Cytoplasmic Acidification Induced by Inorganic Phosphate Uptake in Suspension Cultured Catharanthus roseus Cells

Abstract

Cytoplasmic acidification during inorganic phosphate (Pi) absorption by Catharanthus roseus cells were studied by means of a fluorescent pH indicator, 2',7'-bis-(2-carboxyethyl)-5 carboxyfluorescein (acetomethylester) (BCECF), and 31P-nuclear magnetic resonance spectroscopy. Cytoplasmic acidification measured by decrease in the fluorescence intensity started immediately after Pi application. Within a minute or so, a stable state was attained and no further acidification occurred, whereas Pi absorption was still proceeding. As soon as Pi in the medium was exhausted, cytoplasmic pH started to recover. Coincidentally, the medium pH started to recover toward the original acidic pH. The Pi-induced changes in the cytoplasmic pH were confirmed by 31P-nuclear magnetic resonance study. Maximum acidification of the cytoplasm induced by 1.7 millimolar Pi was 0.2 pH units. Vacuolar pH was also affected by Pi. In some experiments, but not all, pH decreased reversibly by 0.2 to 0.3 pH units during Pi absorption. Results suggest that the cytoplasmic pH is regulated by proton pumps in the plasma membrane and in the tonoplast. In addition, other mechanisms that could consume extra protons in the cytoplasm are suggested.

Cytoplasmic Acidification and Secondary Metabolite Production in Different Plant Cell Suspensions (A Comparative Study)

“…1). As with oxonol VI, any BCECF present in the medium or the vacuole does not fluoresce at low pH values (Sakano et al, 1992). BCECF fluorescence cannot be measured in cell cultures containing large aggregates because of scattering.…”

“…Both probes are very sensitive in that the differences in fluorescence are large and both probes react rapidly. Calibration of the probes with nigericin to obtain actual pH values is not readily possible i n plant cells (Sakano et al, 1992). In our experiments, thereflxe, we have focused on qualitative changes in cytoplasmic pH rather than the actual pH value of the cytoplasm.…”

“…To correct for the resulting differences in concentration of cytoplasmic BCECF, the ratio between the excitation maximum (here 490 nm) and the isosbestic point (439 nm) was calculated (Haugland, 1992). Sakano et al (1992) extensively studied the use of BCECF-AM in Catharanthus roseus cells; from their paper it appeared that BCECF specifically accumulates in the cytoplasm (90% of total BCECF). This was confirmed by our own observations (Fig.…”

“…For comparison we also used BCECF, a widely used pH probe of approximately pH 7 (Haugland, 1992;Sakano et al, 1992). This probe shows fluorescence only after hydrolysis of the AM bond by intracellular esterases.…”

Distúrbios nutricionais induzidos pela adubação de fósforo e ferro em plantas jovens de grápia (<i>Apuleia leiocarpa</i>).

“…Em nível celular, a relação entre P e Fe parece estar relacionada com a variação do pH do meio apoplástico e do potencial eletroquímico, pelo qual a absorção do fósforo inorgânico (H 2 PO 4 -), por ser impulsionada pelo transporte ativo secundário utilizando-se do gradiente de H + , ocasiona o aumento do pH apoplástico (Sakano et al, 1992) e, conseqüentemente, uma imobilização do Fe nesses espaços. Kosegarten e English (1994) encontraram uma relação inversa entre a concentração de clorofila e o pH do apoplasto foliar de soja (Glycine max L).…”