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Measurement of intracellular pH in maize root tissue with α‐methyl fluorinated alanines and in‐vivo 19 F NMR spectroscopy
Author(s) -
Pfeffer Philip E.,
ShacharHill Yair,
Tu ShuI.,
Brauer David
Publication year - 2004
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.2004.00404.x
Subject(s) - vacuole , nuclear magnetic resonance spectroscopy , chemistry , cytoplasm , alanine , intracellular ph , in vivo , intracellular , biochemistry , stereochemistry , amino acid , biology , microbiology and biotechnology
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 (F 2 ALA) and methyl trifluoro alanine (F 3 ALA) with in‐vivo 19 F NMR spectroscopy to measure vacuolar and cytoplasmic pH in maize root tissue. The pH dependence of the chemical shift of F 2 ALA and F 3 ALA is greater than either the commonly used 31 P NMR signal of inorganic phosphate or the 13 C NMR signals of trans‐aconitic acid, which is also found in some plant cells. F 2 ALA and F 3 ALA were also able to detect changes over a greater range of pH. When maize root tissue was incubated in the presence of 0.35 m m F 2 ALA or F 3 ALA, 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 C 2 of trans‐aconitic acid and vacuolar F 3 ALA 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 F 3 ALA. The significance of these values is discussed in terms of the bioenergetics of proton transport across the tonoplast membrane in vivo.