Polyamine Biosynthesis In Plants Research Articles

Cloning and abiotic stress responsive expression analysis of Arginine decarboxylase genes in contrasting rice genotypes

Arginine decarboxylase (ADC) is the first enzyme of polyamine biosynthesis in plants, an important mediator of abiotic stress tolerance. Two genes OsADC1 and OsADC2 were found to be differentially expressed under various abiotic stresses namely salinity, drought, low temperature and high temperature. Significant differences in gene expression were found among contrasting rice genotypes Nerica-L-44 (NL44; tolerant) and Pusa Sugandh 2 (PS2; sensitive). Among the homologs, OsADC2 was induced frequently in abiotic stresses with a higher transcript level than OsADC1. When the stress dependent gene expression was estimated relative to control conditions, PS2 showed a significant and higher level of induction. The estimation of relative gene expression between genotypes for each stress in all shoot tissues showed significantly higher level of expression in NL44 than PS2. In roots, the stress induced expression was higher in the sensitive genotype PS2. Construction of phylogenetic tree provided an insight on the evolution of OsADC gene from lower to higher organisms. The OsADC2 gene was found to be highly diverged from OsADC1 as well as from the counterparts of related and distant taxa. The analysis of amino acid sequence identified the conserved substrate binding, cofactor binding and dimerisation domains essential for enzyme activity.

Expression of SAMDC Gene for Enhancing the Shelf Life for Improvement of Fruit Quality Using Biotechnological Approaches into Litchi (<i>Litchi chinensis</i> Sonn.) Cultivars

Polyamines play an important role in plant response to abiotic stress. S-adenosyl-1-methionine decarboxylase (SAMDC) is one of the key regulatory enzymes in the biosynthesis of polyamines. In order to better understand the effect of regulation of polyamine biosynthesis on the shelf life improvement of litchi fruit, SAMDC cDNA isolated from Datura stramonium cloned in pBI121 was introduced into litchi genome by means of Agrobacterium tumefaciens through zygote disc transformation. Transgene and its expression are confirmed by Southern and Northern blot analyses, respectively. Transgenic plants expressing Datura SAMDC produced 1.7- to 2.4-fold higher levels of spermidine and spermine than wildtype plants under normal environmental condition, which indicated that the transgenic litchi presented an enhanced polyamines synthesis compared to wildtype plants. Our results demonstrated clearly that increasing polyamine biosynthesis in plants may be a means of creating improved fruit shelf life germplasm.

Polyamine biosynthetic diversity in plants and algae

Polyamine biosynthesis in plants differs from other eukaryotes because of the contribution of genes from the cyanobacterial ancestor of the chloroplast. Plants possess an additional biosynthetic route for putrescine formation from arginine, consisting of the enzymes arginine decarboxylase, agmatine iminohydrolase and N-carbamoylputrescine amidohydrolase, derived from the cyanobacterial ancestor. They also synthesize an unusual tetraamine, thermospermine, that has important developmental roles and which is evolutionarily more ancient than spermine in plants and algae. Single-celled green algae have lost the arginine route and are dependent, like other eukaryotes, on putrescine biosynthesis from the ornithine. Some plants like Arabidopsis thaliana and the moss Physcomitrella patens have lost ornithine decarboxylase and are thus dependent on the arginine route. With its dependence on the arginine route, and the pivotal role of thermospermine in growth and development, Arabidopsis represents the most specifically plant mode of polyamine biosynthesis amongst eukaryotes. A number of plants and algae are also able to synthesize unusual polyamines such as norspermidine, norspermine and longer polyamines, and biosynthesis of these amines likely depends on novel aminopropyltransferases similar to thermospermine synthase, with relaxed substrate specificity. Plants have a rich repertoire of polyamine-based secondary metabolites, including alkaloids and hydroxycinnamic amides, and a number of polyamine-acylating enzymes have been recently characterised. With the genetic tools available for Arabidopsis and other model plants and algae, and the increasing capabilities of comparative genomics, the biological roles of polyamines can now be addressed across the plant evolutionary lineage.

