Unraveling the genetics of polyamine metabolism in barley for senescence-related crop improvement.

We explored the polyamine (PA) metabolic pathway genes in barley (Hv) to understand plant development and stress adaptation in Gramineae crops with emphasis on leaf senescence. Bioinformatics and functional genomics tools were utilized for genome-wide identification, comprehensive gene features, evolution, development and stress effects on the expression of the polyamine metabolic pathway gene families (PMGs). Three S-adenosylmethionine decarboxylases (HvSAMDCs), two ornithine decarboxylases (HvODCs), one arginine decarboxylase (HvADC), one spermidine synthase (HvSPDS), two spermine synthases (HvSPMSs), five copper amine oxidases (HvCuAOs) and seven polyamine oxidases (HvPAOs) members of PMGs were identified and characterized in barley. All the HvPMG genes were found to be distributed on all chromosomes of barley. The phylogenetic and comparative assessment revealed that PA metabolic pathway is highly conserved in plants and the prediction of nine H. vulgare miRNAs (hvu-miR) target sites, 18 protein-protein interactions and 961 putative CREs in the promoter region were discerned. Gene expression of HvSAMDC3, HvCuAO7, HvPAO4 and HvSPMS1 was apparent at every developmental stage. SPDS/SPMS gene family was found to be the most responsive to induced leaf senescence. This study provides a reference for the functional investigation of the molecular mechanism(s) that regulate polyamine metabolism in plants as a tool for future breeding decision management systems.

Improvement of cold tolerance in maize (Zea mays L.) using Agrobacterium-mediated transformation of ZmSAMDC gene.

Maize (Zea mays L.) is a food crop sensitive to low temperatures. As one of the abiotic stress hazards, low temperatures seriously affect the yield of maize. However, the genetic basis of low-temperature adaptation in maize is still poorly understood. In this study, maize S-adenosylmethionine decarboxylase (SAMDC) was localized to the nucleus. We used Agrobacterium-mediated transformation technology to introduce the SAMDC gene into an excellent maize inbred line variety GSH9901 and produced a cold-tolerant transgenic maize line. After three years of single-field experiments, the contents of polyamines (PAs), proline (Pro), malondialdehyde (MDA), antioxidant enzymes and ascorbate peroxidases (APXs) in the leaves of the transgenic maize plants overexpressing the SAMDC gene significantly increased, and the expression of elevated CBF and cold-responsive genes effectively increased. The agronomic traits of the maize overexpressing the SAMDC gene changed, and the yield traits significantly improved. However, no significant changes were found in plant height, ear length, and shaft thickness. Therefore, SAMDC enzymes can effectively improve the cold tolerance of maize.

Polyamine synthesis enzyme AMD1 is closely related to the tumorigenesis and prognosis of human breast cancer.

Adenosylmethionine decarboxylase 1 (AMD1) has been implicated in carcinogenesis and tumor progression. However, the potential biomechanism and biological implications of AMD1 in breast cancer (BC) remain unclear. The purpose of this study was to investigate the effect of abnormal expression of AMD1 in BC. The expression of AMD1 in different human BC cell lines was studied by using western blotting and qRT-PCR. In vitro cell proliferation, clone formation, cell cycle and apoptosis assays were performed to explore the effect of AMD1 on cellular proliferation. Xenograft mouse models were established to elucidate the role of AMD1 in BC growth. The expression profiles of AMD1 in 28 pairs of BC tissues and adjacent noncancerous tissues (ANTs) were investigated by using western blotting and immunohistochemistry. The clinical implication and prognostic evaluation of AMD1 in BC were examined by excavating the online database. We found that the expression levels of AMD1 in BC cell lines were significantly higher than those in the normal human breast epithelial cell line MCF-10A. In addition, AMD1 potentiated proliferation, induced cell cycle progression and inhibited apoptosis in BC cells. Subcutaneous tumor xenografts also supported the promotive role of AMD1 in BC growth. We discovered that the level of AMD1 in BC tissues was significantly higher than that in ANTs. Using the online database, increased AMD1 was found to be associated with clinical indicators and predicted a poor prognosis in patients with BC. Our findings indicate that AMD1 elicits potent oncogenic effects on the malignant progression of BC. AMD1 might serve as a promising diagnostic biomarker and therapeutic target for patients with BC.

