The Polyamine Regulator AMD1 Upregulates Spermine Levels to Drive Epidermal Differentiation.

Maintaining tissue homeostasis depends on a balance between cell proliferation, differentiation, and apoptosis. Within the epidermis, the levels of the polyamines putrescine, spermidine, and spermine are altered in many different skin conditions, yet their role in epidermal tissue homeostasis is poorly understood. We identify the polyamine regulator, Adenosylmethionine decarboxylase 1 (AMD1), as a crucial regulator of keratinocyte (KC) differentiation. AMD1 protein is upregulated on differentiation and is highly expressed in the suprabasal layers of the human epidermis. During KC differentiation, elevated AMD1 promotes decreased putrescine and increased spermine levels. Knockdown or inhibition of AMD1 results in reduced spermine levels and inhibition of KC differentiation. Supplementing AMD1-knockdown KCs with exogenous spermidine or spermine rescued aberrant differentiation. We show that the polyamine shift is critical for the regulation of key transcription factors and signaling proteins that drive KC differentiation, including KLF4 and ZNF750. These findings show that human KCs use controlled changes in polyamine levels to modulate gene expression to drive cellular behavior changes. Modulation of polyamine levels during epidermal differentiation could impact skin barrier formation or can be used in the treatment of hyperproliferative skin disorders.

Overexpression of a S-Adenosylmethionine Decarboxylase from Sugar Beet M14 Increased Araidopsis Salt Tolerance.

Polyamines play an important role in plant growth and development, and response to abiotic stresses. Previously, differentially expressed proteins in sugar beet M14 (BvM14) under salt stress were identified by iTRAQ-based quantitative proteomics. One of the proteins was an S-adenosylmethionine decarboxylase (SAMDC), a key rate-limiting enzyme involved in the biosynthesis of polyamines. In this study, the BvM14-SAMDC gene was cloned from the sugar beet M14. The full-length BvM14-SAMDC was 1960 bp, and its ORF contained 1119 bp encoding the SAMDC of 372 amino acids. In addition, we expressed the coding sequence of BvM14-SAMDC in Escherichia coli and purified the ~40 kD BvM14-SAMDC with high enzymatic activity. Quantitative real-time PCR analysis revealed that the BvM14-SAMDC was up-regulated in the BvM14 roots and leaves under salt stress. To investigate the functions of the BvM14-SAMDC, it was constitutively expressed in Arabidopsis thaliana. The transgenic plants exhibited greater salt stress tolerance, as evidenced by longer root length and higher fresh weight and chlorophyll content than wild type (WT) under salt treatment. The levels of spermidine (Spd) and spermin (Spm) concentrations were increased in the transgenic plants as compared with the WT. Furthermore, the overexpression plants showed higher activities of antioxidant enzymes and decreased cell membrane damage. Compared with WT, they also had low expression levels of RbohD and RbohF, which are involved in reactive oxygen species (ROS) production. Together, these results suggest that the BvM14-SAMDC mediated biosynthesis of Spm and Spd contributes to plant salt stress tolerance through enhancing antioxidant enzymes and decreasing ROS generation.

Cotton S-adenosylmethionine decarboxylase-mediated spermine biosynthesis is required for salicylic acid- and leucine-correlated signaling in the defense response to Verticillium dahliae.

