A Na+ uptake-associated vacuolar alkalinization was observed in roots of two barley cultivars (Arivat and the more salt-tolerant California Mariout) by using 23Na and 31p in vivo NMR spectroscopy. A NaCi uptake-associated broadening was also noted for both vacuolar Pi and intracellular Na NMR peaks, consistent with NaI uptake into the same compartment as the vacuolar Pi. A close coupling of Na' with H' transport (presumably the Na+/H' antiport) in vivo was evidenced by qualitative and quantitative correlations between Na+ accumulation and vacuolar alkalinization for both cultivars. Prolongation of the low NaCl pretreatment (30 mM) increased the activity of the putative antiport in Arivat but reduced it in California Mariout. This putative antiport also showed a dependence on NaCl concentration for California Mariout but not for Arivat. No cytoplasmic acidification accompanied the antiporter activity for either cultivar. The response of adenosine phosphates indicated that ATP utilization exceeded the capacity for ATP synthesis in Arivat, but the two processes seemed balanced in California Mariout. These comparisons provide clues to the role of the tonoplast Na'/H' antiport and compensatory cytoplasmic adjustments including pH, osmolytes, and energy phosphates in governing the different salt tolerance of the two cultivars.Though not halophytes, some barley (Hordeum vulgare L.) cultivars are more salt tolerant than are many other crop plants (1-3). Vacuolar Na+ accumulation, part of the response to salt stress (4, 5), is probably mediated through a Na+/H+ antiport that uses the pH gradient between the vacuole and cytoplasm established by means of a H+-ATPase (6). Evidence consistent with the scheme, including the H+-ATPase and Na+/H+ antiporter activities, has been obtained from isolated tonoplast vesicles of barley roots (7-10) and other plant tissues (11-13). In addition, the barley Na+/H+ antiport was shown in isolated tonoplast vesicles to be activated under short-and longer-term NaCl pretreatment (9,14). The relevance of these in vitro findings for plant responses to salt stress needs to be clarified by observation of this process in vivo.One inevitable compensatory consequence of the Na+/H+ exchange process at the tonoplast is at least a transient build-up of H+ in the cytoplasm, which needs to be dissipated for normal functioning of the cytoplasm. The cytoplasm also requires separate osmotic adjustment, Na+ being unsuitable for osmoregulation there (4). In both cases, the "energy" metabolism of the plant will likely be impacted and represent a limiting factor for salinity responses (15, 16). As with the Na+/H+ antiport itself, coordination among compartments is a key feature of the biochemistry accompanying the antiporter activity, hence the need for in vivo experimental approaches that maintain tissue integrity.We have applied the in vivo NMR approach to the saltsensitive barley cultivar Arivat and the more tolerant California Mariout (CM). The advantages of NMR as an noninvasive tool are well docum...