Radical S-Adenosyl Methionine Enzyme BlsE Catalyzes a Radical-Mediated 1,2-Diol Dehydration during the Biosynthesis of Blasticidin S.

The biosynthesis of blasticidin S has drawn attention due to the participation of the radical S-adenosyl methionine (SAM) enzyme BlsE. The original assignment of BlsE as a radical-mediated, redox-neutral decarboxylase is unusual because this reaction appears to serve no biosynthetic purpose and would need to be reversed by a subsequent carboxylation step. Furthermore, with the exception of BlsE, all other radical SAM decarboxylases reported to date are oxidative in nature. Careful analysis of the BlsE reaction, however, demonstrates that BlsE is not a decarboxylase but instead a lyase that catalyzes the dehydration of cytosylglucuronic acid (CGA) to form cytosyl-4'-keto-3'-deoxy-d-glucuronic acid, which can rapidly decarboxylate nonenzymatically in vitro. Analysis of substrate isotopologs, fluorinated analogues, as well as computational models based on X-ray crystal structures of the BlsE·SAM (2.09 Å) and BlsE·SAM·CGA (2.62 Å) complexes suggests that BlsE catalysis likely proceeds via direct elimination of water from the CGA C4' α-hydroxyalkyl radical as opposed to 1,2-migration of the C3'-hydroxyl prior to dehydration. Biosynthetic and mechanistic implications of the revised assignment of BlsE are discussed.

SAMDC3 enhances resistance to Barley stripe mosaic virus by promoting the ubiquitination and proteasomal degradation of viral γb protein.

Posttranslational modifications (PTMs) play important roles in virus-host interplay. We previously demonstrated that Barley stripe mosaic virus (BSMV) γb protein is phosphorylated by different host kinases to support or impede viral infection. However, whether and how other types of PTMs participate in BSMV infection remains to be explored. Here, we report that S-adenosylmethionine decarboxylase 3 (SAMDC3) from Nicotiana benthamiana or wheat (Triticum aestivum) interacts with γb. BSMV infection induced SAMDC3 expression. Overexpression of SAMDC3 led to the destabilization of γb and reduction in viral infectivity, whereas knocking out NbSAMDC3 increased susceptibility to BSMV. NbSAMDC3 positively regulated the 26S proteasome-mediated degradation of γb via its PEST domain. Further mechanistic studies revealed that γb can be ubiquitinated in planta and that NbSAMDC3 promotes the proteasomal degradation of γb by increasing γb ubiquitination. We also found evidence that ubiquitination occurs at nonlysine residues (Ser-133 and Cys-144) within γb. Together, our results provide a function for SAMDC3 in defence against BSMV infection through targeting of γb abundance, which contributes to our understanding of how a plant host deploys the ubiquitin-proteasome system to mount defences against viral infections.

Genomic analysis of the polyamine biosynthesis pathway in duckweed Spirodela polyrhiza L.: presence of the arginine decarboxylase pathway, absence of the ornithine decarboxylase pathway, and response to abiotic stresses.

MAIN CONCLUSION: Identification of the polyamine biosynthetic pathway genes in duckweed S. polyrhiza reveals presence of prokaryotic as well as land plant-type ADC pathway but absence of ODC encoding genes. Their differential gene expression and transcript abundance is shown modulated by exogenous methyl jasmonate, salinity, and acidic pH. Genetic components encoding for polyamine (PA) biosynthetic pathway are known in several land plant species; however, little is known about them in aquatic plants. We utilized recently sequenced three duckweed (Spirodela polyrhiza) genome assemblies to map PA biosynthetic pathway genes in S. polyrhiza. PA biosynthesis in most higher plants except for Arabidopsis involves two pathways, via arginine decarboxylase (ADC) and ornithine decarboxylase (ODC). ADC-mediated PA biosynthetic pathway genes, namely, one arginase (SpARG1), two arginine decarboxylases (SpADC1, SpADC2), one agmatine iminohydrolase/deiminase (SpAIH), one N-carbamoyl putrescine amidase (SpCPA), three S-adenosylmethionine decarboxylases (SpSAMDc1, 2, 3), one spermidine synthase (SpSPDS1) and one spermine synthase (SpSPMS1) in S. polyrhiza genome were identified here. However, no locus was found for ODC pathway genes in this duckweed. Hidden Markov Model protein domain analysis established that SpADC1 is a prokaryotic/biodegradative type ADC and its molecular phylogenic classification fell in a separate prokaryotic origin ADC clade with SpADC2 as a biosynthetic type of arginine decarboxylase. However, thermospermine synthase (t-SPMS)/Aculis5 genes were not found present. Instead, one of the annotated SPDS may also function as SPMS, since it was found associated with the SPMS phylogenetic clade along with known SPMS genes. Moreover, we demonstrate that S. polyrhiza PA biosynthetic gene transcripts are differentially expressed in response to unfavorable conditions, such as exogenously added salt, methyl jasmonate, or acidic pH environment as well as in extreme temperature regimes. Thus, S. polyrhiza genome encodes for complete polyamine biosynthesis pathway and the genes are transcriptionally active in response to changing environmental conditions suggesting an important role of polyamines in this aquatic plant.

