Oat Arginine Decarboxylase Research Articles

Improvement of post-harvest fruit characteristics in tomato by fruit-specific over-expression of oat arginine decarboxylase gene

Over-ripening is the key contributor to post-harvest loss of perishable fruits and is also one of the greatest threats to the growers. Delayed ripening of tomato fruits has been suggested as a possible solution to such post-harvest losses. To overcome post-harvest losses, we engineered tomato fruits to accumulate polyamines (PAs) by over-expression of arginine decarboxylase (ADC) gene of PA biosynthetic pathway. The AsADC transgenic fruits contained elevated levels of putrescine, spermidine and spermine content in comparison to the fruits from untransformed plants. In addition, the expression of several ripening-associated genes was also altered. The transgenic fruits displayed a better shelf-life due to decrease in ethylene levels and respiratory activity. The transgenic tomatoes also exhibited improved key processing features along with the elevated levels of lycopene and vitaminC. Our study highlights that the fortification of PAs is a promising approach for the improvement of post-harvest fruit characteristics together with a high quality of produce.

Overexpression of the Arginine Decarboxylase Gene Improves Tolerance to Salt Stress in Lotus tenuis Plants

To analyse the putative effects of putrescine on the plant response to salinity, micropropagated Lotus tenuis plants from wild-type and transgenic lines harbouring the pRD29A::oat arginine decarboxylase (ADC, EC 4.1.1.19) construct were subjected to a gradual increase in salinity that was applied gradually by means of sodium chloride irrigation (from 0 to 0.3 mol L−1) every 5 days up to the maximum concentration. At the end of the experiment, the transgenic lines were healthier than the wild-type plants and displayed a smaller reduction in shoot biomass and a slight increase in root growth in response to stress. The overexpression of ADC increased osmotic adjustment (5.8-fold) via the release of proline. The salinity treatment doubled the potassium uptake by roots from transgenic ADC stressed plants with a concomitant decrease in the accumulation of sodium, balancing the Na+/K+ ratio. Analysis of gene expression, enzymatic activities and hormone metabolism suggests a crosstalk between polyamines and abscisic acid in response to salinity via modulation of the abscisic acid biosynthesis-related enzyme 9-cis-epoxycarotenoid dioxygenase (EC 1.13.11.51) at the transcriptional level.

Over-expression of Arginine Decarboxylase Gene in Tapetal Tissue Results in Male Sterility in Tomato Plants

Homeostasis of cellular Polyamines (PAs) is very important for proper growth and development of living organisms. Development of male sex organ and gametophyte in plants is a complex process involving many known and unknown pathways. PAs are actively involved in the development of anther and pollen. In the present study, oat arginine decarboxylase (ADC) gene has been over-expressed in tapetum of tomato using tapetum-specific TA29 promoter to perturb the PA homeostasis in order to see its implication on male fertility. Tapetum-specific ADC overexpression resulted in PA accumulation, mainly in free and bound fractions in the anther of transgenics. Transgenic lines displayed abnormal pollen and tapetum development. Pollen grains were aborted and distorted in morphology and tapetum showed premature degeneration in ADC transgenics. These transgenics failed to set seeds, but cross pollination with normal pollen from wild type plants restored the seed setting indicating that s ADC over-expression did not affect female fertility. ADC over-expression also did not affect the growth and morphology of transgenics. The present study suggests that the excess of PAs in anther is lethal for pollen and tapetum development and overexpression of ADC gene can be an effective approach for the engineering of male sterility in plants.

