Mats Björk

Role of carbonic anhydrase in photosynthesis and inorganic-carbon assimilation in the red alga Gracilaria tenuistipitata

The mechanism of inorganic-carbon (Ci) accumulation in the red seaweed Gracilaria tenuistipitata Zhang et Xia has been investigated. Extracellular and intracellular carbonic-anhydrase (CA) activities have been detected. Photosynthetic O2 evolution in thalli and protoplasts of G. tenuistipitata were higher at pH 6.5 than at pH 8.6, where HCO3−is the predominant form of Ci. Dextran-bound sulfonamide (DBS), a specific inhibitor of extracellular CA, reduced photosynthetic O2 evolution at pH 8.6 and did not have any effect at pH 6.5. After inhibition with DBS, O2 evolution was similar to the rate that could be supported by CO2 from spontaneous dehydration of HCO3−. The rate of photosynthetic alkalization of the surrounding medium by the algal thallus was dependent on the concentration of Ci and inhibited by DBS. We suggest that the general form of Ci that enters through the plasma membrane of G. tenuistipitata is CO2. Bicarbonate is utilized mainly by an indirect mechanism after dehydration to CO2, and this mechanism involves extracellular CA.

INDUCIBLE MECHANISMS FOR HCO3– UTILIZATION AND REPRESSION OF PHOTORESPIRATION IN PROTOPLASTS AND THALLI OF THREE SPECIES OF ULVA (CHLOROPHYTA)1

Thalli of Ulva reticulata Forskaal, Ulva rigida C. Ag., and Ulva pulchra Jaasund were incubated at different concentrations of dissolved CO2. Incubation at a high CO2 concentration resulted in decreased oxygen evolution rate and lower affinity for inorganic carbon at high pH conditions, i.e. the ability to use HCO3– as a carbon source was reduced. This effect was reversible, and plants regained this HCO3– uptake capacity when transferred to air concentrations of CO2. The phytosynthetic oxygen evolution rate of plants grown at high CO2 concentration was reduced by high O2 concentrations, whereas thalli and protoplasts from cultures grown at air concentration were not affected. This is interpreted as a deactivation of the carbon‐concentrating mechanism during conditions of high CO2 resulting in high photorespiration when plants are exposed to high O2 concentrations. Protoplasts were not affected by high O2 to the same extent and were not able to utilize HCO3– from the medium. The algae were able to grow at very low CO2 concentrations, but growth was suppressed when an inhibitor of external carbonic anhydrase was present. Assay of carbonic anhydrase activities showed that external and internal CA activities were lower in plants grown at a high CO2 concentration compared to plants grown at a low concentration of CO2. Possible mechanisms for HCO3– utilization in these Ulva species are discussed.

Inorganic-carbon assimilation in the green seaweed Ulva rigida C.Ag. (Chlorophyta)

Mechanisms of carbon assimilation were investigated in thalli and protoplasts of Ulva rigida by measuring HCO3−-dependent O2 evolution at pH 6.5 and 8.6. In thalli, dextran-bound azetazolamide (DBAZ), a specific inhibitor of extracellular carbonic anhydrase (CA), inhibited the rate of O2 evolution at pH 8.6 when HCO3−was the only available form of inorganic carbon (Ci) in the medium. At pH 6.5 when CO2 is accessible, DBAZ did not affect photosynthetic O2 evolution. Inhibition of total CA activity (extracellular and intracellular) by ethoxyzolamide (EZ) inhibited photosynthesis at pH 6.5 and 8.6. During illumination of thalli the medium was alkalized at a rate which increased with increasing light. This alkalization decreased during inhibition of extracellular CA by DBAZ. Protoplasts at pH 6.5 exhibited a higher rate of O2 evolution than in pH 8.6. Addition of CA to protoplasts at pH 8.6 increased the rate of O2 evolution, whereas EZ was inhibitory at both pH 6.5 and 8.6, and DBAZ did not affect photosynthesis at either pH. We suggest that both extracellular and intracellular CA are present and that Ulva rigida assimilates HCO3−by an indirect mechanism. A theoretical scheme for carbon utilization is suggested.

Protoplast isolation from Ulva rigida (Chlorophyta)

High numbers of protoplasts were isolated from wild and cultivated thalli of Ulva rigida. Optimal conditions for protoplast release were obtained with 1·5% Abalone Acetone Powder and 1·5% Cellulysin in 0·4 M mannitol. Treatment for 30 min with hypotonic (0·8 M) solution more than doubled the yield. Growth conditions prior to enzyme treatment also influenced the yield greatly. Protoplasts were photosynthetically active, and 70–90% of the protoplasts were estimated to be viable.

Factors affecting protoplast yield of the carrageenophyte Solieria filiformis (Gigartinales, Rhodophyta)

SummaryThe effect of age, pH of the culture medium, pre-treatment of tissues, enzymes sources and enzymatic adaptability of phycophages fed with a monospecific diet were analyzed on the protoplast yields of the red seaweed Solieria filiformis (Kützing) Gabrielson. New apices from fast growing plants showed the highest protoplast yields. The protoplast yield decreased when the pH of the culture medium increased from 6.0 to 9.0. Crude extracts from the abalone Haliotis coccinea canariensis Nordsieck, fed with Solieria filiformis thalli for three months in combination with cellulysin, released the highest number of viable cells and protoplasts. Yields ranged from 1.0 to 8.5 x 106 protoplasts per gram of fresh weight.

PHOTOSYNTHETIC PROPERTIES OF PROTOPLASTS, AS COMPARED WITH THALLI, OF ULVA FASCIATA (CHLOROPHYTA)1

Protoplasts were prepared from Ulva fasciata Delile, and their photosynthetic performance was measured and compared with that of thalli discs. These protoplasts maintained maximal rates of photosynthesis as high as those of thalli (up to 300 μmol O2·mg chlorophyll−1·h−1) for several hours after preparation and were therefore considered suitable for kinetic studies of inorganic carbon utilization. The photosynthetic K1/2(inorganic carbon) at pH 6.1 was 3.8 μM and increased to 67, 158, and 1410 μM at the pH values 7.0, 7.9, and 8.9, respectively. Compared with these protoplasts, thalli had a much lower affinity for CO2 but approximately the same affinity for HCO3−. Comparisons between rates of photosynthesis and the spontaneous dehydration of HCO3− (at 50 μM inorganic carbon) revealed that photosynthesis of both protoplasts (which lacked apparent activity of extracellular/surface‐bound carbonic anhydrase) and thalli (which were only 25% inhibited by the external carbonic anhydrase inhibitor acetazolamide) could not be supported by CO2 formation in the medium at the higher pH values, indicating HCO3− uptake. Since both protoplasts and thalli were sensitive to 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonate, we suggest that HCO3− transport was facilitated by the membrane‐located anion exchange protein recently reported to function in certain Ulva thalli. These findings suggest that the presence of a cell wall may constitute a diffusion barrier for CO2, but not for HCO3−, utilization under natural seawater conditions.