Analysis of Polyamines in Higher Plants by High Performance Liquid

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) through a 5-,um C18 column, and detection at 254 nm. The method can be directly applied to crude plant extracts, and it is not subject to interference by carbohydrates and phenolics. A good quantitative correlation was found between HPLC analysis of benzoylpolyamines and thin layer chromatography of their dansyl derivatives. With the HPLC method, polyamine titers have been reproducibly estimated for various organs of amaranth, Lemna, oat, pea, Pharbitis, and potato. The analyses correlate well with results of thin layer chromatography determinations. The widespread occurrence of Put,2 Spd, and Spm in bacterial, animal, and plant cells is now well established (6, 10, 26). In recent years, research on these polyamines and their less common homologs (Dap and Cad) of higher plants (26), algae, and bacteria (1, 9) has focused on the suggestive evidence (6, 10, 19) for their involvement in the regulation of growth and development. For example, exogenously added polyamines promote callus formation in Jerusalem artichoke tuber explants (3), prevent leaf senescence (15), and promote macromolecular synthesis and mitosis in oat leaf protoplasts (14). The polyamine biosynthetic enzyme arginine decarboxylase is regulated by phytochrome (7) and GA3 (8) in pea buds and internodes. The evidence accumulated so far needs to be supported by detailed analysis of the endogenous polyamine pools and de novo synthesis from labeled precursors during the developmental processes these compounds seem to affect. The most sensitive and widely used method of polyamine analysis is based on the dansylation of the amino groups under alkaline conditions, separation of the dansyl derivatives by TLC, and quantification by fluorimetry (22, 25). Although originally developed for the analysis of animal and bacterial polyamines, the dansyl method has been adapted for use with higher plant extracts 1 Supported by National Aeronautics and Space Administration and National Science Foundation Grant to A. W. G., and an American Cancer Society Fellowship to H. E. F. 2Abbreviations: Put, putrescine; Spd, spermidine; Sppm, spermine; Dap, diaminopropane; Cad, cadaverine; Agm, agmatine; Homo-Spd, homospermidine; APCad, aminopropylcadaverine; Nor-Spd, norspermidine; dansyl, l-dimethylamino-5-naphthalenesulfonyl; a.u.f.s., absorbance units full scale. (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 Plant Material. The following plant material was grown in controlled environment growth rooms, in plastic pots containing washed vermiculite, subirrigated twice daily with a 1.2 g/L solution ofHyponex (Hydroponics Chemicals Co., Copley, OH): grain and vegetable amaranth (Amaranthus cruentus, A. hypochondriacus; A. tricolor); oat (Avena sativa cv. Victory); Pharbitis nil; and bean (Phaseolus vulgaris cv. Taylor). Plants were grown under a 16-h photoperiod, 25°C, with a 9:1 energy mixture of fluorescent and incandescent light at an energy level of about 17,600 ergs cm sec-1. Cultures of Lemna gibba were a gift from Dr. C. F. Cleland; both fresh cultures and lyophilized samples were analyzed with similar results. Peas (Pisum sativum cv. Alaska) were grown in darkness at 26°C and 70%o RH. Potato tubers (Solanum tuberosum cv. Russet Burbank) were obtained from a local market and kept stored at 5°C until use. Tissues were extracted in 5% cold HC104 at a ratio of about 100 mg/ml HC104. After extraction for 1 h in an ice bath, samples were pelleted at 48,000g x 20 min, and the supernatant phase, containing the 'free' polyamine fraction, was stored frozen at -20°C in plastic vials. HC104 extracts were stable for polyamine analysis by TLC or HPLC for more than 6 months under these conditions, provided excessive refreezing and thawing were avoided. Since polyamines have been shown to bind to glass (18; SS Cohen, personal communication), care was taken, for both plant extracts and standards (see below), to store samples in plastic vials. Chemicals. The following polyamines were obtained as their hydrochlorides (Sigma): Dap, Put, Cad, Spd, and Spm. Agm (Sigma) was obtained as its sulfate. The hydrochlorides of HomoSpd and APCad were gifts from Prof. Seymour S. Cohen (SUNY, Stony Brook, NY). Nor-Spd was used as the free base (Aldrich). One-mM stocks in 0.01 N HCI of these compounds were stored as above, and were stable for at least 6 months at -20°C. Benzoyl chloride (Baker) and dansyl chloride (Sigma) were reagent grade, >95% purity. Dansylation and TLC Analysis. The method of Seiler and Wiechmann (25) as adapted for plant tissues (28) was used with some modifications as follows. Two hundred pl of HC104 extract 701 www.plantphysiol.org on July 21, 2017 Published by Downloaded from Copyright © 1982 American Society of Plant Biologists. All rights reserved.