Ectopic Expression of GhSAMDC3 Enhanced Salt Tolerance Due to Accumulated Spd Content and Activation of Salt Tolerance-Related Genes in Arabidopsis thaliana.

Polyamines (PAs), especially spermidine and spermine (which are involved in various types of abiotic stress tolerance), have been reported in many plant species. In this study, we identified 14 putative S-adenosylmethionine decarboxylase genes (GhSAMDC1-14) in upland cotton. Based on phylogenetic and expression analyses conducted under different abiotic stresses, we selected and transferred GhSAMDC3 into Arabidopsis thaliana. Compared to the wild type, transgenic plants displayed rapid growth and increases in average leaf area and leaf number of 52% and 36%, respectively. In transgenic plants, the germination vigor and rate were markedly enhanced under NaCl treatment, and the plant survival rate increased by 50% under 300 mM NaCl treatment. The spermidine content was significantly increased, possibly due to the synthesis of a series of PAs and oxidant and antioxidant genes, resulting in improved salinity tolerance in Arabidopsis. Various salinity resistance-related genes were upregulated in transgenic plants. Together, these results indicate that ectopic expression of GhSAMDC3 raised salinity tolerance by the accumulation of spermidine and activation of salinity tolerance-related genes in A. thaliana.

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.

The regulation of polyamine pathway proteins in models of skeletal muscle hypertrophy and atrophy: a potential role for mTORC1.

Polyamines have been shown to be absolutely required for protein synthesis and cell growth. The serine/threonine kinase, the mechanistic target of rapamycin complex 1 (mTORC1), also plays a fundamental role in the regulation of protein turnover and cell size, including in skeletal muscle, where mTORC1 is sufficient to increase protein synthesis and muscle fiber size, and is necessary for mechanical overload-induced muscle hypertrophy. Recent evidence suggests that mTORC1 may regulate the polyamine metabolic pathway, however, there is currently no evidence in skeletal muscle. This study examined changes in polyamine pathway proteins during muscle hypertrophy induced by mechanical overload (7 days), with and without the mTORC1 inhibitor, rapamycin, and during muscle atrophy induced by food deprivation (48 h) and denervation (7 days) in mice. Mechanical overload induced an increase in mTORC1 signaling, protein synthesis and muscle mass, and these were associated with rapamycin-sensitive increases in adenosylmethione decarboxylase 1 (Amd1), spermidine synthase (SpdSyn), and c-Myc. Food deprivation decreased mTORC1 signaling, protein synthesis, and muscle mass, accompanied by a decrease in spermidine/spermine acetyltransferase 1 (Sat1). Denervation, resulted increased mTORC1 signaling and protein synthesis, and decreased muscle mass, which was associated with an increase in SpdSyn, spermine synthase (SpmSyn), and c-Myc. Combined, these data show that polyamine pathway enzymes are differentially regulated in models of altered mechanical and metabolic stress, and that Amd1 and SpdSyn are, in part, regulated in a mTORC1-dependent manner. Furthermore, these data suggest that polyamines may play a role in the adaptive response to stressors in skeletal muscle.

AMD1 upregulates hepatocellular carcinoma cells stemness by FTO mediated mRNA demethylation.

