Improved analytical methods for determination of nitrogenous stress metabolites occurring in Limonium species

Abstract Efficient and reliable high-performance liquid chromatographic procedures have been developed for metabolic analyses of amino acids, polyamines and betaines in Limonium species. The adaptive significance of accumulated low-molecular-mass nitrogenous compounds in dry or salt environments is under study. HPLC profiles of dansylated water-soluble polyamines revealed 1,3-diaminopropane and tyramine as the most abundant amines in the species under study whereas common aliphatic di- and polyamines (i.e., putrescine, spermidine and spermine) were poorly represented as their free forms. Nevertheless acylated conjugates of putrescine, 1,3-diaminopropane and spermidine were also characterized, especially in L. vulgare , a halophytic salt marshes species. Direct derivatization of amino acids of the crude aqueous extracts with 6-aminoquinolyl- N -hydroxysuccinimidyl carbamate allowed efficient determinations of most proteinic amino acids as well as non-proteinic ones such as ornithine, γ-aminobutyric acid and β-alanine also related to polyamine metabolism. Analysis of betaines was improved, especially for β-alanine betaine, which is quite uncommon in higher plants whose metabolic routes from β-alanine are poorly understood. β-Alanine betaine was first converted to acrylic acid through trimethylamine β-elimination under alkaline treatment of the crude extracts and then quantified by HPLC. Thus Limonium species ranged from high β-alanine betaine accumulators, to low accumulators and finally to a third group where it was not detected, since β-alanine betaine was replaced by glycine betaine as shown here by 1 H-NMR investigations. Metabolic links between these nitrogenous solutes and the adaptive significance of such adjustments are discussed. Special emphasis is directed towards the possible involvement of aliphatic polyamine oxidative catabolism, which seems to be effective in these species, to precursor recycling for compatible solute biosynthesis.