MAIN CONCLUSION: Cotton S-adenosylmethionine decarboxylase-, rather than spermine synthase-, mediated spermine biosynthesis is required for salicylic acid- and leucine-correlated signaling in the defense response to Verticillium dahliae. Spermine (Spm) signaling is correlated with plant resistance to the fungal pathogen Verticillium dahliae. We identified genes for key rate-limiting enzymes in the biosynthesis of Spm, namely S-adenosylmethionine decarboxylase (GhSAMDC) and Spm synthase (GhSPMS). These were found by screening suppression subtractive hybridization and cDNA libraries of cotton (Gossypium) species tolerant to Verticillium wilt. Both were induced early and strongly by inoculation with V. dahliae and application of plant hormones. Silencing of GhSPMS or GhSAMDC in cotton leaves led to a significant accumulation of upstream substrates and, ultimately, enhanced plant susceptibility to Verticillium infection. Exogenous supplementation of Spm to the silenced cotton plants improved resistance. When compared with the wild type (WT), constitutive expression of GhSAMDC in Arabidopsis thaliana was associated with greater Verticillium wilt resistance and higher accumulations of Spm, salicylic acid, and leucine during the infection period. By contrast, transgenic Arabidopsis plants that over-expressed GhSPMS were unexpectedly more susceptible than the WT to V. dahliae and they also had impaired levels of putrescine (Put) and salicylic acid (SA). The susceptibility exhibited in GhSPMS-overexpressing Arabidopsis plants was partially reversed by the exogenous supply of Put or SA. In addition, the responsiveness of those two transgenic Arabidopsis lines to V. dahliae was associated with an alteration in transcripts of genes involved in plant resistance to epidermal penetrations and amino acid signaling. Together, these results suggest that GhSAMDC-, rather than GhSPMS-, mediated spermine biosynthesis contributes to plant resistance against V. dahliae through SA- and leucine-correlated signaling.

Gene polymorphisms in the ornithine decarboxylase-polyamine pathway modify gastric cancer risk by interaction with isoflavone concentrations.

BACKGROUND: The study aimed to examine the association between genes encoding molecules in the ornithine decarboxylase (ODC)-polyamine pathway (ODC1, AMD1, NQO1, NOS2A, and OAZ2) and gastric cancer risk and whether the gene-phytoestrogen interaction modifies gastric cancer risk. METHODS: Among 76 gastric cancer cases and their 1:4 matched controls within the Korean Multi-center Cancer Cohort, a total of 30 SNPs in five genes involved in the ODC pathway were primarily analyzed. The second-stage genotyping in 388 matched case-control sets was conducted to reevaluate the significant SNPs interacting with phytoestrogens during the primary analysis. The summary odds ratios (ORs) [95 % confidence intervals (CIs)] for gastric cancer were estimated. Interaction effects between the SNPs and plasma concentrations of phytoestrogens (genistein, daidzein, equol, and enterolactone) were evaluated. RESULTS: In the pooled analysis, NQO1 rs1800566 showed significant genetic effects on gastric cancer without heterogeneity [OR 0.83 (95 % CI 0.70-0.995)] and a greater decreased risk at high genistein/daidzein levels [OR 0.36 (95 % CI 0.15-0.90) and OR 0.26 (95 % CI 0.10-0.64), respectively; p interaction < 0.05]. Risk alleles of AMD1 rs1279599, AMD1 rs7768897, and OAZ2 rs7403751 had a significant gene-phytoestrogen (genistein and daidzein) interaction effect to modify the development of gastric cancer. They had an increased gastric cancer risk at low isoflavone levels, but a decreased risk at high isoflavone levels (p interaction < 0.01). CONCLUSIONS: Our findings suggest that common variants in the genes involved in the ODC pathway may contribute to the risk of gastric cancer possibly by modulating ODC polyamine biosynthesis or by interaction between isoflavones and NQO1, OAZ2, and AMD1.

Methionine deficiency does not increase polyamine turnover through depletion of hepatic S-adenosylmethionine in juvenile Atlantic salmon.

During the last few decades, plant protein ingredients such as soya proteins have replaced fishmeal in the diets of aquacultured species. This may affect the requirement and metabolism of methionine as soya contains less methionine compared with fishmeal. To assess whether methionine limitation affects decarboxylated S-adenosylmethionine availability and polyamine status, in the present study, juvenile Atlantic salmon were fed a methionine-deficient plant protein-based diet or the same diet supplemented with dl-methionine for 8 weeks. The test diets were compared with a fishmeal-based control diet to assess their effects on the growth performance of fish. Methionine limitation reduced growth and protein accretion, but when fish were fed the dl-methionine-supplemented diet their growth and protein accretion equalled those of fish fed the fishmeal-based control diet. Methionine limitation reduced free methionine concentrations in the plasma and muscle, while those in the liver were not affected. S-adenosylmethionine (SAM) concentrations were higher in the liver of fish fed the methionine-deficient diet, while S-adenosylhomocysteine concentrations were not affected. Putrescine concentrations were higher and spermine concentrations were lower in the liver of fish fed the methionine-deficient diet, while the gene expression of SAM decarboxylase (SAMdc) and the rate-limiting enzyme of polyamine synthesis ornithine decarboxylase (ODC) was not affected. Polyamine turnover, as assessed by spermine/spermidine acetyltransferase (SSAT) abundance, activity and gene expression, was not affected by treatment. However, the gene expression of the cytokine TNF-α increased in fish fed the methionine-deficient diet, indicative of stressful conditions in the liver. Even though taurine concentrations in the liver were not affected by treatment, methionine and taurine concentrations in muscle decreased due to methionine deficiency. Concomitantly, liver phospholipid and cholesterol concentrations were reduced, while NEFA concentrations were elevated. In conclusion, methionine deficiency did not increase polyamine turnover through depletion of hepatic SAM, as assessed by SSAT activity and abundance.

