Sonia Scaramagli

Polyamine content and translocation in soybean plants

Summary Polyamine content was investigated in all parts of soybean plants ( Glycine max (L.) Merr. cv. Williams) at the fifth or sixth leaf stage. Trichloroacetic acid-soluble free spermidine was the most abundant polyamine found. Apical buds and internodes had a free polyamine content greater than the other parts of the plant. Bound polyamines were also detected, even though their content was quite low. Both free and bound cadaverine and DAO activity, measured in the different organs, were found only in hypocotyls and roots. [1,4- 14 C]-spermidine was supplied to a leaf in order to study the uptake and translocation through the plant; in the same organ the metabolic fate of the labelled spermidine was followed and radioactive putrescine and traces of spermine were also found. Uptake experiments, carried out utilising detached leaves, showed a time-dependent process. In translocation experiments, performed on the whole plant, a gradient of radioactivity distribution along the stem was evident, while the amount of radioactivity present in the leaves was lower than that found in internodes. Label translocation was not polar and its rate in the two internodes under the treated leaf was higher than in the internodes above it.

Auxin and cytokinin modify methyl jasmonate effects on polyamine metabolism and ethylene biosynthesis in tobacco leaf discs

Abstract In several in vitro systems, treatment with methyl jasmonate (MJ) stimulates polyamine biosynthesis as well as accumulation of acid-soluble and -insoluble conjugated polyamines. This effect is attributed to changes in gene expression and enzyme activity of arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and S -adenosylmethionine decarboxylase (SAMDC). In the present study, we used tobacco ( Nicotiana tabacum L. cv. Samsun) leaf discs to investigate the interaction between this MJ-induced response and those triggered by indole-3-acetic acid (IAA), N 6 -benzyladenine (BA) and ethylene. Our results indicate that MJ-induced accumulation of conjugated polyamines is further stimulated by auxin and counteracted by BA. The MJ-induced stimulation of ODC and SAMDC, but not ADC, mRNA levels was diminished by IAA±BA, whereas the corresponding enzyme activities were further enhanced in the presence of hormones. MJ enhanced ethylene production only when combined with hormones, and this trend was reflected in 1-aminocyclopropane-1-carboxylate oxidase transcript levels. Results suggest that the induction by MJ of conjugated polyamine levels, and of the polyamine biosynthetic genes and activities are differentially modulated by hormones, and that ethylene does not seem to be directly involved in this response.

Secondary metabolism in root and callus cultures of Hyoscyamus muticus L.: the relationship between morphological organisation and response to methyl jasmonate

Production of acid-soluble conjugated di- and polyamines, like that of other secondary metabolites, is enhanced by exposure to methyl jasmonate (MJ). We investigated this metabolic response, and activities of enzymes involved in putrescine (Put) and tropane alkaloid biosynthesis, in root cultures of Hyoscyamus muticus and compared it with that of callus cultures. In root cultures, free Put and N-methylputrescine (mPut) increased upon treatment with MJ, whereas in callus cultures mPut levels were not affected. Differently from roots, conjugated amines were scarce or absent in callus cultures, and accumulated only transiently upon treatment with MJ. Arginine decarboxylase, ornithine decarboxylase and diamine oxidase activities in root cultures were strongly stimulated by treatment with MJ, but were inhibited in callus cultures. Exposure to MJ also enhanced putrescine N-methyltransferase activity in root cultures more than in callus cultures. These results are discussed in relation to the different capacity for tropane alkaloid production in the two culture systems.

Morphogenesis in cultured thin layers and pith explants of tobacco. I. Effect of putrescine on cell size, xylogenesis and meristemoid organization

Summary Superficial thin layers and pith explants from vegetative tobacco plants were cultured on a caulogenic medium (1 μmol/L IAA + 10 μmol/L BA), or with IAA alone (1 μmol/L), BA alone (10 μmol/L) or on a hormone-free medium, in the presence or absence of 100 j.l.mollL putrescine. The aim was to investigate histologically possible, and explant type independent, putrescine effects on cytological events specifically induced by the hormone treatments during caulogenesis and callogenesis in vitro. In both types of explants the earliest cytological events (48 h) observed were fragmented or multilobed nuclei and newly formed cell walls, both occurring in IAA and IAA + BA treatments. Effects of putrescine on specific cytological events, induced by IAA or BA in both model systems, were from day 4 to the end of culture. Putrescine strongly counteracted the nuclear fragmentation, cell hypertrophy and xylogenesis specifically induced by IAA during callus growth. Furthermore, the polyamine strongly enhanced BA-induced formation of vegetative meristemoids. However, the extent of putrescine effects declined when the explants were treated with the two hormones in combination, or cultured under hormone-free conditions.

