|Shachar-Hill, Yair - MICHIGAN STATE UNIV|
|Brauer, David - DALE BUMPERS SMALL FARMS|
Submitted to: Physiologia Plantarum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 22, 2004
Publication Date: August 22, 2004
Citation: Pfeffer, P.E., Shachar-Hill, Y., Tu, S., Brauer, D. 2004. Rapid and sensitive measures of intraceulluar ph in maize root tissue by a-methyl fluorinated alanines and (19)f nmr spectroscopy. Physiologia Plantarum. v. 163. pp. 617-627. Interpretive Summary: Phosphate is one of the main nutrients that is transported from the soil by arbuscular mycorrhizal fungi (AMF) to the host plant roots. This important symbiotic relationship enables plants to grow under low input conditions with increased production and minimal impact on the environment. Following movement into the root, phosphate is stored in the cell vacuoles for use by the plant. In order to understand the plant response to the colonization by AMF we need to understand the physiological changes that take place within the plant cells following the symbiotic association and acquisition of phosphate. In this study we have developed spectroscopic probes to examine living roots to determine the pH responses within the cytoplasm and vacuolar compartments. Until now one could not accurately determine the pH responses of the phosphate containing vacuoles since they are too acidic to give reliable values with the probes used. Using the methodology we have developed we will be able to accurately and rapidly measure the physiological responses (vacuolar cytoplasmic pH, membrane potential) of living root cells when they become colonized with AMF.
Technical Abstract: Present methods for the simultaneous measurement of vacuolar and cytoplasmic pH in plant tissue often have severe limitations. The present study explores the use of di-fluorine and tri-fluorine derivatives of ALA, F2ALA and F3ALA, respectively, and 19F NMR spectroscopy to measure vacuolar and cytoplasmic pH in maize root tissue. The pH dependence of the change in chemical shift of F2ALA and F3ALA was greater than that of either 31P as inorganic phosphate or 13C as the trans-aconitic acid. In addition, F2ALA and F3ALA were able to detect changes over a greater range of pH. Maize root tissue accumulated significant concentrations of F2ALA and F3ALA in two compartments differing in pH when incubated in the presence of 0.35 mM of both derivatives. The time course of accumulation and pH environment of these pools were consistent with cytoplasmic and vacuolar localization of both derivatives. The accumulation of F2ALA and F3ALA had no significant effect on growth rate of root tips, indicating that these compounds were non-toxic. The chemical shift of both C2 of trans-aconitic acid and vacuolar F3ALA indicated that the mean vacuolar pH of maize root cells was 4.8 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 the magnitude of these values are discussed in terms of the bioenergetics of proton transport across the tonoplast membrane in vivo.