Helianthus tuberosus and polyamine research: past and recent applications of a classical growth model.

The earliest studies concerning polyamines (PAs) in plants were performed by using in vitro cultured explants of Helianthus tuberosus dormant tuber. This parenchyma tissue was particularly useful due to its susceptibility to several growth substances, including PAs. During tuber dormancy, PA levels are too low to sustain cell division; thus Helianthus represents a natural PA-deficient model. When cultivated in vitro in the presence of auxins, Helianthus tuber dormant parenchyma cells at the G(0) stage start to divide synchronously acquiring meristematic characteristics. The requirement for auxins to induce cell division can be substituted by aliphatic PAs such as putrescine, spermidine or spermine. Cylinders or slices of explanted homogeneous tuber parenchyma were cultured in liquid medium for short-term studies on the cell cycle, or on solid agar medium for long-term experiments. Morphological and physiological modifications of synchronously dividing cells were studied during the different phases of the cell cycle in relation to PAs biosynthesis and oxidation. Long-term experiments led to the identification of the PAs as plant growth regulators, as the sole nitrogen source, as tuber storage substances and as essential factors for morphogenetic processes and cell homeostasis. More recently this system was used to study the effects on plant cell proliferation of platinum- or palladium-derived drugs (cisplatin and platinum or palladium bi-substituted spermine) that are used in human cancer cell lines as antiproliferative and cytotoxic agents. Cisplatin was the most active both in cell proliferation inhibition and on PA metabolism. Similar experiments were performed using three agmatine analogous. Different effects of these compounds were observed on cell proliferation, free PA levels and enzyme activities, leading to a hypothesis of a correlation between their chemical structure and the agmatine metabolism in plants.

Cadaverine turnover in soybean seedlings using 15N-labelled lysine and cadaverine.

The synthesis and translocation of the diamine cadaverine during soybean (Glycine max L. Meer cv. Sakai) germination were studied using 15N-labelled lysine (the cadaverine precursor) and 15N-labelled cadaverine, both under light/dark (12 h/12 h) and total dark germinating conditions. 15N-cadaverine and non-labelled polyamines were simultaneously detected using ionspray ionization-mass spectrometry. Both 15N-cadaverine and 15N-lysine were taken up by soybean. 15N-lysine was transported to the shoot and root and converted into 15N-cadaverine, whereas relatively little 15N-cadaverine was formed from 15N-lysine in the cotyledon. The acropetal translocation of 15N-cadaverine from the cotyledon to the shoot seemed to predominate over basipetal transport to the root. Although no other 15N-derivatised polyamines were found, supplying exogenous 15N-lysine seemed to indirectly affect the metabolism of 14N putrescine, spermidine and spermine, while no significant effect was detected after supplying 15N-cadaverine.

Chitosan treatment induces changes of protein expression profile and stilbene distribution in Vitis vinifera cell suspensions.

Polyphenols, including stilbenes and flavonoids, are an essential part of human diet and constitute one of the most abundant and ubiquitous groups of plant secondary metabolites, and their level is inducible by stress, fungal attack or biotic and abiotic elicitors. Proteomic analysis of Vitis vinifera (L.) cultivar (cv.) Barbera grape cell suspensions, showed that the amount of 73 proteins consistently changed in 50 microg/mL chitosan-treated samples compared with controls, or between the two controls, of which 56 were identified by MS analyses. In particular, de-novo synthesis and/or accumulation of stilbene synthase proteins were promoted by chitosan which also stimulated trans-resveratrol endogenous accumulation and decreased its release into the culture medium. No influence was shown on cis-resveratrol. There was no effect on the accumulation of total resveratrol mono-glucosides (trans- and cis-piceid and trans- and cis-resveratroloside). Throughout the observation period the upregulation of phenylalanine ammonia lyase, chalcone synthase, chalcone-flavanone isomerase (CHI) transcript expression levels well correlated with CHI protein amount and with the accumulation of anthocyanins. Chitosan treatment strongly increased the expression of eleven proteins of the pathogenesis related protein-10 family, as well as their mRNA levels.

Polyamines and salt stress response and tolerance in Arabidopsis thaliana flowers.

In the present study we analysed polyamine metabolism in Arabidopsis thaliana (ecotype Columbia) flowers and stalks collected from plants germinated and grown under increasing salt-stress conditions (0-75 mM NaCl). The expression level of the different isoforms of polyamine biosynthetic enzymes was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR). Spermidine synthase enzyme activity determined both in supernatant and pellet fractions, together with RT-PCR results, led us to hypothesize a different intracellular compartmentation of the isoforms of these enzymes. Free and conjugated polyamines (perchloric acid-soluble and -insoluble) were measured. Free spermidine was the most abundant polyamine and its levels, such as those of free spermine, increased with salt concentration, supporting the hypothesis for a specific role of those polyamines in the response and tolerance to salt stress of Arabidopsis thaliana flowers.

Cloning, functional identification and structural modelling of Vitis vinifera S-adenosylmethionine decarboxylase.