Agrobacterium tumefaciens-mediated transformation of Lotus tenuis and regeneration of transgenic lines

A protocol for the production of transgenic plants was developed for Lotus tenuis via Agrobacterium-mediated transformation of leaf segments. The explants were co-cultivated (for 3 days) with an A. tumefaciens strain harbouring either the binary vector pBi RD29A:oat arginine decarboxylase (ADC) or pBi RD29A:glucuronidase (GUS), which carries the neomycin phosphotransferase II (nptII) gene in the T-DNA region. Following co-cultivation, the explants were cultured in Murashige and Skoog medium supplemented with naphthalenacetic acid (NAA) and benzyladenine (BA) and containing kanamycin (30 μg ml−1) and cefotaxime (400 μg ml−1) for 45 days. The explants were subcultured several times (at 2-week intervals) to maintain the selection pressure during the entire period. About 40% of the explants inoculated with the pBiRD29:ADC strain produced eight to ten adventitious shoots per responsive explant through a direct system of regeneration, whereas 69% of the explants inoculated with the pBi RD29A:GUS strain produced 13–15 adventitious shoots per responsive explant. The selected transgenic lines were identified by PCR and Southern blot analysis. Three ADC transgenic lines were obtained from 30 infected explants, whereas 29 GUS transgenic lines were obtained from 160 explants, corresponding to a transformation efficiency of 10 and 18.1%, respectively. More than 90% of the in vitro plantlets were successfully transferred to the soil. The increase in the activity of arginine decarboxylase from stressed ADC- Lt19 lines was accompanied by a significant rise in the putrescine level. The GUS transgenic line driven by the RD29A promoter showed strong signals of osmotic stress in the leaves and stem tissues. All of the transgenic plants obtained exhibited the same phenotype as the untransformed controls under non-stress conditions, and the stability of the gene introduced into the cloned materials was established.

Post-translational processing, metabolic stability and catalytic efficiency of oat arginine decarboxylase expressed in Trypanosoma cruzi epimastigotes

Trypanosoma cruzi epimastigotes are auxotrophic for polyamines because they are unable to synthesize putrescine de novo. This deficiency is due to the absence of ornithine and arginine decarboxylase genes in the parasite genome. We have been able to obtain transgenic T. cruzi expressing heterologous genes coding for these enzymes. Since arginine decarboxylase normal expression in oat requires a post-translational proteolytic cleavage of an enzyme precursor, we have investigated whether a similar processing occurs inside the transformed protozoa expressing oat arginine decarboxylase or the same enzyme attached to a C-terminal (his)6-tag. We were able to demonstrate that the post-translational processing also takes place inside the transgenic parasites. This cleavage is probably the result of a general proteolytic activity of T. cruzi acting on a protease-sensitive region of the protein. Interestingly, the (his)6-tagged enzyme expressed in the transformed parasites showed considerably increased metabolic stability and catalytic efficiency.

Transgenic wheat plants expressing an oat arginine decarboxylase cDNA exhibit increases in polyamine content in vegetative tissue and seeds

We generated and characterized a transgenic wheat (Triticum aestivum L.) population expressing constitutively an oat arginine decarboxylase (ADC) cDNA. Transgenic wheat plants expressing the transgene had a significant increase in ADC activity with no concomitant changes in ODC activity. We measured 2-fold increases in putrescine, spermidine and spermine content in leaves of transgenic plants compared to wild-type. These plants also exhibited significant increases in the three polyamines in seeds (7-fold putrescine, 2.5-fold spermidine and 1.8-fold spermine). This significant polyamine accumulation was heritable in subsequent generations. In order to characterize molecularly this population we cloned a partial TaADC cDNA. This sequence was used as a probe to ascertain the presence of two distinct ADC transcripts in wheat. Further DNA analyses confirmed the presence of at least two distinct ADC genes in the wheat genome. Our results demonstrate that polyamine levels can be altered in transgenic wheat plants resulting in phenotypically normal and fertile plants. This transgenic wheat population will serve as useful germplasm to embark on further in depth studies to ascertain the role of polyamines in abiotic stress responses.