Morphogenesis in cultured thin layers and pith explants of tobacco. II. Early hormone-modulated polyamine biosynthesis

Summary In order to gain deeper insight into the interrelationships between polyamines and hormones, superficial thin layers and pith explants, excised from the stem of tobacco ( Nicotiana tabacum L.) plants in the vegetative stage, were cultured under various hormonal conditions (IAA, IAA + BA, BA) and in a hormone-free medium, in the presence or in the absence of 100 μmol/L putrescine. In thin layers at 24 h, activation of putrescine biosynthesis via both arginine-(ADC) and ornithine decarboxylases (ODC) as well as free putrescine accumulation were detected in the IAA treatment, and via ADe alone in the presence of IAA + BA. BA caused an inhibition of or had no effect on putrescine biosynthesis. In pith explants, although some free putrescine accumulated in IAA treatment, no increase in its biosynthetic activity was detectable. No DAO activity could be detected in any treatment early in culture either in superficial or deep tissues. Exogenous putrescine did not affect this pattern. The modulation of polyamine biosynthesis by the different hormones in superficial and pith explants is discussed in relation to putrescine accumulation and oxidation and to early cytological events occurring in culture and previously described (Altamura et al., 1995).

Peach (Prunus persicaL.) fruit growth and ripening: transcript levels and activity of polyamine biosynthetic enzymes in the mesocarp

Transcript levels and activities of the polyamine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19), ornithine decarboxylase (ODC, EC 4.1.1.17) and S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), as well as free polyamine titres, were analysed throughout the four growth stages S1-S4 leading up to ripening in the mesocarp from peach fruit (Prunus persica L. Batsch cv. Redhaven) grown under field conditions. SAMDC mRNA, which was northern analysed by using a PCR-generated homologous SAMDC probe, and ADC mRNA levels appeared quite stable during fruit development, while ODC transcript accumulation showed a discontinuous trend. The pattern of transcript levels during growth did not correlate with that of the relative enzyme activity, which instead correlated well with free polyamine levels. Both exhibited maximum levels in S1 and a smaller peak in S3. The behaviour of the polyamine biosynthetic machinery is discussed in relation to the different cell growth rates occurring during fruit development.

Regulation of Rhizogenesis by Polyamines in Tobacco Thin Layers

Summary Tobacco thin layer explants were cultured on a rhizogenic medium for 21 days in the presence or absence of methylglyoxal-bis(guanylhydrazone) (MGBG) or cyclohexylamine (CHA), competitive inhibitors of spermidine synthesis through S-adenosylmethionine decarboxylase and spermidine synthase, respectively. On day 21, explants were transferred to hormone-free medium of the same composition with or without MGBG or CHA, alone or in combination with labelled and unlabelled spermidine. The effects of these treatments on free and bound polyamine levels, on the putrescine biosynthetic activity (ornithine decarboxylase, ODC, and arginine decarboxylase, ADC), on labelled spermidine incorporation and on the rhizogenic response were studied. In MGBG-treated explants rhizogenesis was strongly inhibited while with CHA it was less affected. In the former, the addition of spermidine caused a significant reversion of the rooting inhibition (considering both rooting percentage and the mean number of roots per explant), while not in the latter. MGBG induced strong putrescine and spermidine depletion both in the free and bound forms; CHA induced strong spermidine depletion (especially on day 21), but putrescine accumulation. In both treatments, in the presence of labelled spermidine, labelled free and bound putrescine were also detected. A general enhancement in ADC activity was observed in inhibitor-treated explants; the addition of spermidine to the inhibitors caused a further strong increase in ADC activity. These results are discussed in relation to the different metabolic pathways affected by the inhibitors.

