Effect of auxin on 6-(benzylamino)purine metabolism in suspension cultures.
A review of the literature indicated that the purine cytokinin 6-(benzylamino)purine (SA) may be converted to a wide range of metabolites. Although the functional significance of these metabolites remains obscure, cytokinin physiologists have essentially classed them as either active or inactive. Inactivation of cytokinins is considered to proceed via catabolic oxidation (side-chain cleavage), or N-conjugation with glucose or alanine moieties. The literature survey was hampered by the confusing array of synonyms which have been coined for cytokinin metabolites. Accordingly, a working system of (semi-systematic) abbreviations was devised which accommodated all groups and classes of purine cytokinins. Prior to commencing metabolic interactive studies, it was necessary to resolve the contentious issue associated with the successful extraction of cytokinin nucleotides. Five-week-old soybean callus was fed [8[-14]C]BA and subsequently extracted using four widely used cytokin in extraction techniques. Techniques compared were a modified Bieleski method, 80% ethanol with tissue homogenisation, 80% ethanol without homogenisation, and boiling ethanol. All four procedures produced similar results, showing that all metabolites of SA, including the nucleotide, were adequately extracted. It was concluded that the extraction of nucleotides with Bieleski solvents did not warrant the inconvenience. Auxins have been shown to interact with cytokinins in the regulation of many physiological processes, although little is known of the mechanisms of interaction which proceed at the metabolic level. Previous investigators have shown that auxin promoted cytokinin degradation through catabolic oxidation, Shoot-apex and seed derived cell suspensions of Dianthus zevheri subsp. natalensis were incubated with [8[-14] C]BA for between 30 minutes and 48 hours in the presence of both low (2 mg l-1) and high (4 mg 1¯¹) levels of exogenously supplied 2,4-dichlorophenoxyacetic acid (2,4-D), In both systems, the auxin 2,4-D was shown to promote SA inactivation through 7-glucosylation (N-conjugation). This observation represents the first report of auxin-promoted cytokinin N-conjugate formation. The auxin effect on metabolism was transient in the case of shoot-apex, but not in seed-derived systems over a 48 hour period. Formation of the 7-glucoside of SA was dose-dependent in apex-derived cultures. Further studies were undertaken with indole-3-acetic acid (lAA) and α-naphthaleneacetic acid (NAA). It was found that auxin-promoted 7-glucosylation of SA was only minimally effected by these two auxins. In comparable studies with soybean suspension cultures (Glycine max cv. Acme), 2,4-D-promoted catabolic oxidation was observed between 18 and 48 hours, following application of phytohormones. The main catabolite was tentatively identified as adenosine-5'-monophosphate (AMP), based on chromatographic characteristics. Carrot (Daucus carota) cell suspensions similarly supplied with 2,4-D and SA maintained a large active cytokinin pool. Neither substantial oxidative nor Nconjugative processes were observed. Instead, there was a transient effect by 2,4- D on the relative formation of the riboside and the 7- and 9-glucosides of SA. The effect of auxin on the metabolism of SA thus varied with the species and system investigated. Generally, auxin promoted (rather than inhibited), the formation of inactivated metabolites and catabolites of SA, possibly by the induction of relevant enzyme systems. Transient auxin effects on the metabolism of SA are discussed in relation to the role of the auxin/cytokinin balance in the induction of developmental processes.