Modulation of oat arginine decarboxylase gene expression and genome organization in transgenic Trypanosoma cruzi epimastigotes

We have previously demonstrated that wild-type Trypanosoma cruzi epimastigotes lack arginine decarboxylase (ADC) enzymatic activity as well as its encoding gene. A foreign ADC has recently been expressed in T. cruzi after transformation with a recombinant plasmid containing the complete coding region of the oat ADC gene. In the present study, upon modulation of exogenous ADC expression, we found that ADC activity was detected early after transfection; subsequently it decreased to negligible levels between 2 and 3 weeks after electroporation and was again detected approximately 4 weeks after electroporation. After this period, the ADC activity increased markedly and became expressed permanently. These changes of enzymatic activity showed a close correlation with the corresponding levels of ADC transcripts. To investigate whether the genome organization of the transgenic T. cruzi underwent any modification related to the expression of the heterologous gene, we performed PCR amplification assays, restriction mapping and pulse-field gel electrophoresis with DNA samples or chromosomes obtained from parasites collected at different time-points after transfection. The results indicated that the transforming plasmid remained as free episomes during the transient expression of the foreign gene. Afterwards, the free plasmid disappeared almost completely for several weeks and, finally, when the expression of the ADC gene became stable, two or more copies of the transforming plasmid arranged in tandem were integrated into a parasite chromosome (1.4 Mbp) bearing a ribosomal RNA locus. The sensitivity of transcription to alpha-amanitin strongly suggests involvement of the protozoan RNA polymerase I in the transcription of the exogenous ADC gene.

An Efficient Transformation Method to Regenerate a High Number of Transgenic Plants Using a New Embryogenic Line of Medicago truncatula cv. Jemalong

A simple and efficient regeneration–transformation method was established to obtain transgenic plants of the model legume Medicago truncatula cv. Jemalong. This method takes advantage of a new highly embryogenic line (M9-10a) isolated in our laboratory. Leaflets of in vitro grown M9-10a plants were co-cultured with Agrobacterium tumefaciens EHA105. Plasmid constructs containing the oat arginine decarboxylase gene, Adc and the GUS reporter gene (p35SAdc–Gus) or ELIP-like drought stress protein 22 (DSP22) encoding gene from Craterostigma plantagineum (p35SDsp22) were used. Both constructs include the nptII gene as selection marker. Embryogenic calli (100–97%) were obtained on embryo induction medium containing 100 mg l −1 kanamycin and 500 mg l−1 carbenicillin. Using a two-fold increase in kanamycin concentration, instead of 50 mg l−1 usually used, we reduced the number of emerging false kanamycin-resistant (KanR) embryos, which is an important improvement to the method, making it less laborious and very efficient. Isolation of late torpedo/cotyledonary-stage embryos to lower carbenicillin/agar media reduced secondary embryogenesis and prevents hyperhydricity, improving embryo conversion. Primary transformants (T0) were regenerated within 3–4 months and those that were able to root in a 50 mg l−1 kanamycin medium were transferred to the greenhouse to produce seeds. Southern blot hybridisation analysis confirmed the integration of either the Adc or Dsp22 transgenes in the genome of the T0 transformants. Detection of β-glucuronidase (GUS) activity in Adc–Gus T0 plants demonstrated the expression of the inserted transgene. In average, 1–2 independent transgenic lines are obtained per KanR embryogenic callus, independently of the plasmid construct used for transformation. Inheritance of the transgenes is shown to be stable in the T1 generation.

Heterologous expression of a plant arginine decarboxylase gene in

Wild-type Trypanosoma cruzi epimastigotes lack arginine decarboxylase (ADC) enzymatic activity. However, the transformation of these parasites with a recombinant plasmid containing the oat ADC cDNA coding region gave rise to the transient heterologous expression of the enzyme, suggesting the absence of endogenous mechanisms that could inhibit the expression of a hypothetical own ADC gene or the assay used to measure its enzymatic activity. The foreign ADC enzyme expressed in the transgenic T. cruzi was characterized by identification of the products, the stoichiometry of the catalysed reaction, the specific inhibition by alpha-difluoromethylarginine (DFMA) and the study of its metabolic turnover. The half-life of the heterologous ADC activity in T. cruzi was about 150 min. Bioinformatics studies and polymerase chain reaction (PCR) analyses seem to indicate the absence of ADC-like DNA sequences in the wild-type T. cruzi genome.

