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The effects of two inhibitors of polyamine (spermidine and spermine) biosynthesis, cyclohexylamine (CHA; 5 and 10 mM) and methylglyoxal(bis‐guanylhydrazone) (MGBG; 0.1, 0.5 and 1 mM), on the organogenic response in vegetative bud‐forming tobacco (Nicotiana tabacum L. cv. Samsun) thin layer explants were evaluated micro‐ and macroscopically at different times during culture. The final number of buds formed and the percentage of organogenic explants was significantly reduced by both inhibitors, but much more so by MGBG than CHA. This inhibitory effect was already evident in MGBG‐treated explants on day 5, in terms of the number of meristemoids per explant. On the contrary, in the presence of CHA, the number of meristemoids on day 5 was higher than that in the controls. Between days 9 and 13, meristemoid formation slowed down considerably in inhibitor‐treated explants compared with controls. On day 13, the number of bud primordia was similar in control and CHA‐treated explants, but significantly lower in MGBG‐treated explants. This inhibitor also induced peculiar cytohistological events, such as a reduced formation of oval‐shaped cell aggregates on the explant surface and more frequent cases of nucleolar extrusion, while CHA led to the appearance of hypertrophic epidermal cells; callus formation at the basal end of the explant and xylogenesis were also affected by the inhibitors. Ethylene biosynthesis, measured as [ C]methionine incorporation, was stimulated 2‐ (day 2) to 3‐fold (15 h) by 0.5 mM MGBG, whereas CHA (10 mM) had little effect and aminoethoxyvinylglycine (AVG; 0.1 μM), an ethylene synthesis inhibitor, was strongly inhibitory. In control explants, the incorporation of labelled methionine into ethylene and spermidine followed an inverse trend up to day 8. In these explants, free putrescine increased 32‐fold and spermidine increased about 10‐fold between days 0 and 8. Trichloroacetic acid (TCA)‐soluble conjugated putrescine also accumulated dramatically during culture. While CHA provoked a decline in spermidine levels, MGBG caused an unexpected increase in free spermidine and spermine titres; however, its most conspicuous effect was on the further enhancement of putrescine conjugate accumulation, while CHA and AVG had the opposite effect. Results are discussed in view of establishing a putative link between MGBG‐enhanced ethylene synthesis, increased conjugate titres and inhibition of meristemoid formation.

Cyanide and azide inhibit photosynthesis and catalase activity of isolated, intact spinach (Spinacia okracea) chloroplasts. When chloroplasts are iUuminated in the presence of CN-or N3-, accumulation of H202 is observed, parallel to inhibition of photosynthesis. Photosynthetic 02 evolution is inhibited to the same extent, under saturating light, whether C02 or phosphoglycerate is present as electron acceptor.The illumination of chloroplasts with CN-or N3-inactivates the NADPH-and ATP-dependent phosphoglycerate reduction. This enzyme system can be reactivated by dithiothreitol. In reconstituted, envelope-less chloroplasts, the phosphoglycerate-dependent and the ribose 5-phosphate-dependent 02 evolution are inhibited to the same extent, while electron transport to NADP is unaffected.It is conduded that the inhibition of photosynthesis by CN-and N3-is due to H202 accumulation, which is a consequence of catalase inhibition. Azide and cyanide inhibit both respiration and photosynthesis, in vivo as well as in vitro. While the inhibition of respiration is accounted for by the inhibition of metal-containing terminal oxidases, primarily cyt oxidase, the mechanism of inhibition of photosynthesis is controversial. It is well known that cyanide and azide do not inhibit photosynthetic electron transport from H20 to NADP or to artificial electron acceptors, nor photosynthetic phosphorylation (1, 2). Therefore, the inhibition of photosynthesis by these poisons must be explained by an indirect mechanism of invoking the inhibition of one or more steps of the photosynthetic carbon cycle. Bamberger and Avron (3) have demonstrated that the steps between Rib5P2 and 3PGA are not affected by azide, at concentrations that completely inhibit photosynthetic CO2 fixation in isolated chloroplasts. These authors attribute the inhibition of photosynthesis by N3-to the inhibition of carbonic anhydrase, an enzyme abundant in chloroplast stroma (13), which has been proposed to have a function in the uptake of CO2 into the chloroplast (5, 7). Although this function ' This work was supported by the Consiglio Nazionale delle Richerche of Italy.2 Abbreviations: PEP: phosphoenolpyruvate; 3PGA: 3-phosphoglycerate: Rib-5-P: ribose 5-phosphate; DTT; dithiothreitol.is under dispute (13), the importance of carbonic anhydrase in photosynthesis is beyond doubt. The studies reported here on the mechanism of inhibition of photosynthesis by CN-and N3-lead us to conclude that the inhibition of photosynthesis in isolated chloroplasts by these reagents can be accounted for by their inhibition of catalase and the consequent accumulation of H202, in addition to the inhibition of carbonic anhydrase. It is known that H202 inhibits enzymes dependent on -SH groups for activity.The production of H202 by photosynthetic electron transport has been established by Mehler (1 1), and is the result of reoxidation by 02 of the primary reductant of PSI. On the other hand, no H202 is formed when NADP is available as the electron acceptor. The Mehler reaction has been sh...

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