In this paper we report the cloning and full sequencing of S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) cDNA from Vitis vinifera L. (VV) leaves, an enzyme belonging to the polyamine biosynthetic pathway, which appears to play an important role in the regulation of plant growth and development. The presence of two overlapping ORFs (tiny ORF and small ORF) upstream of the main ORF is reported in the Vitis cDNA. When the Vitis SAMDC cDNA was expressed in yeast without the two upstream ORFs, the resulting activity was about 50 times higher than the activity obtained with the full cDNA. These results demonstrated the strong regulatory activity of the tiny and small ORFs. RT-PCR expression analysis showed evidence of a similar mRNA level in all the tissues tested, with the exception of the petioles. The VV SAMDC was also modelled using its homologues from Solanum tuberosum and Homo sapiens as template. The present work confirmed, for the first time in a woody plant of worldwide economic interest such as grapevine, the presence of a regulatory mechanism of SAMDC, enzyme that has a well-established importance in the modulation of plant growth and development.

Jasmonates and Na-orthovanadate promote resveratrol production in Vitis vinifera cv. Barbera cell cultures.

Here the effect of jasmonic acid, methyljasmonate and Na-orthovanadate on the production of resveratrol was studied in Vitis vinifera cv. Barbera cell suspension cultures. Na-orthovanadate at 0.1 mm and 1 mm concentration was efficient in promoting the production and/or accumulation and release in the culture medium of cis-resveratrol while trans-resveratrol levels were not affected by this treatment. Methyljasmonate was highly effective in stimulating both trans- and cis-resveratrol endogenous accumulation, as well as their release into the culture medium. Cis-resveratrol was absent or detected in very low amounts in the controls. Jasmonic acid was less efficient than methyljasmonate in promoting endogenous resveratrol accumulation, but it stimulated the release in the culture medium especially of cis-resveratrol. Gel analysis was performed on control and 10 microm MeJA treated cell suspensions. Results showed an up-regulation of the stilbene synthase demonstrating that MeJA stimulated the synthesis ex-novo of this protein.

Analysis of polyamine metabolism in soybean seedlings using 15N-labelled putrescine.

The translocation and metabolism of polyamines during soybean germination were studied using 15N-labelled putrescine as a precursor. Both 15N-labelled and unlabelled polyamines were simultaneously detected using a novel application of ionspray ionization-mass spectrometry. 15N-putrescine was rapidly transported to the shoots and roots, where it was converted to spermidine and spermine. The main 15N-polyamine that accumulated in the root was 15N-spermine. It was found that there were differences in the way endogenous putrescine and exogenous 15N-putrescine were metabolized in soybean seedlings.

Effects of spermidine synthase overexpression on polyamine biosynthetic pathway in tobacco plants.

Transgenic tobacco plants overexpressing the Datura stramonium spermidine synthase (EC 2.5.1.16) cDNA were produced in order to understand the role of this gene in the polyamine metabolism and in particular in affecting spermidine endogenous levels. All the analysed transgenic clones displayed a high Level of overexpression of the exogenous cDNA with respect to the endogenous spermidine synthase. No relationship was detected between the mRNA expression level of S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50), which did not change between the negative segregant control and the transgenic plants, and spermidine synthase, suggesting the existence of an independent regulatory mechanism for transcription of the two genes. The determination of enzyme activities indicated an increased spermidine synthase and S-adenosylmethionine decarboxylase activity, with the last being mainly recovered in the particulate fraction. ODC (ODC, EC 4.1.1.17) was the most active enzyme and its activity was equally distributed between the soluble and the particulate fraction, while ADC (ADC, EC 4.1.1.19) activity in the transgenic plants did not particularly change with respect to the controls. In comparison to the controls, the transformed plants displayed an increased spermidine to putrescine ratio in the majority of the clones assayed, white the total polyamine content remained almost unchanged. These findings suggest a high capacity of the transformed plants to tightly regulate polyamine endogenous levels and provide evidence that spermidine synthase is not a limiting step in the biosynthesis of polyamines.

Polyamines and somatic embryogenesis in two Vitis vinifera cultivars.

Polyamine content and activities of enzymes of polyamine biosynthesis were assayed during somatic embryogenesis in Vitis vinifera callus cultures of Chardonnay and Brachetto 'a grappolo lungo' (Brachetto g.l.) cultivars. The analyses were carried out on embryogenic callus samples, embryos at different stages and developing plants. Polyamine content, both in the free and PCA-soluble conjugated form, was higher in Brachetto g.l. than in Chardonnay, and putrescine was present at higher concentrations than the other polyamines. In all samples of both cultivars, ornithine decarboxylase activity (ODC, EC 4.1.1.17) was higher than arginine decarboxylase (ADC, EC 4.1.1.19), with a maximum in developing plant roots. S-Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) activity displayed a similar trend. The activities of all three enzymes were detected both in the supernatant and pellet fractions, indicating for the first time the presence of SAMDC activity in the particulate fraction. Particularly in the Chardonnay cultivar, an increase in the mRNAs expression patterns of ODC and SAMDC during morphogenesis from small embryos to plantlets was detected by northern blot, suggesting a direct correlation with enzymatic activities.