Lotus hairy roots expressing inducible arginine decarboxylase activity.

Biotechnological uses of plant cell-tissue culture usually rely on constitutive transgene expression. However, such expression of transgenes may not always be desirable. In those cases, the use of an inducible promoter could be an alternative approach. To test this hypothesis, we developed two binary vectors harboring a stress-inducible promoter from Arabidopsis thaliana, driving the beta-glucuronidase reporter gene and the oat arginine decarboxylase. Transgenic hairy roots of Lotus corniculatus were obtained with osmotic- and cold-inducible beta-glucuronidase and arginine decarboxylase activities. The increase in the activity of the latter was accompanied by a significant rise in total free polyamines level. Through an organogenesis process, we obtained L. corniculatus transgenic plants avoiding deleterious phenotypes frequently associated with the constitutive over-expression of arginine decarboxylation and putrescine accumulation.

Effects of putrescine accumulation in tobacco transgenic plants with different expression levels of oat arginine decarboxylase

Arginine decarboxylase (ADC; EC 4.1.1.19) is a key enzyme in one of the two possible ways to synthesize putrescine (Put) in plants. In previous work (Masgrau et al. 1997), we observed an altered phenotype (growth inhibition, leaf chlorosis and necrosis) in tobacco transgenic plants (Nicotiana tabacum L. var. Wisconsin-38) containing the oat ADC cDNA under the control of a tetracycline inducible promoter, the severity of which was correlated with Put content. Now we have analysed the T2 generation of a selected transgenic line (line 52), which in previous generations was characterized by presenting a moderate increase in ADC activity and polyamine levels, but no phenotype alterations. Studying two selected individuals, one with a high expression level of the transgene and the other with a moderate expression level, we demonstrate that only the one with increased polyamine content displays the altered (toxic) phenotype. The possible causes of toxicity have been analysed. The results suggest that either Put or its oxidation products, via diamine oxidase (DAO; EC 1.4.3.6), are the responsible factors for the deleterious effects observed in the transgenic plants.

Development of a novel gene transfer system for Cajanus cajan and expression of a monocot arginine decarboxylase cDNA in transformed cell lines

We describe the development of a novel transformation system for pigeonpea ( Cajanus cajan (L.) Millsp.) cell lines using particle bombardment. The aim of our study was not only to develop a transformation system for this important but recalcitrant species but to do so using a gene of interest and to study the behaviour of this transgene in a legume background. The gene we chose for our studies was the heterologous oat arginine decarboxylase cDNA. Transgenic lines engineered with this cDNA had a small, but significant increase in arginine decarboxylase activity (ADC; EC 4.1.1.17). We detected an increase in putrescine levels in all the lines we analysed irrespective of whether major or minor changes in ADC activity were observed. Spermidine and spermine levels were also increased in some of the lines. We confirmed that small changes in the adc message (mRNA) had a significant effect on enzyme activity and more importantly on polyamine levels. This is the first report describing modulation of a gene involved in a metabolic pathway in transgenic pigeonpea tissues.

A transgenic rice cell lineage expressing the oat arginine decarboxylase (adc) cDNA constitutively accumulates putrescine in callus and seeds but not in vegetative tissues.

We introduced the oat adc cDNA into rice under the control of the constitutive maize ubiquitin 1 promoter. We studied molecularly and biochemically sixteen independent transgenic plant lines. Significant increases in mRNA levels, ADC enzyme activity and polyamines were measured in transgenic callus. These increases were not maintained in vegetative tissue or seeds in regenerated plants, with the exception of one lineage. This particular lineage showed very significant increases in putrescine preferentially in seeds (up to 10 times compared to wild type and controls transformed with the hpt selectable marker alone). We have demonstrated that in cereals such as rice, over-expression of the oat adc cDNA results in increased accumulation of polyamines at different stages of development. We have also demonstrated that strong constitutive promoters, such as the maize ubiquitin 1 promoter, are sufficient to facilitate heritable high-level polyamine accumulation in seed. Our results demonstrate that by screening adequate numbers of independently derived transgenic plants, it is possible to identify those individuals which express a desired phenotype or genotype.