Polyamine Accumulation in Transgenic Tomato Enhances the Tolerance to High Temperature Stress

Polyamines play an important role in plant response to abiotic stress. S-adenosyl-l-methionine decarboxylase (SAMDC) is one of the key regulatory enzymes in the biosynthesis of polyamines. In order to better understand the effect of regulation of polyamine biosynthesis on the tolerance of high-temperature stress in tomato, SAMDC cDNA isolated from Saccharomyces cerevisiae was introduced into tomato genome by means of Agrobacterium tumefaciens through leaf disc transformation. Transgene and expression was confirmed by Southern and Northern blot analyses, respectively. Transgenic plants expressing yeast SAMDC produced 1.7- to 2.4-fold higher levels of spermidine and spermine than wild-type plants under high temperature stress, and enhanced antioxidant enzyme activity and the protection of membrane lipid peroxidation was also observed. This subsequently improved the efficiency of CO(2) assimilation and protected the plants from high temperature stress, which indicated that the transgenic tomato presented an enhanced tolerance to high temperature stress (38 degrees C) compared with wild-type plants. Our results demonstrated clearly that increasing polyamine biosynthesis in plants may be a means of creating high temperature-tolerant germplasm.

Regulatory mechanisms of polyamine biosynthesis in plants

Polyamines are small positively charged molecules with a widespread presence in all living organisms. In plants they modulate several aspects of growth and differentiation, and they also participate in the response to abiotic stress. Here we review the molecular mechanisms involved in the regulation of polyamine biosynthesis, which is exerted at different levels including gene expression, protein synthesis, and formation of multienzyme complexes. The importance of polyamines both in development and in stress resistance is also subtended by the phenotype of loss-of-function mutants and of overexpressing lines affecting the different genes that encode polyamine metabolism enzymes.

Modulation of Polyamine Biosynthesis in Transformed Tobacco Plants by Targeting Ornithine Decarboxylase to an Atypical Subcellular Compartment

Ornithine decarboxylase (ODC) is a cytosolic enzyme that catalyses the direct decarboxylation of L-ornithine to putrescine, one of the rate-limiting steps of polyamine biosynthesis in plants. We targeted recombinant human ODC to the cytosol and apoplast of transformed tobacco (Nicotiana tabacum) plants, and evaluated the impact of subcellular compartmentalization on the accumulation of the enzyme and its corresponding metabolic product. Immunoblot analysis showed that human ODC accumulated to high levels in both the cytosol and apoplast of transiently transformed tobacco leaves. In stably transformed tobacco plants with ODC targeted to the apoplast, enzyme activity increased by up to 32- fold (P < 0.001) and putrescine levels increased by up to 8.5-fold (P < 0.05) compared to wild type plants. These results demonstrate that the subcellular targeting of polyamine pathway enzymes may provide a useful strategy to enhance the accumulation and activity of enzymes involved in polyamine biosynthesis and may increase metabolic flux toward desired end products.

Immunomodulation of polyamine biosynthesis in tobacco plants has a significant impact on polyamine levels and generates a dwarf phenotype

Ornithine decarboxylase (ODC) is a cytosolic enzyme that catalyses the direct decarboxylation of l-ornithine to putrescine, one of the rate-limiting steps of polyamine biosynthesis in plants. In the present study, an ODC-specific murine single-chain antibody fragment (scFvODC1) was generated by phage display technology. To evaluate the effect of the recombinant antibody fragment on ODC activity and polyamine levels, we produced transgenic tobacco plants that accumulated scFvODC1 in the cytosol. Expression levels of up to 4% total soluble protein (TSP) were achieved, resulting in the inhibition of up to 90% of endogenous ODC activity. A significant reduction in putrescine, spermidine and spermine levels was observed in transgenic lines producing high levels of scFvODC1. Furthermore, these lines showed developmental abnormalities and a dwarf phenotype. We show that the immunomodulation of enzyme activity is a powerful approach that can be used to alter complex and tightly controlled metabolic pathways, allowing specific steps in the pathway to be blocked and the resulting physiological effects to be investigated.

Modulation of the polyamine biosynthetic pathway in transgenic rice confers tolerance to drought stress.

We have generated transgenic rice plants expressing the Datura stramonium adc gene and investigated their response to drought stress. We monitored the steady-state mRNA levels of genes involved in polyamine biosynthesis (Datura adc, rice adc, and rice samdc) and polyamine levels. Wild-type plants responded to the onset of drought stress by increasing endogenous putrescine levels, but this was insufficient to trigger the conversion of putrescine into spermidine and spermine (the agents that are believed to protect plants under stress). In contrast, transgenic plants expressing Datura adc produced much higher levels of putrescine under stress, promoting spermidine and spermine synthesis and ultimately protecting the plants from drought. We demonstrate clearly that the manipulation of polyamine biosynthesis in plants can produce drought-tolerant germplasm, and we propose a model consistent with the role of polyamines in the protection of plants against abiotic stress.

