JAMES V. MORONEY2, ROBERT K. TOGASAKI, H. DAVID HUSIC3, AND N. E. TOLBERT*. Department ofBiochemistry, Michigan State University, East Lansing, ...
Plant Physiol. (1987) 84, 757-761 0032-0889/87/84/0757/05/$O 1.00/0
Evidence That an Internal Carbonic Anhydrase Is Present in 5% C02-Grown and Air-Grown Chlamydomonas1 Received for publication October 3, 1986 and in revised form February 18, 1987
JAMES V. MORONEY2, ROBERT K. TOGASAKI, H. DAVID HUSIC3, AND N. E. TOLBERT* Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824 (J.V.M., H.D.H.,
N.E.T.), and Department ofBiology, Indiana University, Bloomington, Indiana 47401 (R.K.T.) When Chlamydomonas is grown with elevated concentrations of CO2 it does not exhibit a high affinity for external CQ. These cells have many of the photosynthetic characteristics ofC3 plants, which include a relatively high CO2 compensation point, production of glycolate, and an inhibition of CO2 fixation by 02. High C02-grown cells also have little, if any, carbonic anhydrase activity associated with the periplasmic space. Air-grown cells have much carbonic anhydrase activity in the periplasmic space, as well as some intracellular carbonic anhydrase activity (22). Although the amount of internal carbonic anhydrase activity is low, when the permeant carbonic anhydrase inhibitor, EZ, is present, CO2 fixation at low CO2 concentrations is inhibited (1, 12, 15, 17, 21). The amount of intracellular carbonic anhydrase activity in 5% C02-grown cells is also low, and it is unclear whether the internal carbonic anhydrase is present only in airadapted cells or present in both high and low C02-grown cells. When high C02-grown cells are placed in a low CO2 environment and illuminated, they adapt to the low CO2 conditions in about 3 to 5 h (5). This adaptation includes the biosynthesis of the carbonic anhydrase (4, 21, 24, 29) located in the periplasmic space of Chlamydomonas (10). This adaptation may also include the biosynthesis of other proteins that have not as yet been identified, some of which may be internal isozymes of carbonic anhydrase, as investigated in this report. Recently, several mutants of Chlamydomonas that require high concentrations of CO2 for photoautotrophic growth have been isolated (16, 22, 23). These strains grow poorly with air levels of CO2 but well with elevated concentrations of CO2. One of these mutants, ca-i, is thought to be defective in an internal When Chlamydomonas reinhardtii is grown photoautotroph- CA enzyme (22). A new strain, cia-3, isolated after UV mutaically with low levels of C02, it becomes very efficient in utilizing genesis of Chlamydomonas, which also carries a mutation at the external inorganic carbon for photosynthesis. Under these con- same genetic locus as ca-i (16), is used in this study. By comparditions, cells require only low concentrations of CO2 for growth ing the photosynthetic properties of cia-3 to those of wild-type (3), and CO2 fixation is not inhibited significantly by 02. Badger Chlamydomonas grown with either air or elevated levels of CO2 et al. (1) proposed that Chlamydomonas has the ability to raise and by investigating the effects of the CA inhibitor, EZ, on the the level of CO2 internally by a C.4 accumulation system. Accu- ability of air- or C02-grown cells to accumulate Ci and fix C02, mulation of Ci would increase CO2 fixation and decrease pho- it is concluded that internal CA is involved in CO2 fixation which torespiration (17, 19, 26). However, the mechanism of this Ci indirectly effects the Ci pool. Unlike other enzymes of the C, accumulating system, this internal CA is present in both low and uptake is unclear. high C02-grown, wild-type cells. 'Supported by NSF grant PCM 8005917 and by the McKnight MATERIALS AND METHODS Foundation to N. E. T. and NSF grant PCM 8318174 to R. K. T. Published as article 12070 of the Michigan Agricultural Experiment Strains and Cell Culture Conditions. Chlamydomonas reinStation. hardtii, strain 173 mt+, was used as the wild type. The mutant 2 Present address: Department of Botany, Louisiana State University, strain cia-3 was isolated after UV mutagenesis as previously Baton Rouge, LA 70803. described (5). Cells were grown in liquid culture on minimal 3Present address: Department of Chemistry, Lafayette College, Eas- medium (25), in the light (200.uE/m 2 s-'), and aerated with air ton, PA 18042. or air supplemented with 3 to 5% CO2. Cells were harvested as 4Abbreviations: C,, inorganic carbon (CO2 + HC03-); AZ, acetazol- previously described (14). Strain cia-3 was routinely grown in amide; EZ, ethoxyzolamide; CA, carbonic anhydrase; K0.5(C02), the CO2 minimal medium aerated with 3 to 5% CO2 in air. When airconcentration at which the rate of C02-dependent photosynthetic 02 adapted cia-3 cells were to be used, the culture was transferred evolution is half-maximal. to medium gassed with air alone for 12 to 24 h prior to the 757 ABSTRACT Inorganic carbon (Ci) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C reinhardtii that cannot grow with air levels of CO2. Both air-grown cells, that have a CO2 concentrating system, and 5% COrgrown cells that do not have this system, were used. When the external pH was 5.1 or 73, air-grown, wild-type cells accumulated inorganic carbon (C1) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% C02rgrown cells also accumulated some C,, although not as much as airgrown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO2 fixation by high C02-grown, wild-type cells at both pH 5.1 and 73. These observations imply that 5% CO2grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase.
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experiment. Although cia-3 could not grow on air levels of CO2, external CA was formed when the culture was switched to low CO2 conditions. Measurement of Photosynthetic 02 Evolution. Photosynthesis as C02-dependent 02 evolution was measured with a Rank Brothers 02 electrode (1, 15). Harvested cells were diluted from a concentrated suspension to 25 ug Chl-ml-' in the buffers indicated in the table and figure legends. Buffers were prepared
Plant Physiol. Vol. 84, 1987
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fresh daily and prior to the addition of cells, bubbled with N2 to reduce dissolved CO2 and O2. Cell suspensions were then illuminated with 700 ME/m2 s' of 400 to 700 nm light at 25°C in E E5051the O2 electrode chamber, until the endogenous CO2 and HCO3was consumed, as judged by the cessation of 02 evolution (