Reconstitution of a bacterial/plant polyamine biosynthesis pathway in Saccharomyces cerevisiae.

Polyamine synthesis in most organisms is initiated by the decarboxylation of ornithine to form putrescine via ornithine decarboxylase (ODC). Plants, some bacteria and some fungi and protozoa generate putrescine from arginine, via arginine decarboxylase (ADC) and agmatine ureohydrolase (AUH) or agmatine iminohydrolase. A polyamine-requiring strain of Saccharomyces cerevisiae with a mutation in the gene encoding ODC was transformed with plasmids bearing genes encoding Escherichia coli ADC and AUH. Transformants regained the ability to grow in the absence of exogenous polyamines and contained enzyme activities consistent with the presence of both prokaryotic enzymes. Similar results were obtained when a plasmid containing a gene encoding oat (Avena sativa L.) ADC was substituted for the E. coli gene. These data demonstrate the successful complementation of a yeast biosynthetic polyamine synthesis defect by genes encoding an alternative pathway found in bacteria; they also show that plant ADC can substitute for the bacterial enzyme in this pathway. The recombinant yeast provides a tool for the study of the functional properties of these enzymes and for discovery of compounds that specifically inhibit this pathway.

Over-expression of the oat arginine decarboxylase cDNA in transgenic rice (Oryza sativa L.) affects normal development patterns in vitro and results in putrescine accumulation in transgenic plants

Transgenic rice (Oryza sativa L.) cell lines and plants expressing an oat arginine decarboxylase (Adc) cDNA under the control of the CaMV 35 S promoter were recovered using particle bombardment. Molecular analyses confirmed stable integration of the transgene and active transcription (mRNA). A four- to sevenfold increase in arginine decarboxylase (ADC) activity was observed in transformed plants compared to wild-type controls. Biochemical analysis of cellular polyamines (PAs) indicated up to fourfold increase in putrescine (Put) levels in transgenic callus and regenerated plants. This is the first report which demonstrates an increase in PA levels in plants engineered with the Adc gene. Implications of this increase are discussed in terms of development, physiology and nutrition. We observed a correlation between high levels of Adc gene expression and inability of callus tissue to develop normally into differentiated plants. This correlates well with reports in other species, in which perturbation of the PA pathway using genes involved in PA biosynthesis results in aberrant phenotypes. We have shown for the first time that PA biosynthesis can be manipulated in cereal species using genetic engineering.

Overexpression of arginine decarboxylase in transgenic plants.

The activity of arginine decarboxylase (ADC), a key enzyme in plant polyamine biosynthesis, was manipulated in two generations of transgenic tobacco plants. Second-generation transgenic plants overexpressing an oat ADC cDNA contained high levels of oat ADC transcript relative to tobacco ADC, possessed elevated ADC enzyme activity and accumulated 10-20-fold more agmatine, the direct product of ADC. In the presence of high levels of the precursor agmatine, no increase in the levels of the polyamines putrescine, spermidine and spermine was detected in the transgenic plants. Similarly, the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were unchanged. No diversion of polyamine metabolism into the hydroxycinnamic acid-polyamine conjugate pool or into the tobacco alkaloid nicotine was detected. Activity of the catabolic enzyme diamine oxidase was the same in transgenic and control plants. The elevated ADC activity and agmatine production were subjected to a metabolic/physical block preventing increased, i.e. deregulated, polyamine accumulation. Overaccumulation of agmatine in the transgenic plants did not affect morphological development.

Inducible overexpression of oat arginine decarboxylase in transgenic tobacco plants.