Localization of arginine decarboxylase in tobacco plants.

The lack of knowledge about the tissue and subcellular distribution of polyamines (PAs) and the enzymes involved in their metabolism remains one of the main obstacles in our understanding of the biological role of PAs in plants. Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We have characterized a cDNA coding for ADC from Nicotiana tabacum L. cv. Petit Havana SR1. The deduced ADC polypeptide had 721 amino acids and a molecular mass of 77 kDa. The ADC cDNA was overexpressed in Escherichia coli, and the ADC fusion protein obtained was used to produce polyclonal antibodies. Using immunological methods, we demonstrate the presence of the ADC protein in all plant organs analysed: flowers, seeds, stems, leaves and roots. Moreover, depending on the tissue, the protein is localized in two different subcellular compartments, the nucleus and the chloroplast. In photosynthetic tissues, ADC is located mainly in chloroplasts, whereas in non-photosynthetic tissues the protein appears to be located in nuclei. The different compartmentation of ADC may be related to distinct functions of the protein in different cell types.

Plant C-N Hydrolases and the Identification of a Plant N-Carbamoylputrescine Amidohydrolase Involved in Polyamine Biosynthesis

A nitrilase-like protein from Arabidopsis thaliana (NLP1) was expressed in Escherichia coli as a His(6)-tagged protein and purified to apparent homogeneity by Ni(2+)-chelate affinity chromatography. The purified enzyme showed N-carbamoylputrescine amidohydrolase activity, an enzyme involved in the biosynthesis of polyamines in plants and bacteria. N-carbamoylputrescine amidohydrolase activity was confirmed by identification of two of the three occurring products, namely putrescine and ammonia. In contrast, no enzymatic activity could be detected when applying various compounds including nitriles, amines, and amides as well as other N-carbamoyl compounds, indicating the specificity of the enzyme for N-carbamoylputrescine. Like the homologous beta-alanine synthases, NLP1 showed positive cooperativity toward its substrate. The native enzyme had a molecular mass of 279 kDa as shown by blue-native polyacrylamide gel electrophoresis, indicating a complex of eight monomers. Expression of the NLP1 gene was found in all organs investigated, but it was not induced upon osmotic stress, which is known to induce biosynthesis of putrescine. This is the first report of cloning and expression of a plant N-carbamoylputrescine amidohydrolase and the first time that N-carbamoylputrescine amidohydrolase activity of a recombinant protein could be shown in vitro. NLP1 is one of the two missing links in the arginine decarboxylase pathway of putrescine biosynthesis in higher plants.

Characterization and translational regulation of the arginine decarboxylase gene in carnation (Dianthus caryophyllus L.)

Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We characterized a carnation genomic clone, gDcADC8, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 77.7 kDa. The unusually long 5'-UTR that contained a short upstream open reading frame (uORF) of seven amino acids (MQKSLHI) was predicted to form an extensive secondary structure (free energy of approximately -117 kcal mol-1) using the Zuker m-fold algorithm. The result that an ADC antibody detected two bands of 45 and 33 kDa in a petal extract suggested the full length of the 78 kDa polypeptide precursor converted into two polypeptides in the processing reaction. To investigate the role of the transcript leader in translation, in vitro transcription/translation reactions with various constructs of deletion and mutation were performed using wheat germ extract. The ADC transcript leader affected positively downstream translation in both wheatgerm extract and primary transformant overexpressing ADC gene. It was demonstrated that heptapeptide (8.6 kDa) encoded by the ADC uORF was synthesized in vitro. Both uORF peptide, and the synthetic heptapeptide MQKSLHI of the uORF, repressed the translation of downstream ORF. Mutation of the uORF ATG codon alleviated the inhibitory effect. ORF translation was not affected by either a frame-shift mutation in uORF or a random peptide. To our knowledge, this is the first report to provide evidence that a uORF may inhibit the translation of a downstream ORF, not only in cis but also in trans, and that the leader sequence of the ADC gene is important for efficient translation.