BACKGROUND: S-adenosylmethionine decarboxylase proenzyme (AMD1) is a key enzyme involved in the synthesis of spermine (SPM) and spermidine (SPD), which are associated with multifarious cellular processes. It is also found to be an oncogene in multiple cancers and a potential target for tumor therapy. Nevertheless, the role AMD1 plays in hepatocellular carcinoma (HCC) is still unknown. METHODS: HCC samples were applied to detect AMD1 expression and evaluate its associations with clinicopathological features and prognosis. Subcutaneous and orthotopic tumor mouse models were constructed to analyze the proliferation and metastasis of HCC cells after AMD1 knockdown or overexpression. Drug sensitive and tumor sphere assay were performed to investigate the effect of AMD1 on HCC cells stemness. Real-time quantitative PCR (qRT-PCR), western blot, immunohistochemical (IHC) and m6A-RNA immunoprecipitation (Me-RIP) sequencing/qPCR were applied to explore the potential mechanisms of AMD1 in HCC. Furthermore, immunofluorescence, co-IP (Co-IP) assays, and mass spectrometric (MS) analyses were performed to verify the proteins interacting with AMD1. RESULTS: AMD1 was enriched in human HCC tissues and suggested a poor prognosis. High AMD1 level could promote SRY-box transcription factor 2 (SOX2), Kruppel like factor 4 (KLF4), and NANOG expression of HCC cells through obesity-associated protein (FTO)-mediated mRNA demethylation. Mechanistically, high AMD1 expression increased the levels of SPD in HCC cells, which could modify the scaffold protein, Ras GTPase-activating-like protein 1 (IQGAP1) and enhance the interaction between IQGAP1 and FTO. This interaction could enhance the phosphorylation and decrease the ubiquitination of FTO. CONCLUSIONS: AMD1 could stabilize the interaction of IQGAP1 with FTO, which then promotes FTO expression and increases HCC stemness. AMD1 shows prospects as a prognostic predictor and a therapeutic target for HCC.

AMD1 is required for the maintenance of leukemic stem cells and promotes chronic myeloid leukemic growth.

Polyamines are critical elements in mammals, but it remains unknown whether adenosyl methionine decarboxylase (AMD1), a rate-limiting enzyme in polyamine synthesis, is required for myeloid leukemia. Here, we found that leukemic stem cells (LSCs) were highly differentiated, and leukemia progression was severely impaired in the absence of AMD1 in vivo. AMD1 was highly upregulated as chronic myeloid leukemia (CML) progressed from the chronic phase to the blast crisis phase, and was associated with the poor prognosis of CML patients. In addition, the pharmacological inhibition of AMD1 by AO476 treatment resulted in a robust reduction of the progression of leukemic cells both in vitro and in vivo. Mechanistically, AMD1 depletion induced loss of mitochondrial membrane potential and accumulation of reactive oxygen species (ROS), resulting in the differentiation of LSCs via oxidative stress and aberrant activation of unfolded protein response (UPR) pathway, which was partially rescued by the addition of polyamine. These results indicate that AMD1 is an essential element in the progression of myeloid leukemia and could be an attractive target for the treatment of the disease.

Biosynthesis of a Novel Bioactive Metabolite of Spermidine from Bacillus amyloliquefaciens: Gene Mining, Sequence Analysis, and Combined Expression.

Spermidine is a biologically active polyamine with extensive application potential in functional foods. However, previously reported spermidine titers by biosynthesis methods are relatively low, which hinders its industrial application. To improve the spermidine titer, key genes affecting the spermidine production were mined to modify Bacillus amyloliquefaciens. Genes of S-adenosylmethionine decarboxylase (speD) and spermidine synthase (speE) from different microorganisms were expressed and compared in B. amyloliquefaciens. Therein, the speD from Escherichia coli and speE from Saccharomyces cerevisiae were confirmed to be optimal for spermidine synthesis, respectively. Gene and amino acid sequence analysis further confirmed the function of speD and speE. Then, these two genes were co-expressed to generate a recombinant strain B. amyloliquefaciens HSAM2(PDspeD-SspeE) with a spermidine titer of 105.2 mg/L, improving by 11.0-fold compared with the control (HSAM2). Through optimization of the fermentation medium, the spermidine titer was increased to 227.4 mg/L, which was the highest titer among present reports. Moreover, the consumption of the substrate S-adenosylmethionine was consistent with the accumulation of spermidine, which contributed to understanding its synthesis pattern. In conclusion, two critical genes for spermidine synthesis were obtained, and an engineering B. amyloliquefaciens strain was constructed for enhanced spermidine production.

