Nello Bagni

Biosynthesis, oxidation and conjugation of aliphatic polyamines in higher plants

Summary. This chapter will focus on polyamine biosynthesis, oxidation, conjugation processes, mainly to hydroxycinnamic acids, and compartmentation of enzymes, substrates and products, giving an overview about recent results especially in higher plants. New research advances regarding the cloning of the main cDNA encoding for polyamine biosynthetic and oxidative enzymes, will be taken into consideration.

Polyamines: A new class of growth substances

This review on polyamines attempts to present evidence in favour of their role as plant growth substances. Many results suggest that polyamines are implicated in a number of growth and developmental processes in plants, ranging from cell division, embryogenesis, senescence and flowering. However, due to their cationic nature and low molecular weight, polyamines also display effects similar to those of inorganic cations that will be only marginally considered in this short review Finally, their changes in response to stress phenomena and their utilization for alkaloid synthesis are not dealt with and the reader should refer to recent reviews covering these aspects in considerable detail (Bachrach and Heimer, 1989; Flores et al., 1990).

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 μg/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.

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.

Polyamine Pattern and Biosynthesis in Zygotic and Somatic Embryo Stages of Vitis vinifera

Summary A comparative study of free polyamine levels, arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) activities was carried out in the different stages of somatic embryos of Vitis vinifera cv. Grenache noir, and in zygotic embryos of the same cultivar, before and after germination. Somatic embryos showed a high level of free polyamines and a high putrescine/ spermidine ratio. On a per unit basis, an accumulation of putrescine (Put) and spermidine (Spd) occurred in the late torpedo stage, which coincided with the beginning of abnormal growth and disorganized cell proliferation. In globular, heart-shaped and torpedo so matic embryos, ADC activity was higher than ODC activity. Later, in giant somatic embryos, a very high ADC and an even higher ODC activity occurred, except when expressed on a dry weight basis. By contrast, zygotic embryos showed a low level of free polyamines and a Put/Spd ratio approximately equal to one. These data suggest that the abnormal behaviour of grape somatic embryos and their low rate of development into plantlets (

Polyamines and nucleic acids during the first cell cycle of Helianthus tuberosus tissue after the dormancy break

Polyamines (spermine, spermidine, and putrescine) and nucleic acids were studied during the first cell cycle after the break of dormancy of tuber slices of Helianthus tuberosus L., cv. OB1. Immediately after the break of dormancy, a marked decrease in stored arginine and glutamine and a corresponding increase of polyamines were observed. This first synthesis of polyamines were observed. This firs synthesis of polyamines occurred very early during the G1 phase, concomitant to the synthesis of RNAs. A RNA, probably messenger-like RNA, was synthesized very actively only during the first hours of activation in the culture medium plus 2,4-dichlorophenoxyacetic acid, or in water. At the onset of the S phase, after 12h of activation, an incorporation of [3H] thymidine was also detected. A second putrescine synthesis and polyamine accumulation began during the progression of the S phase. During the progression of mitosis, there was a decrease of polyamine synthesis and accumulation.

Long-distance translocation of polyamines in phloem and xylem of Ricinus communis L. plants

Abstract. Polyamine content and enzyme activities in the biosynthetic and degradative pathways of polyamine metabolism were investigated in sieve-tube sap, xylem sap and tissues of seedlings and adult plants of Ricinus communis L. Polyamines were present in tissues and translocation fluids of both seedlings and adult plants in relatively high amounts. Only free polyamines were translocated through the plant, as indicated by the finding that only the free form was detected in the phloem and the xylem sap. Removal of the endosperm increased the polyamine content in the sieve-tube exudate of seedlings. The level and pattern of polyamines in tissue of adult leaves changed during leaf age, but not, however, in the sieve-tube sap. Xylem sap was relatively poor in polyamines. Polyamine loading in the phloem was demonstrated by incubating cotyledons with [14O]putrescine and several unlabelled polyamines. Feeding cotyledons with cadaverine and spermidine led to a decrease in the level of putrescine in sieve-tube sap, indicating a competitive effect. Comparison of polyamine content in the tissue and export rate showed that the export would deplete the leaves of polyamines within 1–3 d, if they were not replenished by biosynthesis. Polyamine biosynthesis in Ricinus proceeds mostly via arginine decarboxylase, which in vitro is 100-fold more active than ornithine decarboxylase. The highest arginine decarboxylase, ornithine decarboxylase and diamine oxidase activities were detected in cotyledons, while in sieve-tube sap only a slight arginine decarboxylase activity was found.

Evidence for the Subcellular Localization of Polyamines and their Biosynthetic Enzymes in Plant Cells

Summary In the present work ornithine-, arginine- and S-adenosylmethionine decarboxylase activities were found in chloroplasts of Pinus radiata D. Don cotyledons and in isolated mitochondria from activated slices of Helianthus tuberosus L. tubers. Buffer molarity and pH optima were determined for each enzyme. In organelles different ratios between free polyamine contents were found with respect to the entire cell. Most of the S-adenosylmethionine decarboxylase activity was found in the 26,000 g supernatant of both plants where arginine and ornithine decarboxylase activities were markedly lower. Arginase activity was also detected in mitochondria. The different ratios between the enzyme activities in the various subcellular fractions of both systems may be an indication that these are effectively compartmentalized.

Daucus carota Cell Cultures: Polyamines and Effect of Polyamine Biosynthesis Inhibitors in the Preembryogenic Phase and Different Embryo Stages

Summary Polyamine content, ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (EC 4.1.1.19) activities, as well as the effects of two inhibitors of putrescine synthesis, were studied during the preembryogenic phase and different stages of somatic embryogenesis in suspension cultures carrots ( Daucus carota L.). In the preembryogenic phase α-difluoromethylornithine at 5mM, an irreversible inhibitor of ornithine decarboxylase, does not reduce growth but causes inhibition of putrescine accumulation; canavanine, a competitive inhibitor of arginine decarboxylase, completely blocks growth in the same phase. Difluoromethylornithine seems to prolong the commitment of the cells to embryogenesis during the preembryogenic phase, while canavanine inhibits the subsequent embryo differentiation. In general, a decrease in polyamine content and higher ornithine decarboxylase as compared to arginine decarboxylase activity is observed during the preembryogenic phase. Difluoromethylornithine does not affect ornithine decarboxylase in vivo , while both difluoromethylornithine and canavanine inhibit ornithine and arginine decarboxylase, respectively, in vitro . Spermidine and spermine contents, expressed on a per embryo basis, increase during the progression from globular to heart and torpedo stages of embryogenesis. While putrescine is the main polyamine in the heart stage, spermidine is more abundant in the torpedo stage. Embryos treated with difluoromethylornithine for 36h develop into normal plantlets at a higher frequency, and they are consistently larger than controls; the canavanine treatment blocks any further development of the embryos, which are always abnormal.

In vitro interactions between polyamines and pectic substances

Putrescine, spermidine and spermine induce a decrease in the pH value of 1 mM polygalacturonic acid or pectin solutions; spermidine and spermine also cause the precipitation of the polymers. The association constants between polyamines and polygalacturonic acid were in the order of 10(5) for putrescine and spermidine, and 10(6) for spermine. The number of galacturonic units per binding sites are proportional to the number of positive charges on the polyamine molecule. Low affinity binding sites appear at high polyamine concentrations. Calcium ions seem to compete weakly with spermine by lowering the association constant 4- to 6-fold. Two natural pectins tested, showed that methylation of the carboxylic groups influences only the number of galacturonic units per site but not the association constant.