Modulation of SAMDC expression in Arabidopsis thaliana alters in vitro shoot organogenesis

S -Adenosylmethionine decarboxylase (SAMDC, EC 4.1.4.50) is a rate-limiting enzyme in the biosynthesis of polyamines (PAs) from putrescine. To gain more insight into the role of PAs in shoot organogenesis, a reverse genetic approach has been used to study in vitro shoot organogenesis by manipulating SAMDC expression in Arabidopsis . Up- and downregulation of SAMDC expression was achieved by transferring sense, antisense and double-stranded Arabidopsis SAMDC complementary DNA constructs back into Arabidopsis via Agrobacterium tumefaciens . Results show that the biosynthesis of PAs and ethylene is mutually antagonistic by manipulation of SAMDC expression. Further results demonstrate that increased shoot organogenesis seems to be directly related to PA accumulation. This effect of PA may be further enhanced with reduced ethylene. It also suggests that spermidine is involved in the process of acquiring organogenesis competence through downregulation of ethylene production and shoot organogenesis, which might result from the concerted action of PAs and plant hormones such as auxin, cytokinins and ethylene.     

Bis(guanylhydrazones) negatively affect in vitro germination of kiwifruit pollen and alter the endogenous polyamine pool

Bis(guanylhydrazones) are a class of compounds known to interfere with the metabolism of polyamines (PAs). Among them, the methylglyoxal derivative (MGBG) has been studied most thoroughly. Because PAs and their biosynthetic enzymes are strongly involved in pollen tube organization, emergence and elongation, a number of these inhibitors have been studied in the present work for their effects on the in vitro performance of kiwifruit (Actinidia deliciosa) pollen. Increasing concentrations of several bis(guanylhydrazones) in the range 0.05-1 mM were checked for their effect on pollen germination. Most of the compounds tested showed a dose-dependent inhibitory effect on tube emergence, which was established very early during incubation. At 0.5 mM, the methylpropylglyoxal derivative (MPGBG) had a stronger inhibitory effect than MGBG. To verify whether the inhibitors reached their metabolic target, PA levels and S-adenosylmethionine decarboxylase (SAMDC) activity were determined in pollen germinated in the presence or absence (controls) of 0.5 mM bis(guanylhydrazones). Spermidine (Spd) content was significantly reduced in the treated pollen, and this effect was more pronounced after treatment with MGBG than with MPGBG. An early and strong reduction in SAMDC activity was observed after exposure to either inhibitor. Inhibition of pollen germination by MGBG or MPGBG could not be reversed by the addition of exogenous Spd, which per se was inhibitory. Taken together, our results suggest that bis(guanylhydrazones) alter PA metabolism and negatively affect kiwifruit pollen germination, even though a strict cause-effect relationship could not be established, and other mechanisms, unrelated to PA activity, must be involved.     

Expression of an antisense Datura stramonium S-adenosylmethionine decarboxylase cDNA in tobacco: changes in enzyme activity, putrescine-spermidine ratio, rhizogenic potential, and response to methyl jasmonate

S-adenosylmethionine decarboxylase activity (SAMDC; EC 4.1.1.21) leads to spermidine and spermine synthesis through specific synthases which use putrescine, spermidine and decarboxylated S-adenosylmethionine as substrates. In order to better understand the regulation of polyamine (PA), namely spermidine and spermine, biosynthesis, a SAMDC cDNA of Datura stramonium was introduced in tobacco (Nicotiana tabacum L. cv. Xanthi) in antisense orientation under the CaMV 35S promoter, by means of Agrobacterium tumefaciens and leaf disc transformation. The effect of the genetic manipulation on PA metabolism, ethylene production and plant morphology was analysed in primary transformants (R0), and in the transgenic progeny (second generation, R1) of self-fertilised primary transformants, relative to empty vector-transformed (pBin19) and wild-type (WT) controls. All were maintained in vitro by micropropagation. Primary transformants, which were confirmed by Southern and northern analyses, efficiently transcribed the antisense SAMDC gene, but SAMDC activity and PA titres did not change. By contrast, in most transgenic R1 shoots, SAMDC activity was remarkably lower than in controls, and the putrescine-to-spermidine ratio was altered, mainly due to increased putrescine, even though putrescine oxidising activity (diamine oxidase, EC 1.4.3.6) did not change relative to controls. Despite the reduction in SAMDC activity, the production of ethylene, which shares with PAs the common precursor SAM, was not influenced by the foreign gene. Some plants were transferred to pots and acclimatised in a growth chamber. In these in vivo-grown second generation transgenic plants, at the vegetative stage, SAMDC activity was scarcely reduced, and PA titres did not change. Finally, the rhizogenic potential of in vitro-cultured leaf explants excised from antisense plants was significantly diminished as compared with WT ones, and the response to methyl jasmonate, a stress-mimicking compound, in terms of PA conjugation, was higher and differentially affected in transgenic leaf discs relative to WT ones. The effects of SAMDC manipulation are discussed in relation to plant generation, culture conditions and response to stress.     

The polycyclic aromatic hydrocarbon phenanthrene causes oxidative stress and alters polyamine metabolism in the aquatic liverwort Riccia fluitans L

The polycyclic aromatic hydrocarbon (PAH) phenanthrene (PHEN) is a highly toxic pollutant, commonly found in aquatic environments, the effects of which on aquatic plants have not been studied in depth. As PAHs are known to induce oxidative stress and recent studies have shown that polyamines (PAs) participate in the defence reactions protecting plants against environmental stresses, PA metabolism and oxidative damage were investigated in the aquatic form of the liverwort Riccia fluitans L. exposed to PHEN. Exposure of Riccia fluitans plants to PHEN at concentrations of 0.5 microm or less induced oxidative stress, but at a level from which plants could recover. Despite increased levels of enzymatic and non-enzymatic antioxidants, recovery appeared, at least in part, due to increased synthesis of PAs, achieved via increased activities of the enzymes arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC). Chemical inhibition of these enzymes inhibited plant recovery, while treatment with PAs aided recovery. Finally, as chloroplasts and the plasma membrane appeared to be key targets for PHEN-induced damage, the potential roles of PAs in protecting these cellular components were considered. How PAs could protect plant cells from serious environmental pollutants such as PHEN and could prevent oxidative stress is discussed.     