Effect of Methionine on AMD1 Gene Expression in Prostate Cancer Cells.

OBJECTIVE: To investigate the effect of AMD1 gene expression on prostate cancer cells (PC-3M), explore the mechanism of AMD1 action in cancer cells, and examine the regulation of AMD1 gene expression by methionine (MET). METHODS: Quantitative PCR (qPCR) and western blot analysis (WB) approaches were used to detect and measure gene expression. The cell apoptotic rate was determined by flow cytometric (FCM) analysis. RESULTS: qPCR and WB assays showed that both AMD1 gene expression and cell apoptotic rate were associated with MET. CONCLUSION: MET has a significant regulatory effect on the expression of the AMD1 gene and a certain amount of MET can promote the expression of the AMD1 gene. This provides a health guideline for a low-methionine diet for prostate cancer patients and scientific evidence for prostate cancer prevention.

In Silico Prediction of Metabolic Fluxes in Cancer Cells with Altered S-adenosylmethionine Decarboxylase Activity.

This paper investigates the redistribution of metabolic fluxes in the cell with altered activity of S-adenosylmethionine decarboxylase (SAMdc, EC: 4.1.1.50), the key enzyme of the polyamine cycle and the common target for antitumor therapy. To address these goals, a stoichiometric metabolic model was developed that includes five metabolic pathways: polyamine, methionine, methionine salvage cycles, folic acid cycle, and the pathway of glutathione and taurine synthesis. The model is based on 51 reactions involving 57 metabolites, 31 of which are internal metabolites. All calculations were performed using the method of Flux Balance Analysis. The outcome indicates that the inactivation of SAMdc results in a significant increase in fluxes through the methionine, the taurine and glutathione synthesis, and the folate cycles. Therefore, when using therapeutic agents inactivating SAMdc, it is necessary to consider the possibility of cellular tumor metabolism reprogramming. S-adenosylmethionine affects serine methylation and activates serine-dependent de novo ATP synthesis. Methionine-depleted cell becomes methionine-dependent, searching for new sources of methionine. Inactivation of SAMdc enhances the transformation of S-adenosylmethionine to homocysteine and then to methionine. It also intensifies the transsulfuration process activating the synthesis of glutathione and taurine.

Understanding the influence of trenbolone acetate and polyamines on proliferation of bovine satellite cells.

Approximately 90% of beef cattle on feed in the United States receive at least one anabolic implant, which results in increased growth, efficiency, and economic return to producers. However, the complete molecular mechanism through which anabolic implants function to improve skeletal muscle growth remains unknown. This study had 2 objectives: (1) determine the effect of polyamines and their precursors on proliferation rate in bovine satellite cells (BSC); and (2) understand whether trenbolone acetate (TBA), a testosterone analog, has an impact on the polyamine biosynthetic pathway. To address these, BSC were isolated from 3 finished steers and cultured. Once cultures reached 75% confluency, they were treated in 1% fetal bovine serum (FBS) and/or 10 nM TBA, 10 mM methionine (Met), 8 mM ornithine (Orn), 2 mM putrescine (Put), 1.5 mM spermidine (Spd), or 0.5 mM spermine (Spe). Initially, a range of physiologically relevant concentrations of Met, Orn, Put, Spd, and Spe were tested to determine experimental doses to implement the aforementioned experiments. One, 12, or 24 h after treatment, mRNA was isolated from cultures and abundance of paired box transcription factor 7 (Pax7), Sprouty 1 (Spry), mitogen-activated protein kinase-1 (Mapk), ornithine decarboxylase (Odc), and S adenosylmethionine (Amd1) were determined, and normalized to 18S. No treatment × time interactions were observed (P ≥ 0.05). Treatment with TBA, Met, Orn, Put, Spd, or Spe increased (P ≤ 0.05) BSC proliferation when compared with control cultures. Treatment of cultures with Orn or Met increased (P ≤ 0.01) expression of Odc 1 h after treatment when compared with control cultures. Abundance of Amd1 was increased (P < 0.01) 1 h after treatment in cultures treated with Spd or Spe when compared with 1% FBS controls. Cultures treated with TBA had increased (P < 0.01) abundance of Spry mRNA 12 h after treatment, as well as increased mRNA abundance of Mapk (P < 0.01) 12 h and 24 h after treatment when compared with 1% FBS control cultures. Treatment with Met increased (P < 0.01) mRNA abundance of Pax7 1 h after treatment as compared with 1% FBS controls. These results indicate that treatments of BSC cultures with polyamines and their precursors increase BSC proliferation rate, as well as abundance of mRNA involved in cell proliferation. In addition, treatment of BSC cultures with TBA, polyamines, or polyamine precursors impacts expression of genes related to the polyamine biosynthetic pathway and proliferation.