[Molecular cloning and characterization of S-adenosyl-L-methionine decarboxylase gene (DoSAMDC1) in Dendrobium officinale].

S-Adenosyl-L-methionine decarboxylase (SAMDC) is a key enzyme in the polyamines biosynthesis, thus is essential for basic physiological and biochemical processes in plant. In the present study, a full length cDNA of DoSAMDC1 gene was obtained from symbiotic germinated seeds of an endangered medicinal orchid species Dendrobium officinale, using the rapid amplification of cDNA ends (RACE)-PCR technique for the first time. The full length cDNA was 1 979 bp, with three open reading frames, i.e. tiny-uORF, small-uORF and main ORF (mORF). The mORF was deduced to encode a 368 amino acid (aa) protein with a molecular mass of 40.7 kD and a theoretical isoelectric point of 5.2. The deduced DoSAMDC1 protein, without signal peptide, had two highly conserved function domains (proenzyme cleavage site and PEST domain) and a 22-aa transmembrane domain (89-110). Multiple sequence alignments and phylogenetic relationship analyses revealed DoSAMDC1 had a higher level of sequence similarity to monocot SAMDCs than those of dicot. Expression patterns using qRT-PCR analyses showed that DoSAMDC1 transcripts were expressed constitutively without significant change in the five tissues (not infected with fungi). While in the symbiotic germinated seeds, the expression level was enhanced by 2.74 fold over that in the none-germinated seeds, indicating possible involvement of the gene in symbiotic seed germination of D. officinale.

RNAi silencing of three homologues of S-adenosylmethionine decarboxylase gene in tapetal tissue of tomato results in male sterility.

Polyamines play very important role in various cellular metabolic functions, including floral induction, floral differentiation and fertility regulation. In the present study, S-adenosylmethionine decarboxylase (SAMDC), a key gene involved in polyamine biosynthesis, has been targeted in tapetal tissue of tomato using RNAi to examine its effect on tapetum development and pollen viability. The target SAMDC gene fragments of three homologues were cloned in a hairpin RNA construct under the control of tapetal-specific A9 promoter, which was used to generate several RNAi tomato plants. These RNAi lines expressed the intended small interfering RNAs in the anther and showed the aborted and sterile pollen exhibiting shrunken and distorted morphology. These RNAi tomato plants having sterile pollen, failed to set fruits but female fertility of the plants remained unaffected as cross pollination resulted in fruit setting. Expression profiling of SAMDC genes showed considerable decrease in transcripts of SAMDC1 (5-8 fold) and SAMDC2 and SAMDC3 (2-3 fold) in the anthers of RNAi plants. The other polyamine biosynthesis genes, ADC and SPDSYN exhibited ~1.5 fold decrease in their transcript levels. Presence of siRNA molecules specific to SAMDC homologues in anther and tapetal-specific activity of A9 promoter as shown with GUS reporter system of RNAi plants suggested down-regulation of the target genes in tapetum by RNAi. These observations indicate the importance of SAMDC, in turn polyamines in pollen development, and thus tapetum-specific down-regulation of SAMDC genes using RNAi can be used for developing male sterile plants.

Identification of the primary structure and post-translational modification of rat S-adenosylmethionine decarboxylase.

