Rates of cellular uptake of CO 2 and HCO 3 Ϫ during steady-state photosynthesis were measured in the marine diatoms Thalassiosira weissflogii and Phaeodactylum tricornutum, acclimated to CO 2 partial pressures of 36, 180, 360, and 1,800 ppmv. In addition, in vivo activity of extracellular (eCA) and intracellular (iCA) carbonic anhydrase was determined in relation to CO 2 availability. Both species responded to diminishing CO 2 supply with an increase in eCA and iCA activity. In P. tricornutum, eCA activity was close to the detection limit at higher CO 2 concentrations. Simultaneous uptake of CO 2 and HCO 3 Ϫ was observed in both diatoms. At air-equilibrated CO 2 levels (360 ppmv), T. weissflogii took up CO 2 and HCO 3Ϫ at approximately the same rate, whereas CO 2 uptake exceeded HCO 3 Ϫ uptake by a factor of two in P. tricornutum. In both diatoms, CO 2 : HCO 3 Ϫ uptake ratios progressively decreased with decreasing CO 2 concentration, whereas substrate affinities of CO 2 and HCO 3 Ϫ uptake increased. Half-saturation concentrations were always Յ5 M CO 2 for CO 2 uptake and Ͻ700 M HCO 3 Ϫ for HCO 3 Ϫ uptake. Our results indicate the presence of highly efficient uptake systems for CO 2 and HCO 3 Ϫ in both diatoms at concentrations typically encountered in ocean surface waters and the ability to adjust uptake rates to a wide range of inorganic carbon supply.Primary production by marine phytoplankton takes place in an environment that is characterized by high and relatively constant HCO 3 Ϫ concentrations (ϳ2 mM) but low and variable concentrations of molecular dissolved CO 2 [CO 2,aq ] (ϳ5-25 M). Variation in [CO 2,aq ] of ocean surface waters is mainly caused by intense photosynthesis during phytoplankton blooms, differences in water temperature, or mixing with deep water of different CO 2 content. On longer timescales, rising CO 2 concentrations in the upper layers of the ocean are expected in response to the present increase in atmospheric CO 2 partial pressure (pCO 2 ; Houghton et al. 1996). Because these changes in [CO 2,aq ] are always accompanied by changes in pH, concentrations of HCO 3 Ϫ vary much less because of concomitant shifts in the relative proportions of the inorganic carbon (C i ) species.The response of phytoplankton growth to changes in CO 2 supply is largely determined by the mechanism of C i uptake. Several studies indicate that both CO 2 and HCO 3 Ϫ in the bulk seawater are utilized by marine eukaryotic microalgae (e.g., Colman and Rotatore 1995;Rotatore et al. 1995;Korb et al.
Mass-spectrometric disequilibrium analysis was applied to investigate CO 2 uptake and HCO 3 ؊ transport in cells and chloroplasts of the microalgae Dunaliella tertiolecta and Chlamydomonas reinhardtii, which were grown in air enriched with 5% (v/v) CO 2 (high-Ci cells) or in ambient air (low-Ci cells). High-and low-Ci cells of both species had the capacity to transport CO 2 and HCO 3 ؊ , with maximum rates being largely unaffected by the growth conditions. In high-and low-Ci cells of D. tertiolecta, HCO 3 ؊ was the dominant inorganic C species taken up, whereas HCO 3 ؊ and CO 2 were used at similar rates by C. reinhardtii. The apparent affinities of HCO 3 ؊ transport and CO 2 uptake increased 3-to 9-fold in both species upon acclimation to air. Photosynthetically active chloroplasts isolated from both species were able to transport CO 2 and HCO 3 ؊ . For chloroplasts from C. reinhardtii, the concentrations of HCO 3 ؊ and CO 2 required for half-maximal activity declined from 446 to 33 M and 6.8 to 0.6 M, respectively, after acclimation of the parent cells to air; the corresponding values for chloroplasts from D. tertiolecta decreased from 203 to 58 M and 5.8 to 0.5 M, respectively. These results indicate the presence of inducible highaffinity HCO 3 ؊ and CO 2 transporters at the chloroplast envelope membrane.
SummaryAs nitric oxide (NO) is a key messenger in many organisms, reliable techniques for the detection of NO are essential. Here, it is shown that a combination of membrane inlet mass spectrometry (MIMS) and restriction capillary inlet mass spectrometry (RIMS) allows for the fast, speci®c, and non-invasive online detection of NO that has been emitted from tissue cultures of diverse organisms, or from whole plants. As an advantage over other NO assays, MIMS/RIMS discriminates nitrogen isotopes and simultaneously measures NO and O 2 (and other gases) from the same sample. MIMS/RIMS technology may thus help to identify the source of gaseous NO in cells, and elucidate the relationship between primary gas metabolism and NO formation. Using RIMS, it is demonstrated that the novel fungicide F 500 j triggers NO production in plants.
Abstract. Mass-spectrometric measurements of 180 exchange from 13ClSO 2 were used to follow changes in the intracellular carbonic anhydrase (CA) activity of cells of Chlamydomonas reinhardtii Dang. wild type and the ca-I mutant during adaptation to air. With intact cells as well as with crude homogenates total intracellular CA activity in wild-type cells increased six to tenfold within 4 h after transferring cells from 5% CO2 (high inorganic carbon, CO to ambient air (air adapted). After that time the activity slowly declined to a level similar to that observed with cells which had been continuously grown in air (low-C~ grown). In the ca-1 mutant, total CA was induced to a similar extent during 4 h of adaptation; however, absolute activities were two to three times lower in ca-1 than in the wild type regardless of the CO 2 supply. When crude extracts from wild-type cells were separated into soluble and insoluble fractions, each fraction contained about half of the internal CA activity. Within 4 h of adaptation, both forms of CA activity were simultaneously enhanced by nine to tenfold, reaching levels similar to those found in low-Ci-grown cells. In contrast, in the ca-1 mutant the soluble CA activity was only enhanced by about eightfold while the level of insoluble CA was very low even in low-C~ cells. After isolation of intact chloroplasts from wild-type cells and further subfractionation, around 70--80% of total chloroplastic CA activity was found to be in Both chloroplastic CA activities were inducible within the first 4 h of adaptation to air, with each of them being eight to ten times higher than in high-Ci algae. After that time their activities were similar to the corresponding CA values in low-Ci-grown cells. In contrast, plastids from high-C i cells of the ca-1 mutant showed 40% less insoluble-CA activity compared to the wild type and this insoluble-CA activity was not increased at all by transferring algae to air. In addition, no soluble-CA activity was detected in chloroplasts from high-Ci and air-adapted ca-1 cells. These results indicate the presence of three intracellular CA activities in high-Ci air-adapted and low-C~ cells of the wild type and that two of them are associated with the chloroplasts. All three activities are completely induced within the first 4 h of adaptation to air in wild-type cells. In contrast, it was not possible to induce any of the chloroplastic CA activities in the ca-1 mutant. The possibility that the soluble chloroplastic CA represents a pyrenoid-located CA is discussed.
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