Isolation of Mesophyll Cells from Mulberry Leaves for Photosynthetic Studies

A procedure was developed for isolating mesophyll cells frommulberry leaves that have consistently high photosynthetic activity.To obtain photosynthetically active cells from some plants,their petioles, immersed in water, had to be kept in the darkfor 3–4 days prior to cell isolation. (Received May 6, 1983; Accepted August 15, 1983)     

Effects of growth temperature on photosynthetic carbon metabolism in green plants II. Photsoynthetic 14CO2-incorporation in plants acclimatized to varied temperatures

During photosynthetic ¹⁴CO₂-fixation, leaves of plants suchas wheat, the broad bean and spinach, which had been acclimatizedto high temperature (20–25?C), incorporated much moreradioactivity into sucrose, and less into glycine and serinein comparison with similar plants grown in the cold (mean temperature,5–7?C). Radioactivities incorporated into glycine and serine greatlydescreased on the addition of -hydroxyethylsulfonate or on theremoval of oxygen from the atmosphere, indicating that thesecompounds are synthesized through the glycolate pathway. In leaves of wheat grown under low temperatures, relativelyhigh radioactivity was detected in ribulose 1,5-diphosphateamong the photosynthetic ¹⁴CO₂-fixation products, whereas practicallyno radioactivity was detected in this compound in leaves ofwheat which had been acclimatized to high temperatures. We assumedthat the carboxylation reaction of ribulose 1,5-diphosphateis suppressed in plants acclimatized to low temperatures. It was further inferred that the C-2 and C-2 moiety of ribulose1,5-diphosphate accumulating as a result of suppression of carboxylationis converted to glycine and serine through the glycolate pathway. The possibility was also discussed that during photosyntheticCO₂-fixation in wheat leaves at least a part of the C₆-compoundformed by the carboxylation of ribulose 1,5-diphosphate is directlyconverted to sugar phosphate. ¹Part of this investigation was reported at the 2nd InternationalCongress on Photosynthesis Research at Stresa, Italy, June 1971.This paper is based on a dissertation submitted by S.S. to theFaculty of Science, the University of Tokyo, in partial fulfilmentof the requirements for a Ph.D. degree. ²Present address: Department of Botany, Faculty of Science,University of Tokyo, Tokyo, Japan. (Received July 20, 1973; )     

Effects of growth temperature on photosynthetic carbon metabolism in green plants III. Differences in structure, photosynthetic activities and activities of ribulose diphosphate carboxylase and glycolate oxidase in leaves of wheat grown under varied temperatures

The rate of ferricyanide photoreduction in broken chloroplastsisolated from leaves of wheat acclimatized to a low temperature(mean temperature, 5–7?C) was similar to that in chloroplastsfrom wheat acclimatized to a high temperature (20–25?C). There was no practical difference in glycolate oxidase activityin leaf extracts of wheat plants grown at low and high temperatures.In contrast, the ribulose diphosphate carboxylase activity ofchloroplasts from low temperature sample was less than halfthat for the high temperature sample. Chloroplasts having a high rate of photosynthetic CO₂-fixationwere obtained from wheat acclimatized to a low temperature,whereas the CO₂-fixation activity in chloroplasts isolated fromhigh temperature-acclimatized wheat was very low. Electron microscopy revealed that chloroplasts in high temperature-acclimatizedwheat were ellipsoidal, electron dense and contained starchgranules. Those in low temperature-acclimatized leaves wereround and did not contain starch granule. ²Present address: Department of Botany, Faculty of Science,University of Tokyo, Tokyo, Japan (Received August 7, 1973; )     

Photosynthetic metabolism of 14CO2 in the process of the "glucose-bleaching" of Chlorella protothecoides

Changes in photosynthetic carbon metabolism during the glucosebleaching of Chlorella protothecoides cells were investigatedusing NaH¹⁴CO₃ as tracer. Several hours after incubating thegreen algal cells in the glucose medium in the dark, the ratesof ¹⁴C-incorporation into glucose polymers and sucrose decreasedand the incorporation into the lipid fraction (fatty acids)greatly increased. At this stage, the rate of photosynthetic¹⁴CO₂ fixation and the chlorophyll content were practicallythe same as in the starting green cells. Afterwards, the photosyntheticcapacity and chlorophyll content continued to decrease throughoutthe experimental period. In contrast, when photosynthetic ¹⁴CO₂fixation of green cells was carried out in the medium containingglucose, the rate of ¹⁴C-incorporation into glucose polymersincreased, though there was no change in the incorporationsinto sucrose and the lipid fraction. ¹Part of this investigation was reported at the Conference "ComparativeBiochemistry and Biophysics of Photosynthesis" (Japan-U.S. CooperativeScience Program) held at Hakone, Japan in 1967. ²Present address: Faculty of Agriculture, Tamagawa University,Machida-shi, Tokyo, Japan. (Received June 10, 1974; )     

Localization of Carbonic Anhydrase in Mesophyll Cells of Terrestrial C3 Plants in Relation to CO2 Assimilation

