the cell wallis stretched, rather than ruptured, during the development ofdaughter autospores, which are thus prevented from being released. ¹ Present address: Research & Development Authority, Ben-GurionUniversity of the Negev, P.O. Box 1025, Beer-Sheva 84110, Israel. (Received June 16, 1981; Accepted October 22, 1981)     

Photosynthetic Characteristics and Carbonic Anhydrase Activity in Cells Cultured Photoautotrophically and Mixotrophically and Cells Isolated from Leaves

Cultured green cells of Nicotiana tabacum var. Samsun, Cytisusscoparius Link and Hyoscyamus niger which were grown photoautotrophicallyunder a stream of air enriched with 1% CO₂ or mixotrophicallyin the presence of 3% sucrose and ordinary air showed very lowcarbonic anhydrase activity, which was only 0–9% of thatin the respective intact leaves. The CO₂ compensation pointfor photosynthesis of autotrophically and mixotrophically culturedgreen cells of tobacco was higher than 0.3 mM NaHCO₃ at pH 7.8,but that of the cells isolated from tobacco leaves was lowerthan 0.1 mM NaHCO₃ at pH 7.8. The fact that the cultured cellscannot grow photoautotrophically under ordinary air is due toa high CO₂ compensation point in photosynthesis. The dark respiratoryactivity in both photoautotrophically and mixotrophically culturedtobacco cells was more than 7-fold that in the cells isolatedfrom tobacco leaves. We therefore could not conclude whetherthe high CO₂ compensation point in the cultured cells is dueto the low carbonic anhydrase activity or simply reflects thehigh respiratory activity. (Received July 10, 1980; Accepted November 25, 1980)     

Identification of Distinct Internal and External Isozymes of Carbonic Anhydrase in Chlorella saccharophila

External carbonic anhydrase (CA) was detected in whole cells of alkaline-grown Chlorella saccharophila but was suppressed by growth at acid pH or growth on elevated levels of CO2. Internal CA activity was measured potentiometrically as an increase in activity in cell extracts over that of intact cells. Cells grown under all conditions had equal levels of internal CA activity. Two isozymes were identified after electrophoretic separation of soluble proteins on cellulose acetate plates. The fast isozyme was found in cells grown under all conditions, whereas the slow isozyme was found only in cells grown at alkaline pH. Western blot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis using antibodies produced against the periplasmic form of CA from Chlamydomonas reinhardtii revealed a single band at 39 kD, which did not change in intensity between growth conditions and was associated only with proteins eluted from the fast band. The slow isozyme was inactivated by incubation of cell extract at 30[deg]C and by incubation in 10 mM dithiothreitol, whereas the internal form was unaffected. These results indicate that external and internal forms of CA differ in structure and their activities respond differently to environmental conditions.     

Effects of Temperature and CO2 Concentration on Induction of Carbonic Anhydrase and Changes in Efficiency of Photosynthesis in Chlorella vulgaris 11h

The increase in carbonic anhydrase (CA) activity and the decreasein apparent K_m(CO₂) for photosynthesis induced by reducing CO₂concentration during the growth of Chlorella vulgaris 11h cellswere followed under different temperatures. Both changes wereaccelerated by raising the temperature and reached an optimumat 32–37?C. When the CO₂ concentration was lowered from3 to 0.04%, the rate of photosynthetic O₂ evolution at limitingCO₂ concentrations increased and reached a stationary levelafter 3 h. Under such conditions, the concentration of CO₂ dissolvedin the algal suspension decreased logarithmically (t_1/2=10 min)and reached a concentration in equilibrium with 0.04% CO₂ inair after ca. 2 h. When high-CO₂ cells grown with 3% CO₂ in air were transferredto various lower CO₂ concentrations, CA activity and apparentK_m(CO₂) for photosynthesis changed depending on the CO₂ concentration.The CO₂ concentration which gives one-half the maximum valuefor K_m(CO₂) and one-half minimum value foi CA activities wasabout 0.5%. The inverse relationship observed for the changesin CA activity and the affinity for CO₂ in photosynthesis supportsthe theory that CA loweres the apparent K_m(CO₂) for photosynthesisin Chlorella vulgaris 11h. (Received August 27, 1984; Accepted February 8, 1985)     