The coding region nucleotide sequences of rat, hamster, and bovine S-adenosylmethionine decarboxylase (AdoMetDC) cDNA exhibit over 90% homology with the human sequence. No N-terminal amino acid could be detected when either bovine or rat AdoMetDC was subjected to Edman degradation, suggesting that the beta-subunit must be blocked since the pyruvate residue is located at the amino terminus of the alpha-subunit. In this study, we present the primary structure, including post-translational modification, of rat prostate AdoMetDC. Our strategy was to compare the molecular masses of peptides produced by five specific cleavage methods with peptides expected from the known cDNA-derived amino acid sequence of rat AdoMetDC using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). All AdoMetDC peptide fragments produced by the five cleavage methods could be assigned to theoretical peptides based on the rat cDNA sequence except for the peptides containing the N-terminus of the beta- and alpha-subunits. The N-terminus of the alpha-subunit was assigned as pyruvoyl peptide. Liberation of acetylmethionine was demonstrated when the peptide containing the beta-subunit N-terminal amino acid obtained by lysylendopeptidase digestion was reacted with acylamino acidreleasing enzyme. Furthermore, N-terminal acetylation of the beta-subunit was confirmed by MALDI-post source decay analysis. In conclusion, the results of the present study on amino acid full sequence of rat prostate AdoMetDC determined by the combination of five specific cleavage methods demonstrate that the N-terminus of the beta-subunit is acetylated, and the expected amino acid sequence based on the rat AdoMetDC cDNA sequence is correct.

Polyamine biosynthesis and control of the development of functional pollen in kiwifruit.

The role of polyamines (PAs) in plant reproduction, especially pollen development and germination has been demonstrated in several higher plants. The aim of the present research was to investigate PA involvement in pollen development and germination in dioecious kiwifruit (Actinidia deliciosa). Differences in PA content, level and gene expression for PA biosynthetic enzymes, and the effect of PA biosynthetic inhibitors were found during pollen development (or abortion in female flowers). Whereas PAs, especially spermidine (Spd), remained high throughout the development of functional pollen, the levels collapsed by the last stage of development of sterile pollen. Mature and functional pollen from male-fertile anthers showed S-adenosyl methionine decarboxylase activity (SAMDC; involved in Spd biosynthesis) throughout microgametogenesis, with high levels of soluble SAMDC found starting from the late uninucleate microspore stage. Soluble SAMDC was absent in male-sterile anthers. Arginine decarboxylase [ADC; for putrescine (Put) biosynthesis] showed little difference in functional vs sterile pollen; ornithine decarboxylase [ODC; also for putrescine (Put) biosynthesis] was present only in sterile pollen. Ultrastructural studies of aborted pollen grains in male-sterile flowers showed that cytoplasmic residues near the intine contain vesicles, extruding towards the pollen wall. Very high SAMDC activity was found in the wall residues of the aborted pollen. The combined application in planta of competitive inhibitors of S-adenosylmethionine decarboxylase (MGBG) and of spermidine synthase (CHA), or of D-arginine (inhibitor of Put synthesis), to male-fertile plants led to abnormal pollen grains with reduced viability. The importance of PAs during male-fertile pollen germination was also found. In fact, PA biosynthetic enzymes (ADC and, mainly, SAMDC) were active early during pollen hydration and germination in vitro. Two different SAMDC gene transcripts were expressed in germinating pollen together with a lower level of ADC transcript. Gene expression preceded PA enzyme activity. The application of PA inhibitors in planta drastically reduced pollen germination. Thus, low free Spd can lead either to degeneration or loss of functionality of kiwifruit pollen grains.

Transcriptional regulation of the rice arginine decarboxylase (Adc1) and S-adenosylmethionine decarboxylase (Samdc) genes by methyl jasmonate.

