A sensitive (0.01-1 nmol) method has been developed for the analysis of polyamines in higher plant extracts based on high performance liquid chromatography (HPLC) of their benzoyl derivatives (Redmond, Tseng 1979 J Chromatogr 170: 479-481). Putrescine, cadaverine, agmatine, spermidine, spermine, and the less common polyamines nor-spermidine and homospermidine can be completely resolved by reverse phase HPLC, isocratic elution with methanol:water (64%, v/v) (27,28). HPLC analysis of dansyl-polyamines has also been reported (23,24). The dansyl reagent is not specific for amino groups, and the presence of carbohydrates and phenolics in plant cells causes substantial interference and reduces the accuracy of quantitative analyses.A recent report (20) showed the separation of polyamine standards (Put, Cad, Spd, Spm) based on the Schotten-Baumann benzoylation reaction, and subsequent analysis by reverse-phase HPLC. We have extended the range of compounds that can be resolved by this procedure and developed the method for an accurate estimation of polyamine titer in a variety of crude higher plant extracts. These results are reported below, and the correlation with the analysis of dansyl-polyamines is discussed. MATERIALS AND METHODS

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The diamine putrescine, the triamine spermidine, and the tetramine spermine are ubiquitous in plant cells, while other polyamines are of more limited occurrence. Their chemistry and pathways of biosynthesis and metabolism are well characterized.They occur in the free form as cations, but are often conjugated to small molecules like phenolic acids and also to various macromolecules. Their titer varies from approximately micromolar to more than millimolar, and depends greatly on environmental conditions, especially stress. In cereals, the activity of one of the major polyamine biosynthetic enzymes, arginine decarboxylase, is rapidly and dramatically increased by almost every studied external stress, leading to 50-fold or greater increases in putrescine titer within a few hours. The physiological significance of this increase is not yet clear, although most recent work suggests an adaptive, protective role. Polyamines produced through the action of ornithine decarboxylase, by contrast, seem essential for DNA replication and cell division. The application of exogenous polyamines produces effects on patterns of senescence and morphogenesis, suggesting but not proving a regulatory role for polyamines in these processes. The evidence for such a regulatory role is growing.

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