Carbonic Anhydrase Activity and CO2-Transfer Resistance in Zn-Deficient Rice Leaves

It has been reported that carbonic anhydrase (CA) activity in plant leaves is decreased by Zn deficiency. We examined the effects of Zn deficiency on the activity of CA and on photosynthesis by leaves in rice plants (Oryza sativa L.). Zn deficiency increased the transfer resistance from the stomatal cavity to the site of CO₂ fixation 2.3-fold and, consequently, the value of the transfer resistance relative to the total resistance in the CO₂-assimilation process increased from 10% to 21%. This change led to a reduced CO₂ concentration at the site of CO₂ fixation, resulting in an increased gradient of CO₂ between the stomatal cavity and this site. The present findings support the hypothesis that CA functions to facilitate the supply of CO₂ from the stomatal cavity to the site of CO₂ fixation. We also showed that the level of mRNA for CA decreased to 13% of the control level during Zn deficiency. This decrease resembled the decrease in CA activity, suggesting the possible involvement of the CA mRNA level in the regulation of CA activity.     

植物叶片和角果的碳酸酐酶与光合速率的关系研究

以3种植物为材料,对其叶片、角果的碳酸酐酶活性日变化和相应的净光合速率日变化以及不同叶位的叶片和不同长度角果的CA活性和净光合速率关系进行研究.结果表明,在日变化中,油菜和诸葛菜的CA活性与其净光合速率之间存在显著性正相关,羽衣甘蓝则不明显.但3种植物不同叶位的叶片和不同长度角果的CA活性与净光合速率之间都呈显著正相关,说明无论是植物的营养器官叶片,还是生殖器官角果,CA活性的高低对净光合速率都有较大的影响.     

Photosynthetic Activity during the Division Cycle in Synchronized Euglena gracilis

Axenic populations of the photosynthetic protozoan Euglena gracilis, grown with autotrophic nutrition, were synchronized with respect to cell division by culture on an alternating light-dark cycle. No cell divisions occurred in the light periods; approximately 100% of the cells divided in the dark periods. In such cultures, the synthesis of photosynthetic pigments and accumulation of polysaccharide were confined to the light periods. The capacity for photosynthesis, however, increased continuously over the entire light-dark cycle, and is thus not directly correlated with pigment content. A correlation was seen between photosynthetic capacity and protein content, suggesting that enzymatic mechanisms of the photosynthetic apparatus might be the limiting factor. Estimates of total photosynthetic activity indicate that about 5 x 10(-6) calories are required for the synthesis of a new cell.     

Purification and properties of a lytic enzyme from the cell wall of Chlorella ellipsoidea C-87

Cell wall lytic activity was detected in the culture medium and cell wall of 1AM Chlorella ellipsoidea C-87. The enzymes of both fractions had their highest activity at pH 5. The lytic activity bound to the cell wall consisted of a polysaccharide releasing enzyme, an exo-type enzyme releasing disaccharide, and glucosidase; but only the polysaccharide releasing enzyme was solubilized by lithium chloride. A polysaccharide releasing enzyme with a molecular weight around 40 kDa was isolated from the culture medium. Hemicellulose is degraded by the polysaccharide releasing enzyme, and the rigid wall by the exo-type enzyme.     

Factors Controlling Induction of Carbonic Anhydrase and Efficiency of Photosynthesis in Chlorella vulgaris llh Cells

When Chlorella vulgaris llh cells which had been grown in airenriched with 2–4% CO₂ (high-CO₂ cells) were bubbled withair containing ca. 400 ppm CO₂, illumination at an intensityas low as the light compensation point (350 lux) was sufficientto increase the photosynthetic rate under limiting CO₂ concentrations.The same treatment induced carbonic anhydrase (CA) activity.The induction of CA activity and increase in photosyntheticrate at limiting CO₂ concentrations were observed in the presenceof 10 µM DCMU which completely inhibits photosynthesis.These results indicate that photosynthetic electron transportis not involved in CA induction in Chlorella vulgaris llh cells.The parallelism between the changes in CA activity and the rateof photosynthesis under limiting CO₂ concentrations agree withthe previous conclusion that the transport of CO₂ from outsideto the site of CO₂ fixation is facilitated by CA and hence lowersthe apparent K_m(CO₂) for photosynthesis. (Received December 24, 1982; Accepted May 10, 1983)