We investigated the effect of methyl jasmonate (MeJa) treatment on the expression of two genes in the rice polyamine biosynthesis pathway and on the polyamine content in wild type plants and transgenic rice plants expressing a Datura stramonium (Ds) Adc cDNA, the latter accumulating up to three-fold the normal level of putrescine. Exogenous MeJa transiently inhibited the expression of OsAdc1, OsSamdc and Spermidine synthase (OsSpds) genes in the polyamine biosynthesis pathway, probably through transcriptional repression. There was also a similar negative impact on the DsAdc transgene in transgenic plants, even though a constitutive promoter was used to drive transgene expression. The free putrescine content was reduced significantly in the leaves of both wild type and transgenic plants in response to MeJa, although the magnitude of the effect was greater in wild type plants. We discuss our findings with respect to the previously proposed threshold model of polyamine metabolism in plants subjected to abiotic stress.

Overexpression of carnation S-adenosylmethionine decarboxylase gene generates a broad-spectrum tolerance to abiotic stresses in transgenic tobacco plants.

Polyamines (PAs), such as putrescine, spermidine, and spermine, are present in all living organism and implicate in a wide range of cellular physiological processes. We have used transgenic technology in an attempt to evaluate their potential for mitigating the adverse effects of several abiotic stresses in plants. Sense construct of full-length cDNA for S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in PA biosynthesis, from carnation (Dianthus caryophyllus L.) flower was introduced into tobacco (Nicotiana tabacum L.) by Agrobacterium tumefaciens-mediated transformation. Several transgenic lines overexpressing SAMDC gene under the control of cauliflower mosaic virus 35S promoter accumulated soluble total PAs by 2.2 (S16-S-4) to 3.1 (S16-S-1) times than wild-type plants. The transgenic tobacco did not show any difference in organ phenotype compared to the wild-type. The number and weight of seeds increased, and net photosynthetic rate also increased in transgenic plants. Stress-induced damage was attenuated in these transgenic plants, in the symptom of visible yellowing and chlorophyll degradation after all experienced stresses such as salt stress, cold stress, acidic stress, and abscisic acid treatment. H2O2-induced damage was attenuated by spermidine treatment. Transcripts for antioxidant enzymes (ascorbate peroxidase, manganase superoxide dismutase, and glutathione S-transferase) in transgenic plants and GUS activity transformed with SAMDC promoter::GUS fusion were induced more significantly by stress treatment, compared to control. These results that the transgenic plants with sense SAMDC cDNA are more tolerant to abiotic stresses than wild-type plants suggest that PAs may play an important role in contributing stress tolerance in plants.

Molecular cloning and functional characterization of two apple S-adenosylmethionine decarboxylase genes and their different expression in fruit development, cell growth and stress responses.

Two full-length S-adenosylmethionine decarboxylase (SAMDC) cDNAs, MdSAMDC1 and MdSAMDC2, were isolated from apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. Both cDNAs encoded tiny and small ORFs in addition to the SAMDC ORFs, and genomic sequences of MdSAMDC1 and MdSAMDC2 contained two or three introns in the 5' upstream regions, respectively. Yeast complementation experiment indicated that two MdSAMDCs encoded functional proteins, and that the tiny and small ORFs possibly repressed their translation efficiency. RNA gel blot analysis showed that MdSAMDC1 were differentially regulated in fruits depending on the developmental stage and in cell suspension during the culture period, but MdSAMDC2 did not. In contrast, MdSAMDC2 was positively induced by cold and salt stresses, but MdSAMDC1 was not. These results suggest that MdSAMDC1 is mainly involved in fruit development and cell growth while MdSAMDC2 in stress responses, compared with their respective counterpart.

A leader intron and 115-bp promoter region necessary for expression of the carnation S-adenosylmethionine decarboxylase gene in the pollen of transgenic tobacco.

The expression of CSDC9 encoding S-adenosylmethionine decarboxylase (SAMDC) is developmentally and spatially regulated in carnation. To examine the regulation of the SAMDC gene, we analyzed the spatial expression of CSDC9 with a 5'-flanking beta-glucuronidase fusion in transgenic tobacco plants. GUS was strongly expressed in flower, pollen, stem and vein of cotyledons. Expression in both anther and stigma was under developmental control; analysis of a series of mutants with deletions of the 5'-flanking region demonstrated differential activation in petal, anther, stigma and pollen grains. All the major cis-regulatory elements required for pollen-specific transcription were located in the upstream region between -273 and -158. This region contains four putative elements related to gibberellin induction (pyrimidine boxes, TTTTTTCC and CCTTTT) and pollen-specific expression (GTGA and AGAAA). In addition, the first 5'-leader intron was necessary for tissue-specific expression.