Spermidine-binding proteins. Purification and expression analysis in maize.

Polyamine-binding proteins have been identified in a wide range of organisms, including mammals, yeasts, and bacteria. In this work, we have investigated specific spermidine binding to plant membrane proteins purified from microsomes of etiolated maize (Zea mays) coleoptiles. In the final purification step, specific spermidine-binding activity (K(d) 6.02 10(-7) M) was eluted from a HiTrapQ fast-protein liquid chromatography column at about 0.25 M NaCl, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most active fraction showed a major polypeptide of about 60 kD and another copurifying 18-kD protein. Competition experiments, performed on HiTrapQ active fractions, confirmed the specificity of the binding. Upon Sephadex G-100 gel filtration, spermidine binding was associated almost exclusively with the 18-kD protein. On the basis of the N-terminal sequences, degenerate oligonucleotide probes were designed and used to isolate, by reverse transcriptase-polymerase chain reaction and polymerase chain reaction, cDNA fragments of about 1 kb for the 60-kD protein, and 0.9 kb for the 18-kD protein. Northern-blot analysis performed on etiolated coleoptiles and different tissues from 10-d-old maize plants indicated the presence of two different mRNAs of 1.7 and 0.7 kb. Southern-blot analysis indicated that the genes encoding the 60- and 18-kD proteins are probably derived from differential processing of the same precursor mRNA. Using rabbit polyclonal antibodies raised against these proteins, affinity purification and dot-blot experiments detected analogous membrane proteins in monocot and dicot plants.

Alkaloid production in Duboisia hybrid hairy root cultures overexpressing the pmt gene.

Putrescine:SAM N-methyltransferase (PMT) catalyses the N-methylation of the diamine putrescine to form N-methylputrescine, the first specific precursor of both tropane and pyridine-type alkaloids, which are present together in the roots of Duboisia plants. The pmt gene of Nicotiana tabacum was placed under the regulation of the CaMV 35S promoter and introduced into the genome of a scopolamine-rich Duboisia hybrid by a binary vector system using the disarmed Agrobacterium tumefaciens strain C58C1 carrying the rooting plasmid pRiA4. The presence of the foreign gene in kanamycin-resistant hairy roots and its overexpression were confirmed by polymerase chain reaction and Northern blot analysis respectively. The N-methylputrescine levels of the resulting engineered hairy roots increased (2-4-fold) compared to wild type roots, but there was no significant increase in either tropane or pyridine-type alkaloids.

Ozone-response mechanisms in tobacco: implications of polyamine metabolism.

• Polyamines have been suggested to counteract oxidative damage in plants. Here, we present a detailed analysis of polyamine accumulation and its relationship to photosynthetic parameters in two tobacco (Nicotiana tabacum) cultivars (ozone-sensitive Bel W3 and ozone-tolerant Bel B) after a single ozone pulse and after a 1-month exposure in the open air. • Free putrescine accumulated in undamaged tissue of both cultivars, whereas putrescine conjugated to soluble and cell-wall bound components accumulated predominantly in tissue undergoing cell death in Bel W3 plants. Accumulation was caused by a redirection of the conjugation pathway, as well as by a transient increase in arginine decarboxylase and ornithine decarboxylase specific activity. This increase seemed to be regulated at post-transcriptional level. • Measurements of chlorophyll content and fluorescence showed that, in addition to visible necrotic lesions, Bel W3 plants suffered considerable photosynthetic damage in other parts of the leaf. • Accumulation of conjugated putrescine is part of the ozone-induced programmed cell death response in Bel W3 plants. Ozone-induced synthesis of free putrescine is not correlated with ozone-resistance in Bel B plants, which are apparently impaired in signal transduction pathways that are necessary to control the cellular redox state. However, Bel B plants are able to perceive ozone stress and to induce a series of defense mechanisms without activating hypersensitive cell death.

Endogenous polyamine content and metabolism in the ectomycorrhizal fungus Paxillus involutus.

Endogenous polyamine content of the ectomycorrhizal fungus Paxillus involutus, as well as the activity of its biosynthetic enzymes in relation to mycelia ageing were investigated in this work. Polyamines in free, PCA-soluble and insoluble conjugated forms, are present in Paxillus involutus mycelia in relatively high amounts and the ratio of putrescine to spermidine is age-dependent. Both arginine- and ornithine-decarboxylases are present, but putrescine biosynthesis proceeds mostly via ornithine decarboxylase and decreases with the age of mycelia. There was a large release of free polyamines from mycelia which showed age-dependent features. Clear polyamine uptake was observed in 2-wk-old mycelia and no competition between putrescine and cadaverine was detected. Putrescine uptake seems to reduce ornithine decarboxylase activity, but does not affect arginine decarboxylase.