Polyamine conjugate levels and ethylene biosynthesis: inverse relationshipwith vegetative bud formation in tobacco thin layers

The effects of two inhibitors of polyamine (spermidine and spermine) biosyn thesis, cyclohexylamine (CHA; 5 and 10 mM) and methylglyoxal(bis-guanylhydr azone) (MGBG; 0.1, 0.5 and 1 mM), on the organogenic response in vegetative bud-forming tobacco (Nicotiana tabacum L. cv. Samsun) thin laver explants were evaluated micro- and macroscopically at different times during culture . The final number of buds formed and the percentage of organogenic explant s was significantly reduced bg both inhibitors, but much more so by MGBG th an CHA. This inhibitory effect mas already evident in MGBG-treated explants on day 5, in terms of the number of meristemoids per explant. On the contr ary, in the presence of CHA, the number of meristemoids on day 5 aas higher than that in the controls. Between days 9 and 13, meristemoid formation sl owed down considerably in inhibitor-treated explants compared with controls . On dag 13, the number of bud primordia nas similar in control and CHA-tre ated explants but significantly lower in MGBG-treated explants. This inhibi tor also induced peculiar cytohistological events, such as a reduced format ion of oval-shaped cell aggregates on the explant surface and more frequent cases of nucleolar extrusion, while CHA led to the appearance of hypertrop hic epidermal cells; callus formation at the basal end of the explant and x ylogenesis were also affected by the inhibitors. Ethylene biosynthesis, mea sured as [C-14]methionine incorporation, nas stimulated 2- (day 2) to 3-fol d (15 h) by 0.5 mM MGBG, whereas CHA (10 mM) had little effect and aminetho xyvinylglycine (AVG; 0.1 mM), an ethylene synthesis inhibitor, was strongly inhibitory. In control er;plants, the incorporation of labelled methionine into ethylene and spermidine followed an inverse trend up to day 8. In the se explants, free putrescine increased 32-fold and spermidine increased abo ut 10-fold between days 0 and 8. Trichloroacetic acid (TCA)-soluble conjuga ted putrescine also accumulated dramatically during culture. While CHA prov oked a decline in spermidine levels, MGBG caused an unexpected increase in free spermidine and spermine titres; however, its most conspicuous effect m as on the further enhancement of putrescine conjugate accumulation, while C HA and AVG had the opposite effect. Results are discussed in,view of establ ishing a putative link between MGBG-enhanced ethylene synthesis, increased conjugate titres and inhibition of meristemoid formation.