Evolutionary links as revealed by the structure of Thermotoga maritima S-adenosylmethionine decarboxylase.

S-adenosylmethionine decarboxylase (AdoMetDC) is a critical regulatory enzyme of the polyamine biosynthetic pathway and belongs to a small class of pyruvoyl-dependent amino acid decarboxylases. Structural elucidation of the prokaryotic AdoMetDC is of substantial interest in order to determine the relationship between the eukaryotic and prokaryotic forms of the enzyme. Although both forms utilize pyruvoyl groups, there is no detectable sequence similarity except at the site of pyruvoyl group formation. The x-ray structure of the Thermatoga maritima AdoMetDC proenzyme reveals a dimeric protein fold that is remarkably similar to the eukaryotic AdoMetDC protomer, suggesting an evolutionary link between the two forms of the enzyme. Three key active site residues (Ser55, His68, and Cys83) involved in substrate binding, catalysis or proenzyme processing that were identified in the human and potato AdoMet-DCs are structurally conserved in the T. maritima AdoMetDC despite very limited primary sequence identity. The role of Ser55, His68, and Cys83 in the self-processing reaction was investigated through site-directed mutagenesis. A homology model for the Escherichia coli AdoMetDC was generated based on the structures of the T. maritima and human AdoMetDCs.

Genomic characterization of the S-adenosylmethionine decarboxylase genes from soybean.

A full-length gene GmSAMDC1, encoding the S-adenosylmethionine decarboxylase (SAMDC), a key enzyme involved in polyamine biosynthesis, was identified from soybean expressed sequence tags and was characterized. GmSAMDC1 encoded a peptide of 355 amino acids. When compared with other plant SAMDCs, the GmSAMDC1 protein had several highly conserved regions including a putative pro-enzyme cleavage site and a PEST sequence. The 5' leader sequence of the the GmSAMDC1 mRNA contained two additional open reading frames (ORFs), which may regulate the translational process. The genomic sequence of the GmSAMDC1 gene contained three introns in the 5' leader sequence, but no intron in the 3'-UTR or the main pro-enzyme ORF. A simple sequence repeat (SSR) was found in intron 2, and the GmSAMDC1 gene was mapped to linkage group D1 using this SSR. The genomic organization of the GmSAMDC1 gene in the subgenus Glycine and the subgenus Soja was found to be different by Southern-blot and PCR analysis. A pseudogene, GmSAMDC2, was also identified. This gene contained no intron and lost its two uORFs. Northern-blot analysis showed that the GmSAMDC1 gene expression was induced by salt, drought and cold, but not induced by wounding; suggesting that the gene was implicated in response to multiple-stress conditions.

Structural roles of cysteine 50 and cysteine 230 residues in Arabidopsis thaliana S-adenosylmethionine decarboxylase.

The Arabidopsis thaliana S-Adenosylmethionine decarboxylase (AdoMetDC) cDNA (GenBank U63633) was cloned. Site-specific mutagenesis was performed to introduce mutations at the conserved cysteine Cys(50), Cys(83), and Cys(230), and lys(81) residues. In accordance with the human AdoMetDC, the C50A and C230A mutagenesis had minimal effect on catalytic activity, which was further supported by DTNB-mediated inactivation and reactivation. However, unlike the human AdoMetDC, the Cys(50) and Cys(230) mutants were much more thermally unstable than the wild type and other mutant AdoMetDC, suggesting the structural significance of cysteines. Furthermore, according to a circular dichroism spectrum analysis, the Cys(50) and Cys(230) mutants show a higher a-helix content and lower coiled-coil content when compared to that of wild type and the other mutant AdoMetDC. Also, the three-dimensional structure of Arabidopsis thaliana AdoMetDC could further support all of the data presented here. Summarily, we suggest that the Cys(50) and Cys(230) residues are structurally important.

Expression of a heterologous S-adenosylmethionine decarboxylase cDNA in plants demonstrates that changes in S-adenosyl-L-methionine decarboxylase activity determine levels of the higher polyamines spermidine and spermine.

