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World-wide salinity and drought problems necessitate the understanding of biological adaptation to water deficit. Osmotic adjustment via organic solutes is a common strategy for organisms to deal with water deficit problems. Numerous water-soluble organic metabolites across several chemical classes are commonly utilized as osmolytes, including betaines, sulfonium and sulfonate compounds, amino acids, carbohydrates, and polyols. To deal with the complexity and variability in osmolyte composition, we have devised an analytical approach that combines high-resolution 1H NMR and GLC to provide both structure identification and quantification of a broad spectrum of compounds. This combined approach also facilitated direct analyses of crude tissue extracts without extensive sample preparation, making it well-suited for a convenient screening of potential osmolytes. The structures of known osmolytes were confirmed from two-dimensional total correlation 1H NMR spectra, which also yielded structural information about unknown compounds. Five each terrestrial plant and marine animal species were examined for 41 metabolites, including osmolyte candidates glycinebetaine, dimethylsulfoniopropionate, taurine, proline, glycine, asparagine, alanine, glutamine, glucose, and sucrose. The osmotic function of glycinebetaine, proline, asparagine, glutamine, glucose, and sucrose was also demonstrated in leaves of Distichlis spicata under different salinity treatments.

The cytoplasmic pH, concentrations of phosphate metabolites, and rate of ATP synthesis were measured in vivo in excised rice shoot tips under normoxic and hypoxic conditions using 31P NMR. When supplied with glucose, the shoot tips grew rapidly and were relatively unaffected by hypoxia. The cytoplasmic pH decreased transiently by only 0.2 units during hypoxia, and the concentration of ATP was maintained to at least 90% of the normoxic level. Most importantly, the unidirectional rate constant of ATP synthesis from free phosphate decreased less than 25% during hypoxia. This is in contrast to other actively growing tissues such as the maize root tip. gamma-Aminobutyrate was the major nonvolatile fermentation end product after 22 h of hypoxia. Other hypoxia-induced changes included a modest increase in [Ala] and [succinate] as well as a substantial decrease in [malate].

Thirty-three metabolites were observed in perchloric acid extracts of four different tissues by in vitro 1H-NMR, GC-MS and alcohol dehydrogenase assay, and the information was used to interpret an in vivo two-dimensional nuclear Overhauser effect 1H-NMR spectrum. The metabolite profiles of the different tissues indicate a number of potential tissue-specific markers: N-acetylaspartate and gamma-aminobutyric acid for rat brain, glutamine/glutamic acid ratio for dog heart, arginine and sucrose for carrot, and t-aconitate, sucrose, asparagine/aspartic acid concentration ratios for corn roots. gamma-Aminobutyric acid and malate can be regarded as metabolic indicators for stressed corn roots. Concentrations of threonine and valine in corn roots were constant under hypoxic and salt stress, and can serve as internal standards for both in vivo and in vitro NMR studies. The in vitro information was further used to identify 12 compounds from the in vivo 1H-NMR spectra (including the two-dimensional nuclear Overhauser effect spectrum) of a carrot cylinder by correlating the chemical shift and nuclear Overhauser effect information. Thus, our choice of methods with a capability for structural determination allows the characterization of complex tissue extracts with minimum sample preparation, and supports, as well as complements, in vivo 1H-NMR investigations of metabolism.

We have used a coaxial superfusion system to obtain physiologically interpretable in vivo 1H NMR spectra at 500 MHz of carrot roots, maize roots, and rice shoots in water (no 2H2O). The superfusion system was constructed from common laboratory parts, required no modification of the probe and sample loading procedure, and was inherently leak resistant. The assignment and quantitation of the in vivo 1H NMR resonances were achieved by performing two-dimensional NMR experiments in vivo, and by in vitro analysis including NMR and gas chromatography-mass spectrometry. The in vivo spectra were dominated by resonances arising from sugars, organic acids, amino acids, and ethanol. In vivo measurements of spin-lattice relaxation times and chemical shifts of beta protons of malate in carrot roots suggested that malate was located in a relatively viscous and acidic compartment. In rice shoots, the hypoxic time courses of 9 metabolites were established in vivo, and 23 in vitro. In both cases, accumulation of lactate, ethanol, Ala, and gamma-aminobutyrate as well as a decrease in Gln and Asn concentrations were observed. These findings are consistent with accelerated glycolysis and decreased tricarboxylic acid cycle activity.

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 NaCl uptake-associated broadening was also noted for both vacuolar Pi and intracellular Na NMR peaks, consistent with Na+ 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.