Peanut (Arachis hypogaea L.) S-adenosylmethionine decarboxylase confers transgenic tobacco with elevated tolerance to salt stress.

Polyamines play an important role in stress response. In the pathway of polyamines synthesis, S-adenosylmethionine decarboxylase (SAMDC) is one of the key enzymes. In this study, a full length cDNA of SAMDC (AhSAMDC) was isolated from peanut (Arachis hypogaea L.). Phylogenetic analysis revealed high sequence similarity between AhSAMDC and SAMDC from other plants. In peanut seedlings exposed to sodium chloride (NaCl), the transcript level of AhSAMDC in roots was the highest at 24 h that decreased sharply at 72 and 96 h after 150 mM NaCl treatment. However, the expression of AhSAMDC in peanut leaves was significantly inhibited, and the transcript levels in leaves were not different compared with control These results implied the tissue-specific and time-specific expression of AhSAMDC. The physiological effects and functional mechanism of AhSAMDC were further evaluated by overexpressing AhSAMDC in tobaccos. The transgenic tobacco lines exhibited higher germination rate and longer root length under salt stress. Reduced membrane damage, higher antioxidant enzyme activity, and higher proline content were also observed in the transgenic tobacco seedlings. What's more, AhSAMDC also led to higher contents of spermidine and spermine, which can help to scavenge reactive oxygen species. Together, this study suggests that AhSAMDC enhances plant resistance to salt stress by improving polyamine content and alleviating membrane damage.

Podocyte healthy self-eating boosted by a spermidine meal?

The mechanisms sustaining a high level of autophagy in podocytes are not well delineated. Seminal studies had unraveled that the polyamine pathway is involved in the regulation of aging and autophagy. Polyamines (e.g., spermine, spermidine, and putrescine) are ubiquitous molecules essential for the physiological processes, including cell growth, development, and differentiation. Liang et al. examined the role of ornithine decarboxylase, and spermidine synthase, and demonstrated that endogenous spermidine is required to maintain intact podocyte autophagy.

Ectopic expression of GhSAMDC1 improved plant vegetative growth and early flowering through conversion of spermidine to spermine in tobacco.

Polyamines play essential roles in plant development and various stress responses. In this study, one of the cotton S-adenosylmethionine decarboxylase (SAMDC) genes, GhSAMDC1, was constructed in the pGWB17 vector and overexpressed in tobacco. Leaf area and plant height increased 25.9-36.6% and 15.0-27.0%, respectively, compared to the wild type, and flowering time was advanced by 5 days in transgenic tobacco lines. Polyamine and gene expression analyses demonstrated that a decrease in spermidine and an increase in total polyamines and spermine might be regulated by NtSPDS4 and NtSPMS in transgenic plants. Furthermore, exogenous spermidine, spermine and spermidine synthesis inhibitor dicyclohexylamine were used for complementary tests, which resulted in small leaves and dwarf plants, big leaves and early flowering, and big leaves and dwarf plants, respectively. These results indicate that spermidine and spermine are mainly involved in the vegetative growth and early flowering stages, respectively. Expression analysis of flowering-related genes suggested that NtSOC1, NtAP1, NtNFL1 and NtFT4 were upregulated in transgenic plants. In conclusion, ectopic GhSAMDC1 is involved in the conversion of spermidine to spermine, resulting in rapid vegetative growth and early flowering in tobacco, which could be applied to genetically improve plants.

Polyamine biosynthetic pathways and their relation with the cold tolerance of maize (Zea mays L.) seedlings.