The activity and intracellular compartmentation of carbonicanhydrase was examined in mesophyll protoplasts of several C₃terrestrial species including wheat, since this enzyme may facilitatediffusion of inorganic carbon in solution by converting CO₂to bicarbonate. Carbonic anhydrase was located in the mesophyllchloroplast with little or no activity in the cytosolic fraction.In wheat, carbonic anhydrase was absent in etiolated leavesand increased in the light during greening. Thus the enzymemay have a role in photosynthesis in the chloroplast but notin the cytosol of mesophyll cells of higher C₃ plants. The amount of CO₂ required for half maximum rates of photosynthesis(under low O₂) was about two-fold higher for isolated protoplaststhan with isolated chloroplasts of wheat. The form of inorganiccarbon taken up by protoplasts, like that of chloroplasts, isCO₂. The results are discussed in relation to a possible resistanceto CO₂ transfer in the cytosol of mesophyll cells. (Received February 25, 1985; Accepted May 7, 1985)     

Effect of solar near ultraviolet radiation on growth of Chlorella cells

Cells of Chlorella regularis (Artari) Oltmanns (S-50) were grown under solar radiation in Tokyo, using a newly constructed outdoor culture system. The maximum specific growth rate (log₂ unit h⁻¹) was about 0.3, which was a little lower than the highest value reported using artificial light in the laboratory. The near ultraviolet light, consisting mostly of UV-A, inhibited the growth from 10 to 40% in summer mornings; the inhibition was not significant in the afternoon. The percentage inhibition was correlated with the total dose of the ultraviolet radiation.     

THE EFFECT OF SODIUM CHLORIDE ON CARBONIC ANHYDRASE ACTIVITY IN MARINE MICROALGAE1

The effects of NaCl on the internal and external carbonic anhydrase (CA) activity of several marine microalgae were studied. Unlike freshwater microalgae in which CA activity is generally inhibited by NaCl, marine microalgae exhibited considerable species-dependent variation when exposed to NaCl. CA activity in Phaeodactylum tricornutum, a diatom, was inhibited, whereas it was activated in the coccolithophorid Pleurochrysis carterae. CA activity in the chlorophyte Dunaliella primolecta was not significantly affected by NaCl. In Dunaliella salina and Dunaliella parva, NaCl inhibited external CA without affecting the internal activity, whereas in Chlorella vulgaris C-133 and Dunaliella peircei only the internal CA was inhibited. Internal CA of Dunaliella tertiolecta was not affected by NaCl, but the external enzyme was significantly enhanced. Salt substitution experiments revealed that chloride (Cl^-) is the ion affecting CA activity; the effect of Cl^- can be replaced by bromide ion. Cl^- affects external CA activity while also affecting the apparent affinity for inorganic carbon during photosynthesis. Microalgae whose internal CA activity was enhanced by Cl^- showed higher intracellular Cl^- concentrations than those species that were inhibited.     

Changes in Photosynthetic Characteristics Induced by Transferring Air-Grown Cells of Chlorococcum littorale to High-CO2 Conditions

When air-grown cells of Chlorococcum littorale was enrichedwith CO₂, growth was enhanced after a lag period of one to twodays at 20% CO₂, and 3 to 6 days at 40% CO₂. Changes in therate of photosynthesis measured as oxygen evolution and CO₂fixation, were similar to those observed for growth. Duringthe initial inhibition of photosynthesis in 40% CO₂, the activityof PSII was suppressed. In contrast, PSI activity was greatlyenhanced. Air-grown cells of C. littorale possessed comparatively highcarbonic anhydrase (CA) activity which was localized insidethe cells and on the cell surface. Under high CO₂ concentrationsextracellular CA activity was greatly suppressed and intracellularactivity almost completely abolished. Phosphoenol pyruvate carboxylaseactivity was also suppressed in high CO₂-grown cells. Ribulose-l,5-bisphosphatecarboxylase activity was higher in high-CO₂ grown cells thanin air-grown cells. The above results indicated that the lagphase induced by 40% CO₂ was due to suppression of PSII activity. ¹Part of this work was reported in the International PhotosynthesisCongress, Nagoya, 1992.     

Carbonic Anhydrase of a Unicellular Red Alga Porphyridium cruentum R-l. I. Purification and Properties of the Enzyme

Cells of Porphyridium cruentum R-l, a unicellular red alga,grown under ordinary air (0.04% CO₂) showed much higher activityof carbonic anhydrase (CA) than those grown under CCvenrichedair (2% CO₂). CA activity was not detected in a suspension ofintact cells, and was detectable only after the cells had beenhomogenized, indicating that this enzyme was localized onlywithin the algal cells. After partial purification of Porphyridium CA, its mol wt wasestimated as 59 kDa by SDS-PAGE and 55 kDa by gelfiltration.This suggests that the native enzyme is a monomer. Its activitywas not affected by benzensulfonamides, potent inhibitors ofCAs isolated from Chlamydomonas and other organisms. Chloride(or bromide) ions was essential for CA activity. CA activitymarkedly decreased when the cell extract had been incubatedat pH lower than 7 before assay. Upon readjusting the pH ofthe preincubation medium to 9 or higher, the enzyme activitywas restored, indicating that the inactivation is reversible. (Received April 17, 1987; Accepted July 21, 1987)     

Accumulation of Inorganic Carbon in the Cells of Euglena gracilis Z Grown Photoautotrophically in Ordinary Air

Affinity for inorganic carbon in photosynthesis of Euglena gracilisZ was higher in the cells grown in ordinary air than in thosegrown in 4% CO₂. The cells grown in ordinary air accumulatedinorganic carbon in the cells to the level far in excess ofthat expected from passive diffusion due to pH gradient acrossthe cell surface membrane. (Received April 5, 1986; Accepted June 25, 1986)