Pyrrolizidine alkaloid biosynthesis in Phalaenopsis orchids: developmental expression of alkaloid-specific homospermidine synthase in root tips and young flower buds

Pyrrolizidine alkaloids (PAs) are typical compounds of plant secondary metabolism and are believed to be part of the plant's chemical defense. Within the monocotyledonous plants, PAs have been described in only a few genera, mainly orchids, including Phalaenopsis. Because phylogenetic analyses suggest an independent origin of PA biosynthesis within the monocot lineage, we have analyzed the developmentally regulated expression of homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis, at the cell level. HSS is expressed in the tips of aerial roots exclusively in mitotically active cells. Raphide crystal idioblasts present within the root apical meristem do not show HSS expression. In addition, young flower buds, but not mature flowers, express HSS and have been shown by tracer feeding experiments to be able to catalyze PAs. This second site of PA biosynthesis ensures high concentrations of PAs in the reproductive structures of the Phalaenopsis flower, even after the flower opens. Thus, in spite of its identical function in PA biosynthesis, HSS shows in Phalaenopsis a completely different spatial and developmental expression pattern in comparison to other PA-producing species. These results show that the proverbial diversity of plant secondary metabolism is not just a matter of structural diversity, but is also multifaceted in terms of pathway regulation and expression.     

Pyrrolizidine alkaloids: different acquisition and use patterns in Apocynaceae and Solanaceae feeding ithomiine butterflies (Lepidoptera: Nymphalidae)

Pyrrolizidine alkaloids (PAs) often serve as chemical mediators of plant-herbivore-predator interactions. Butterflies (Danainae and Ithomiinae) and moths (Arctiidae) usually acquire PAs from plant sources (larval host plants, flowers or withered leaves visited by adults—pharmacophagy) and thereby become chemically protected against predators; they also use PAs as pheromone precursors. Study by GC-MS of PAs in three species of Ithomiinae butterflies, their larval host plants and adult alkaloid sources showed three different acquisition patterns: (1) larvae of the primitive Tithorea harmonia sequester PAs from their food plant Prestonia acutifolia (Apocynaceae: Echitoideae), and adults may also acquire these alkaloids from plant sources; (2) larvae of the more derived Aeria olena feed on Prestonia coalita , in whose leaves no PAs were detected, but freshly emerged adults sometimes contain PAs and males intensively seek and sequester these alkaloids in plant sources; and (3) larvae of the still more advanced Mechanitis polymnia feed on several PA-free Solanum species, and adult males sequester the alkaloids from various plant sources. Males and females of all three species contain mostly two PAs, the diastereoisomeric retronecine monoesters lycopsamine and intermedine, stored in the N-oxide form. Larval host plants and adult plant sources showed a large array of PA structures, the most abundant and frequent being lycopsamine and its diastereoisomers intermedine, echinatine, rinderine and indicine, and the deoxy-analogues supinine and amabiline. Bioassays with wild caught and freshly emerged adults suggest that protection against predation by the orb weaving spider Nephila clavipes may be dependent on PA concentration and maybe some spider idiosyncrasies, but freshly emerged Aeria olena without PAs are also liberated by Nephila , suggesting other protective compounds. The role of this spider as a selective pressure for PA acquisition by ithomiines is not clear.     

Frequent gain and loss of pyrrolizidine alkaloids in the evolution of Senecio section Jacobaea (Asteraceae)

Pyrrolizidine alkaloids (PAs) of the macrocyclic senecionine type are secondary metabolites characteristic for most species of the genus Senecio (Asteraceae). These PAs are deterrent and toxic to most vertebrates and insects and provide plants with a chemical defense against herbivores. We studied the PA composition of 24 out of 26 species of Senecio section Jacobaea using GC-MS. The PA profiles of eight of these species have not been studied before and additional PAs were identified for most other species that were included in previous studies. With one exception (senecivernine) all 26 PAs identified in sect. Jacobaea can be regarded as derivatives of the biosynthetic backbone structure senecionine. Based on the PA profiles of the species of sect. Jacobaea and the results of previous tracer studies, we constructed two hypothetical biosynthetic scenarios of senecionine diversification. Both scenarios contain two major reactions: the conversion of the necine base moiety retronecine into the otonecine moiety and site-specific epoxidations within the necic acid moiety. Further reactions are site-specific hydroxylations, sometimes followed by O-acetylations, site-specific dehydrogenations, E, Z-isomerizations, and epoxide hydrolysis and chlorolysis. The GC-MS data and both biosynthetic scenarios were subsequently used to study the evolution of PA formation in sect. Jacobaea by reconstructing the evolutionary history of qualitative PA variation in this section. This was achieved by optimizing additive presence/absence data of PAs and types of enzymatic conversions on a maximum parsimony cladogram of section Jacobaea inferred from DNA sequence and morphological data. Besides showing large intra- and interspecific variation, PA distribution appears to be largely incidental within the whole clade. These results together with the finding that all but one of the PAs identified in sect. Jacobaea are also present in species of other sections of Senecio indicate that differences in PA profiles in Senecio can not be explained by the gain and loss of PA specific genes, but rather by a transient switch-off and switch-on of the expression of genes encoding PA pathway-specific enzymes.