Polyamines and flowering: Spermidine biosynthesis in the different whorls of developing flowers of Nicotiana tabacum L

Abstract The regulation of S‐adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.1), a key enzyme of polyamine biosynthesis, was analysed at the level of transcript accumulation, enzyme activity, and free and conjugated polyamine accumulation during floral development in tobacco (Nicotiana tabacum L., cv. Samsun). SAMDC was investigated at five different stages of flower development and in each floral whorl. Using a PCR‐derived homologous SAMDC probe, a transcript of approximately 2.0 kb was detected. In floral whorls, transcript accumulation was lower in anthers than in ovaries. In the latter, SAMDC activity did not parallel transcript accumulation. Although the SAMDC transcript was relatively abundant in pollen, no enzyme activity could be detected. Hybridisation with a spermidine synthase (SPDsyn; EC 2.5.1.16) cDNA probe from the closely related species Datura stramonium indicated that the pattern of accumulation of the 1.4 lib tobacco spermidine synthase mRNA was similar to that of SAMDC. The levels of ...

Ethylene involvement in vegetative bud formation in tobacco thin layers

SummaryThe role of ethylene in vegetative bud regeneration was studied in cultured tobacco (Nicotiana tabacum L. cvSamsun) thinlayer expiants. The experimental approach consisted in supplementing the bud-inducing medium with an inhibitor of ethylene biosynthesis, aminoethoxyvinylglycine (AVG), an ethylene antagonist, silver thiosulphate (STS), or an ethylene-releasing compound, 2-chloroethylphosphonic acid (CEPA), at various concentrations. The organogenic response was assessed both macroscopically (percentage of bud-forming expiants, final number of buds per expiant) and cytohistologically (number, characteristics, and localisation of meristemoids and bud primordia). The time course of ethylene production during culture was also evaluated. At the end of culture (day 27) all the expiants treated with these compounds had a lower number of buds compared to controls. STS was detrimental to meristemoid initiation at all the concentrations tested. In contrast, 0.5 μM AVG, which strongly inhibited ethylene production, provoked a large increase in the formation of meristemoids early in culture and the appearance of anomalous (“twin”) buds. CEPA reduced meristemoid formation but, at the lower concentrations (1 and 10 μM) speeded up bud emergence. On the whole it mainly favoured disorganised growth and xylogenesis. The results of this work highlight the contrasting effects of ethylene in relation to the two critical stages of the organogenic process, i.e., meristemoid formation and bud primordium development.

Acclimation to low water potential in potato cell suspension cultures leads to changes in putrescine metabolism

Changes in levels and biosynthesis of di- and polyamines are associated with stress responses in plant cells. The involvement of these molecules was investigated here in cultured potato (Solanum tuberosum L.) cells grown in medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin, and acclimated or not to low water potential. The diamine (putrescine) and polyamine (spermidine and spermine) status in cells gradually acclimated to increasing concentrations (up to 20 %, w/v) of polyethylene glycol (PEG) Mr 8000, was compared with that of unacclimated cells abruptly exposed (shocked) or not (controls) to 20 % (w/v) PEG. After a 72-h subculture, the free and perchloric acid (PCA)-soluble conjugated di- and polyamine pattern in acclimated cells was not dramatically different from that of controls, but PCA-insoluble conjugated putrescine was 14-fold higher than in controls. In shocked cells, a strong reduction in free putrescine and spermidine/spermine titres occurred. Arginine (ADC, EC 4.1.1.19) and ornithine (ODC, EC 4.1.1.17) decarboxylase activities were not substantially altered in shocked cells compared with controls, while in PEG-acclimated cell populations they increased about 3-fold, both in the soluble and particulate fractions. S-Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21) and diamine oxidase (DAO, EC 1.4.3.6) activities followed a similar pattern to each other in that their activities were enhanced 2- and 3-fold, respectively, in acclimated cells over unacclimated controls. Ethylene production was also enhanced in acclimated cells. These results indicate that, with respect to di- and polyamines, acquired tolerance to low water potential in potato cells leads principally to changes in putrescine biosynthesis and conjugation which may be involved in ensuring cell survival.