BACKGROUND: The present study was designed to investigate the inhibition role of two polyamine biosynthesis inhibitors, i.e., D-arginine (D-Arg) and DL-α-difluoromethylornithine (DFMO), in polyamine biosynthesis under chilling stress in different tissues of two maize inbred lines - Huang C (chilling-tolerance) and Mo17 (chilling-sensitive). RESULTS: The results showed that exposure to the lower concentration of polyamine biosynthesis inhibitors improved seedlings growth, such as the root length, root and shoot fresh weight, chlorophyll a (chl a). The effectiveness of 10 µM D-Arg treatments was more prominent than those of 10 µM DFMO. However, the higher concentration of inhibitors suppressed seedlings growth, and the exposure to 100 µM DFMO caused stronger decreases in the photosynthetic pigments, such as chlorophyll a (chl a), chlorophyll b (chl b), total chlorophyll and carotenoids, than the other treatments. Meanwhile, the inhibitor treatments caused the lower content of putrescine (Put) in roots, mesocotyls and coleoptiles in both maize inbred lines as compared with untreated plants. However, the lower concentration (10 µM) of polyamine biosynthetic inhibitors improved the Spd content, except 10 µM D-Arg in root of Huang C, and 10 µM DFMO in coleoptiles of both Mo17 and Huang C. The correlation analysis found that Spd was positively significantly correlated with root length and shoot fresh weight of seedling. CONCLUSION: It was showed that the Spd played an important role in seedling growth improvement. At the same concentration of polyamine biosynthetic inhibitors, the Put contents in different tissues of the seedlings treated with DFMO were generally lower than those treated with D-Arg, except for Put contents in root of Mo17 with 10 µM treatment. Moreover, the treatments of 100 µM were more prominent than those of 10 µM treatments. Exposure to 100 µM D-Arg and 100 µM DFMO could each decrease the activities of Arginine decarboxylase (ADC), Ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) in all maize tissues. However, the decrease of the ADC activity was more prominent in 100 µM D-Arg-treated seedlings, while the decrease of SAMDC and ODC activities was prominent in 100 µM DFMO-treated seedlings. Genes involved in polyamine biosynthesis, such as ADC, ODC, SAMDC, and PAO, showed different expression patterns in response to chilling stress and polyamine biosynthesis inhibitors. This study suggested that Put was synthesized via both the ADC and ODC pathways after chilling stress, with the ODC pathway being the major one.

Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides.

Stop codon readthrough (SCR) occurs when the ribosome miscodes at a stop codon. Such readthrough events can be therapeutically desirable when a premature termination codon (PTC) is found in a critical gene. To study SCR in vivo in a genome-wide manner, we treated mammalian cells with aminoglycosides and performed ribosome profiling. We find that in addition to stimulating readthrough of PTCs, aminoglycosides stimulate readthrough of normal termination codons (NTCs) genome-wide. Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context all influence the likelihood of SCR. In comparison to NTCs, downstream stop codons in 3'UTRs are recognized less efficiently by ribosomes, suggesting that targeting of critical stop codons for readthrough may be achievable without general disruption of translation termination. Finally, we find that G418-induced miscoding alters gene expression with substantial effects on translation of histone genes, selenoprotein genes, and S-adenosylmethionine decarboxylase (AMD1).

Many genes provide a set of instructions needed to build a protein, which are read by structures called ribosomes through a process called translation. The genetic information contains a short, coded instruction called a stop codon which marks the end of the protein. When a ribosome finds a stop codon it should stop building and release the protein it has made. Ribosomes do not always stop at stop codons. Certain chemicals can actually prevent ribosomes from detecting stop codons correctly, and aminoglycosides are drugs that have exactly this effect. Aminoglycosides can be used as antibiotics at low doses because they interfere with ribosomes in bacteria, but at higher doses they can also prevent ribosomes from detecting stop codons in human cells. When ribosomes do not stop at a stop codon this is called readthrough. There are different types of stop codons and some are naturally more effective at stopping ribosomes than others. Wangen and Green have now examined the effect of an aminoglycoside called G418 on ribosomes in human cells grown in the laboratory. The results showed how ribosomes interacted with genetic information and revealed that certain stop codons are more affected by G418 than others. The stop codon and other genetic sequences around it affect the likelihood of readthrough. Wangen and Green also showed that sequences that encourage translation to stop are more common in the area around stop codons. These findings highlight an evolutionary pressure driving more genes to develop strong stop codons that resist readthrough. Despite this, some are still more affected by drugs like G418 than others. Some genetic conditions, like cystic fibrosis, result from incorrect stop codons in genes. Drugs that promote readthrough specifically in these genes could be useful new treatments.