Many cellular events depend on a tightly compartmentalized distribution of H ⁺ ions across membrane-bound organelles. However, measurements of organelle pH in living cells have been scarce. Several mutants of the Aequorea victoria green fluorescent protein (GFP) displayed a pH-dependent absorbance and fluorescent emission, with apparent pKa values ranging from 6.15 (mutations F64L/S65T/H231L) and 6.4 (K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L) to a remarkable 7.1 (S65G/S72A/T203Y/H231L). We have targeted these GFPs to the cytosol plus nucleus, the medial/trans-Golgi by fusion with galactosyltransferase, and the mitochondrial matrix by using the targeting signal from subunit IV of cytochrome c oxidase. Cells in culture transfected with these cDNAs displayed the expected subcellular localization by light and electron microscopy and reported local pH that was calibrated in situ with ionophores. We monitored cytosolic and nuclear pH of HeLa cells, and mitochondrial matrix pH in HeLa cells and in rat neonatal cardiomyocytes. The pH of the medial/trans-Golgi was measured at steady-state (calibrated to be 6.58 in HeLa cells) and after various manipulations. These demonstrated that the Golgi membrane in intact cells is relatively permeable to H ⁺ , and that Cl ⁻ serves as a counter-ion for H ⁺ transport and likely helps to maintain electroneutrality. The amenability to engineer GFPs to specific subcellular locations or tissue targets using gene fusion and transfer techniques should allow us to examine pH at sites previously inaccessible.

Summary: Cytoplasmic free calcium ([Ca²⁺]_cyt) acts as a stimulus‐induced second messenger in plant cells and multiple signal transduction pathways regulate [Ca²⁺]_cyt in stomatal guard cells. Measuring [Ca²⁺]_cyt in guard cells has previously required loading of calcium‐sensitive dyes using invasive and technically difficult micro‐injection techniques. To circumvent these problems, we have constitutively expressed the pH‐independent, green florescent protein‐based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al. 1999; Proc. Natl. Acad. Sci. USA 96, 2135–2140). This yellow cameleon calcium indicator was expressed in guard cells and accumulated predominantly in the cytoplasm. Fluorescence ratio imaging of yellow cameleon 2.1 allowed time‐dependent measurements of [Ca²⁺]_cyt in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic acid (ABA) induced repetitive [Ca²⁺]_cyt transients in guard cells. [Ca²⁺]_cyt changes could be semi‐quantitatively determined following correction of the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive [Ca²⁺]_cyt transients with peak values of up to approximately 1.5 μM, whereas ABA‐induced [Ca²⁺]_cyt transients had peak values up to approximately 0.6 μM. These values are similar to stimulus‐induced [Ca²⁺]_cyt changes previously reported in plant cells using ratiometric dyes or aequorin. In some guard cells perfused with low extracellular KCl concentrations, spontaneous calcium transients were observed. As yellow cameleon 2.1 was expressed in all guard cells, [Ca²⁺]_cyt was measured independently in the two guard cells of single stomates for the first time. ABA‐induced, calcium‐induced or spontaneous [Ca²⁺]_cyt increases were not necessarily synchronized in the two guard cells. Overall, these data demonstrate that that GFP‐based cameleon calcium indicators are suitable to measure [Ca²⁺]_cyt changes in guard cells and enable the pattern of [Ca²⁺]_cyt dynamics to be measured with a high level of reproducibility in Arabidopsis cells. This technical advance in combination with cell biological and molecular genetic approaches will become an invaluable tool in the dissection of plant cell signal transduction pathways.