We posed the question of whether steady-state levels of the higher polyamines spermidine and spermine in plants can be influenced by overexpression of a heterologous cDNA involved in the later steps of the pathway, in the absence of any further manipulation of the two synthases that are also involved in their biosynthesis. Transgenic rice (Oryza sativa) plants engineered with the heterologous Datura stramonium S-adenosylmethionine decarboxylase (samdc) cDNA exhibited accumulation of the transgene steady-state mRNA. Transgene expression did not affect expression of the orthologous samdc gene. Significant increases in SAMDC activity translated to a direct increase in the level of spermidine, but not spermine, in leaves. Seeds recovered from a number of plants exhibited significant increases in spermidine and spermine levels. We demonstrate that overexpression of the D. stramonium samdc cDNA in transgenic rice is sufficient for accumulation of spermidine in leaves and spermidine and spermine in seeds. These findings suggest that increases in enzyme activity in one of the two components of the later parts of the pathway leading to the higher polyamines is sufficient to alter their levels mostly in seeds and, to some extent, in vegetative tissue such as leaves. Implications of our results on the design of rational approaches for the modulation of the polyamine pathway in plants are discussed in the general framework of metabolic pathway engineering.

S-adenosylmethionine decarboxylase from Leishmania infantum promastigotes: molecular cloning and differential expression.

S-Adenosylmethionine decarboxylase (AdoMetDC), an enzyme involved in the synthesis of polyamines as well as in the cell methylation processes, has been considered in trypanosomes as a specific drug target. We have cloned by RT-PCR a DNA fragment of 1,364 bp which contains the open reading frame and the 5' end fragment of the AdoMetDC encoding gene from the parasite protozoon Leishmania infantum. The 1,197 bp ORF encodes for a 392 amino acid residue polypeptide. The sequence comparison with AdMetDC from different species showed a high level of homology, around 80%. with the American and African trypanosomes and a certain distance from the polypeptides of higher eukaryotes. AdoMetDC has been cloned in a pQE32 vector and overexpressed in a M15 Escherichia coli strain. The gene expression shows variations between the distinct phases of the parasite, being higher in the most infective one. This fact may be related to the multiple defense mechanism of the protozoon against the macrophage action.

Expression of the pea S-adenosylmethionine decarboxylase gene is involved in developmental and environmental responses.

A cDNA, able to complement the S-adenosyl-L-methionine decarboxylase (SAMdC; EC 4.1.1.50)-defective yeast strain Y342, has been isolated from pea (Pisum sativum L.). Expression of the SAMdC gene was characterised during pea development. Northern analysis showed a differential expression of the pea SAMdC gene in vegetative and reproductive tissues. The highest SAMdC mRNA levels were found in undifferentiated callus and tissues with high rates of cell division, and at the onset of fruit development. SAMdC expression was also induced in senescing ovaries, probably in relation to an accumulation of spermine during ovary senescence. Finally, the levels of SAMdC transcripts in leaves and shoots were differentially affected by ozone treatment.

Gene structure and chromosomal localization of mouse S-adenosylmethionine decarboxylase.

The structure of the mouse S-adenosylmethionine decarboxylase (AdoMetDC) gene has been determined. The mouse gene (AMD1) consisted of eight exons and seven introns, similar to the rat AdoMetDC gene, and was mapped to chromosome 10. The characteristics of AMD1 gene were as follows: (1) The region of the promoter necessary for maximal transcriptional activity was located about 400 nucleotides upstream of the transcriptional initiation point, and contained a TATA box and two GC boxes. The transcriptional activity of the promoter was nearly equal to that of the SV40 promoter. (2) Two polyadenylation signals for transcription were observed, and the larger AdoMetDC mRNA, which is the dominant form of mRNA, corresponded to mRNA that is generated using the second polyadenylation signal. (3) Using stable transfectants, we confirmed that the upstream open reading frame (uORF) in the 5'-untranslated region (5'-UTR) of AdoMetDC mRNA functioned as a negative regulatory element. Lower concentrations of polyamines affect both stimulation and inhibition of AdoMetDC synthesis, through the uORF in the mRNA, than affect general protein synthesis.