Control of plant disease by perturbation of fungal polyamine metabolism

The diamine putrescine and the polyamines spermidine and spermine are ubiquitous in nature and are essential for cell proliferation. Since polyamine biosynthesis in plants can start from either ornithine or arginine, while fungal polyamine biosynthesis appears to utilise only the ornithine route, it was suggested that specific inhibition of fungal polyamine biosynthesis should be lethal. Indeed, inhibitors of polyamine biosynthesis, e.g. the ornithine decarboxylase inhibitor α‐difluoromethylornithine, have been shown to inhibit fungal growth in vitro and to control fungal infections on a variety of plants under glasshouse and field conditions. It is now known that polyamine analogues can perturb polyamine metabolism leading to powerful antiproliferative effects in cancer cells. This paper reviews the results of a research programme focused on the synthesis and evaluation of putrescine analogues as novel fungicides. A number of aliphatic, alicyclic and cyclic diamines have been shown to possess considerable fungicidal activity, but although many of these compounds perturb polyamine metabolism in fungal cells, such changes are not considered sufficient to account for the observed antifungal effects. More recent work on spermidine analogues is also described.

Regulation of arginine decarboxylase by spermine in osmotically‐stressed oat leaves

Biosynthesis of polyamines in plants is controlled primarily by the enzymes ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (ADC: EC 4.1.1.19), which are responsible for the production of putrescine, and S‐adenosyl‐L‐methionine (SAM) decarboxylase (EC 4.1.1.50) that is necessary for the formation of spermidine and spermine (Spm). Little is known about the metabolic or molecular mechanisms regulating the synthesis of these enzymes. We have studied the regulation of ADC synthesis by Spm in osmotically‐stressed oat (Avena sativa L. ev. Victory) leaves, using a polyclonal antibody to oat ADC and a cDNA clone encoding oat ADC. Treatment with Spm in combination with osmotic stress resulted in increased steady‐state levels of ADC mRNA, yet the levels of ADC activity decreased. This absence of correlation is explained by the fact that Spm inhibits processing of the ADC proenzyme, which results in increased levels of this inactive ADC form and a consequent decrease in the ADC‐processed form. Spermine treatment leads to delayed loss of chlorophyll in dark‐incubated and osmotically‐treated oat leaves. Thus, post‐translational regulation of ADC synthesis by Spm may be important in explaining its anti‐senescence properties.

Modulation of cellular polyamines in tobacco by transfer and expression of mouse ornithine decarboxylase cDNA

In an attempt to modulate the metabolism of polyamines in plants, Agrobacterium tumefaciens strains were produced which contained either a full-length or a 3'-truncated mouse ornithine decarboxylase (ODC) cDNA under the control of the cauliflower mosaic virus 35S promoter. Plants of Nicotiana tabacum cv. Xanthi were used for transformation with these two strains of Agrobacterium. Transformations were confirmed by Southern hybridization and amplification by polymerase chain reaction. Two plants containing the full-length cDNA (ODC-12 and ODC-30) and two containing the truncated cDNA (12701-2 and 12701-31) were selected for further experiments. Northern blot analysis indicated that transcription was occurring and western blot analysis detected a polypeptide of ca. 50 kDa that was unique to the plants transformed with truncated ODC cDNA. In order to distinguish between the native and the mouse ODC in the transformed tissues, enzyme activity was assayed at pH optima for the two enzymes, i.e. pH 8.2 and 6.8, respectively. Substantially higher levels of ODC activity were seen at pH 6.8 (optimum for mouse ODC) in the transformants as compared to the controls. This ODC activity was inhibited by alpha-difluoromethylornithine and anti-mouse ODC antisera in a manner consistent with that reported for the mouse ODC. Analysis of cellular polyamines showed significantly elevated levels (4-12-fold) of putrescine in callus derived from transformed plant tissues as compared to the controls. The modulation of polyamine biosynthesis in plants by these techniques should allow us to further analyze the role of these ubiquitous compounds in plant growth and development.

Polyamines and Somatic Embryogenesis in carrot II. The Effects of Cyclohexylammonium phosphate

Summary The effects of cyclohexylammonium phosphate (CHAP), a potent inhibitor of spermidine synthase, on Somatic Embryogenesis and cellular polyamine content were studied using carrot (Daucus carota L.) cell cultures. Somatic embryogenesis was only delayed somewhat in the presence of 0.5 mM CHAP. While there was a substantial reduction in the cellular spermidine levels, an increase in putrescine was observed during the 6-day culture period. Spermine was less affected by CHAP than spermidine during the first 24 h, but a significant increase in this polyamine was seen in longer treatments. The activity of arginine decarboxylase was higher in cells treated with CHAP. The effects of CHAP on polyamines and arginine decarboxylase were similar both in the presence and the absence of 2,4-D. The results are in agreement with previous reports on the effects of CHAP on polyamine biosynthesis in plants and animals.