The cucumber cotyledon expansion test was used as a model system to study a possible relationship between cytokinin and polyamines. When kinetin was applied to excised cotyledons incubated in the dark it caused a marked increase in the activity of arginine decarboxylase. As a result of ADC action, putrescine content also rose markedly, whereas the level of spermidine and spermine decreased. However, inhibition of putrescine biosynthesis with D-arginine did not affect cytokinin promotion growth. Applied alone, putrescine had no significant effect on growth. These results indicate that the large increase in putrescine content that derives from cytokinin treatment cotyledons is not essential for cytokinin-induced expansion of cotyle-dons. Addition of K and Ca ions to the cotyledons incubated with cytokinin caused a marked reduction in the putrescine level and ADC activity.The higher level of putrescine (35 %) and spermine (62 %) bound to chromatin and the large increase (174 %) in spermidine content bound to ribosomes which derive from cytokinintreated cotyledons in relation to literature data can indicate that these polyamines may play an important role in gene expression during cytokinin-stimulated expansion of cucumber cotyledons.The inhibition of cytokinin effect, viz. enlargement of the cotyledons by inhibitors of spermidine biosynthesis, additionally suggessted a possible involvement of polyamines in cytokinin action.List of abbreviations: ADC -arginine decarboxylase: DCHA -dicyclohexylamine; MGBG -methylglyoxal bis-(quanylhydrazone); PAs -polyamines; Put -putrescine; SAM -S-adenosylmethionine; Spd -spermidine; Spm -spermine; TLC -thin layer chromatography.
Tissue culture of Dianthus caryophyllus L. (cv. William Sire.) obligatory requiring N6-bermyladenine for greening provides a good system to study the interactions between cytokinins and polyamines. Polyamines were analyzed as dansyl derivatives which are separated by thin layer chromatography and detected by fluorescence spectrophotometry.Green callus growing on benzyladenine -containing medium showed decrease in the contents of free, conjugated and bound putrescine and spermidine in comparison to chlorophyll-less callus (control callus) growing on cytokinin-free medium. The level of spermine free, conjugated and bound forms increased about 6 %, 77 % and 28 % respectively in tissue culture growing in the presence of cytokinin.Spermidine was dominant polyamine bound to ehromatin isolated from control callus. Chromatin isolated from green-callus was characterized by a lower level of each polyamine in comparison to chlorophyll-less callus.Polyamines were found in plastid membrane fraction isolated from chlorophyll-less and green callus. A significant increase the levels of polyamines (putrescine, spermidine and spermine) bound to plastid membranes in green callus (+ benzyladenine) in comparison to chlorophyll-less callus (-benzyladenine) was observed. Additionaly, methylglyoxal-bis(guanylhydrazone) an inhibitor of S-adenosylmethionine decarboxylase depressed the greening process.Our results suggest that cytokinin-induced chloroplast differentiation in carnation tissue culture may be partly mediated through the polyamines bound to thylakoid membranes. A possible role of polyamines during cytokinin-induced formation of photosynthetic apparatus is discussed.
Key words: Ceratodon purpureus, chromatin, g a m e t o p h o r e buds, kinetin, polyamines, ribosomes, inhibitors of polyamine biosynthesis PAs-polyamines, PCA-perchloric acid, Put-putrescine, S A M D C -S -a d e n o s y l m e t h i o n i n e decarboxylase, Spd-spermidine, Spm-spermine AbstractCytokinin-induced gametophore bud formation in the protonema of Ceratodon purpureus was used as a model system to study a possible relationship between cytokinin and polyamines. The levels of free, conjugated and bound to chromatin and ribosomes putrescine, spermidine and spermine in moss growing in a medium with or without kinetin was investigated. Cytokinin elevated (about 2-fold) the level of free, conjugated and bound to the PCA-insoluble fraction putrescine and it was correlated with a 2-fold increase in arginine decarboxylase activity. A higher level of spermine bound to chromatin and spermidine bound to ribosomes m cytokinin-treated protonema can play an important role in cytokinin-evoked stimulation of transcription and translation processes (Gwd£d~ and Szweykowska 1982, Schneider et al. 1988). This suggests that polyamines may be involved in cytokinin mode of action during the induction of gametophore bud formation.This suggestion was additionaly supported by the experiments with inhibitors of putrescine synthesis, D-arginine and canavanine as well as spemfidine synthesis, MGBG, which when added to the medium together with kinetin, reduced or inhibited cytokinin-stimulated gametophore bud formation.
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