Measurement of internal pH in the coccolithophoreEmiliania huxleyi using 2?,7?-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein acetoxymethylester and digital imaging microscopy

Abstract

Internal pH (pHi) was determined inEmiliania huxleyi (Lohmann) using the probe 2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluoresceinacetoxymethylester (BCEF-AM) and digital imaging microscopy. The probe BCECF-AM was taken up and hydrolysed to the free acid by the cells. A linear relationship was established between pHi and the 490/450 fluorescence ratio of BCECF-AM over the pH range 6.0 to 8.0 using the ionophore nigericin. Two distinct pH domains were identified within the cell, the cytoplasmic domain (approx. pH 7.0) and the chloroplast domain (approx. pH 8.0). The average pHi was 7.29 (±0.11) for cells in the presence of 2 mM HCO 3 (-) . In the absence of HCO 3 (-) the pHi was decreased by 0.8 pH unit. The importance of these changes in pHi is considered in relation to inorganic-carbon uptake.

Carbon dioxide availability, intracellular pH and growth rate of the coccolithophore Emiliania huxleyi

“…Points represent the average of 3 replicate readmgs surement of the spatial and temporal variation in pH of a single cell and the rapid change in pH, in response to external conditions. Cytoplasmic pH in the high-calcifying cells of Emiliania huxleyi (Table 1) is more acidic than that reported for other plant cells (Smith 1979, Raven 1980 and other non-calcifying marine microalgae (Raven & Smith 1980, Burns & Beardall 1987 but is similar to values found for the low-calcifying cells (Dixon et al 1989) with a pH, near neutrality. This suggests that the optimum pH, for cellular metabolic pathways in E. huxleyi may be different from some other algae.…”

“…Measurement of pH, using 2'7'-bis(2-carboxyethyl)-5 (and -6) carboxyfluorescein acetomethylester (BCECF-AM): This was as described previously (Dixon et al 1989). The final intracellular concentration of BCECF-AM was 5.0 pM.…”

“…In cells growing at external pH 7.0 the pH, was 6.19 in mid growth phase, declined to 6.07 by late growth phase and was maintained at this value throughout the stationary phase. At external pH 7.8, pHi was 6.52 at mid growth phase, declined to 6.41 by late growth phase and to 6.32 by the end of Growth media lacking H C 0 i "Intracellular pH monitored as the average (* SE) 4801450 excitation ratio of several (10) E. huxleyi cells loaded with the ratiometric pH indicator BCECF (Dixon et al 1989). Cells were immobilized on 0.01 % poly-L-lysine coated coverslips bPoints represent the average (* SE) of 3 replicate readings Points represent the average of 3 replicate readings stationary phase.…”

“…The pH, in cyanobacteria (Falkner et al 1976, Coleman & Colman 1981 and green algae (Lane & Burns 1981, Tromballa 1983, Goyal & Gimmler 1989, has been measured using 5,5-dimethyl-2['4C]oxalidine-2,4-dione (DMO), while the fluorescent probe 2',7'-bis-(2 carboxyethy1)-5 (and -6) carboxyfluorescein acetoxymethyl ester (BCECF-AM) has been used in conjunction with dual wavelength fluorescence microscopy to measure the pH, in lowcalcifying cells of Emiliania huxleyl (Dixon et al 1989). In the present study the 2 methods are compared and used to investigate possible potential interactions between inorganic carbon availability, pH, and growth.…”

Light acclimation and pH perturbations affect photosynthetic performance in Chlorella mass culture

“…This strain can successfully grow at pH 2.5 (Beardall 1981), which shows its immense internal pH regulation capacity. Internal pH is generally maintained within narrow limits above ~pH 7 in Chlorella (Smith & Raven 1979, Beardall & Raven 1981, Sianoudis et al 1987) and other taxa (Dixon et al 1989, Kurkdjian & Guern 1989. Homeostasis is maintained by 2 main processes: proton binding and H + translocation between cell organelles and the external medium by active, energy-demanding, or passive ion exchange mechanisms (Smith & Raven 1979).…”