Measurement of intracellular pH in maize root tissue with a-methyl fluorinated alanines and in-vivo F NMR spectroscopy

Methods for the simultaneous measurement of vacuolar and cytoplasmic pH in plant tissues currently have significant limitations. This study demonstrates the usefulness of methyl difluoro alanine (F2ALA) and methyl trifluoro alanine (F3ALA) with in-vivo F NMR spectroscopy to measure vacuolar and cytoplasmic pH in maize root tissue. The pH dependence of the chemical shift of F2ALA and F3ALA is greater than either the commonly used P NMR signal of inorganic phosphate or the C NMR signals of trans-aconitic acid, which is also found in some plant cells. F2ALA and F3ALA were also able to detect changes over a greater range of pH. When maize root tissue was incubated in the presence of 0.35 mM F2ALA or F3ALA, these accumulated to significant concentrations in two compartments of different pH with no significant effect on growth rate of root tips. The time course of accumulation and the pH of the two compartments were consistent with one being the cytoplasm and the other the vacuole. The chemical shift of both C2 of trans-aconitic acid and vacuolar F3ALA indicated that the mean vacuolar pH of maize root cells was 4.6 and that the pH gradient across the tonoplast membrane was about 2.8 units. Under a variety of conditions, there was considerable heterogeneity in the pH of the vacuoles in maize root tissue as indicated by the peak width of the signal from F3ALA. The significance of these values is discussed in terms of the bioenergetics of proton transport across the tonoplast membrane in vivo.