Flower formation in stem explants was chosen as an experimental system for the study of the function of hydroxycinnamoyl amides (HCAs) in plants. The explants, derived from flowering and non‐flowering Nicotiana tabacum L. var. Xanthi nc., differentiated to two types of callus and afterwards to flower buds. A novel reversed‐phase high performance liquid chromatography method, following sample clean‐up on CM‐Fractogel columns, enabled us to examine HCA concentrations in small tissue samples. Two different groups of HCA could be distinguished during in vitro flower formation: Firstly, feruloyl‐ and diferuloyputrescine, the major detectable HCAs that accumulated during callus proliferation; secondly, caffeoylputrescine, which accumulated during the later stage of flower differentiation and reached higher concentrations than feruloylputrescine.

The distribution and formation of hydroxycinnamoyl amides (HCA) in in vitro grown stem explants from day‐neutral Nicotiana tabacum L. var. Xanthi no were investigated using [14C]‐labelled precursors. Feruloylputrescine (FP) (labelled from [14C]‐putrescine) was continually formed with a decreasing formation rate during a culture period of 35 days. The specific radioactivity, however, remained constant. In contrast, caffeoylputrescine (CP) was labelled in the second part of culture only. [UC]‐Label from putrescine, spermidine, spermine, and cinnamic acid was incorporated into FP and p‐coumaroylspermidine. CP was labelled from [14C]‐putreseine and [14C]‐spermidine only, while diferuloylputrescine (diFP) failed to be labelled by any of the precursors used. Tissue investigations showed that FP is dominant in cortex during formation of cortical callus, while CP showed a sequential gradient from pith to cortical callus to floral buds. Experiments with photoperiodical Nicotiana species were used to investigate the pattern of HCAs in induced and non‐induced plants. The origin of explants had no influence on the pattern of FP and CP but resulted in different growth responses. FP is a marker of cortical callus formation in all explants. CP does not trigger the growth processes observed in the expants since CP also increased in photoperiodically non‐induced explants of tobacco, which stopped growing after the formation of cortical callus.

The role of feruloylputrescine (FP) and of caffeoylputrescine (CP) was investigated in an explant system of stem explants from day-neutral Nicotiana tabacum L. var Xanthi nc. Previously, a correlation between cortical callus formation and increase in FP content, as well as between in vitro flower formation and increase in CP content had been shown. During the explant growth in vitro, the increase of both FP and CP was inhibited by 4-fluor-(1-amino-2-phenylethyl)phosphonic acid and 2-amino-indene-2-phosphonic acid, both inhibitors of phenylalanine ammonia-lyase (EC 4.3.1.5). DL-a-difluoromethylarginine, an inhibitor of arginine decarboxylase (ADC, EC 4.1.1.19), prevented only the increase in FP, while DL-a-difluoromethylornithine, an inhibitor of ornithine decarboxylase (EC 4.1.1.17), reduced only that of CP. Increase in dry weight and the formation of cortical callus and of floral buds of explants were not affected by any of the inhibitors. We conclude, in contrast to earlier hypotheses, that FP and CP do not trigger growth and differentiation in the explants. It seems more likely that FP and CP increase in response to auxin and cytokinin in the media.HCA' are widely distributed in the plant kingdom (12) and are particularly abundant in plant cell and callus cultures (6,15,25). Suggestions pertaining to their role in plants include virus resistance, stress response, and reproduction (15,25). The latter role was forwarded mainly on the grounds that HCA accumulate in floral parts of plants and are absent from cytoplasmic sterile anthers in Zea mays (5, 12-16, 18, 21). HCA were therefore proposed to play a role in induction and differentiation of flowers in plants.The majority of investigators examined the role of HCA as a part of the action of PA. They hydrolyzed plant extracts with HCI and derivatized the conjugated PA with dansylchloride. This method has been used for thin cell layer explants (29) of tobacco to compare in vitro growth responses such as generative or vegetative shoot formation with patterns of free and conjugated PA, the latter including HCA. Torrigiani et