To test the possible interaction of polyamines in plant growth responses, transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase (ADC) gene under the control of a tetracycline-inducible promoter were generated. Inducible overexpression of oat ADC in transgenic tobacco led to an accumulation of ADC mRNA, increased ADC activity and changes in polyamine levels. Transgenic lines, induced during vegetative stage, displayed different degrees of an altered phenotype, the severity of which was correlated with putrescine content. These phenotypic changes were characterized by short internodes, thin stems and leaves, leaf chlorosis and necrosis, as well as reduced root growth. This is the first report to show altered phenotypes as a consequence of polyamine changes under tetracycline-induction in in vivo conditions. Interestingly, overexpression of oat ADC in tobacco resulted in similar detrimental effects to those observed by ADC activation induced by osmotic stress in the homologous oat leaf system. In the context of the role of specific polyamines in plant growth and development, the present results indicate that activation of the ADC pathway leading to high levels of endogenous putrescine (or its catabolytes) is toxic for the vegetative growth of the plant. In contrast, no visible phenotypic effects were observed in flowering plants following tetracycline induction. Further characterization of the different transgenic lines may shed light on the action of specific polyamines in different plant developmental processes.

Arginine Decarboxylase Is Localized in Chloroplasts.

Plants, unlike animals, can use either ornithine decarboxylase or arginine decarboxylase (ADC) to produce the polyamine precursor putrescine. Lack of knowledge of the exact cellular and subcellular location of these enzymes has been one of the main obstacles to our understanding of the biological role of polyamines in plants. We have generated polyclonal antibodies to oat (Avena sativa L.) ADC to study the spatial distribution and subcellular localization of ADC protein in different oat tissues. By immunoblotting and immunocytochemistry, we show that ADC is organ specific. By cell fractionation and immunoblotting, we show that ADC is localized in chloroplasts associated with the thylakoid membrane. The results also show that increased levels of ADC protein are correlated with high levels of ADC activity and putrescine in osmotically stressed oat leaves. A model of compartmentalization for the arginine pathway and putrescine biosynthesis in active photosynthetic tissues has been proposed. In the context of endosymbiote-driven metabolic evolution in plants, the location of ADC in the chloroplast compartment may have major evolutionary significance, since it explains (a) why plants can use two alternative pathways for putrescine biosynthesis and (b) why animals do not possess ADC.

Arginine decarboxylase of oats is activated by enzymatic cleavage into two polypeptides.

Oat arginine decarboxylase is synthesized as a 66-kDa proenzyme, but the soluble enzyme is found in oats as a complex of 42- and 24-kDa polypeptide fragments, both derived from the 66-kDa precursor. We report here that this proteolytic cleavage is the result of a processing enzyme, distinct from arginine decarboxylase itself, that leads to activation of the arginine decarboxylase. The proteolysis is resistant to a standard set of protease inhibitors, but is inhibited by high concentrations of Zn2+, as is the activation of arginine decarboxylase. Agmatine, putrescine, spermidine, and spermine, as well as the arginine decarboxylase inhibitor difluoromethylarginine, each had no effect on the reaction. Oat arginine decarboxylase is thus similar to some other amino acid decarboxylases in requiring a proteolytic cleavage for activation; however, it differs in that the other examples are auto-catalytic self-processing.

Mechanisms of polyamine action during senescence responses induced by osmotic stress

The peeled Avena sativa L. leaf-system has been used as a simple and rapid model, which allows the study of the mechanisms of polyamine action during senescence responses induced by osmotic stress. The use of guazatine, an inhibitor of polyamine oxidase activity, has demonstrated the existence of a positive correlation between high levels of spermidine and spermine and delay of senescence in oat leaves and mesophyll protoplasts. The availability of antibodies specific for key polypeptides of thylakoid membranes in combination with ultrastructural studies have led to the discovery that spermine stabilizes the molecular composition and preserves the integrity of thylakoid membranes. The analyses of malondialdehyde and lipoxygenase levels has revealed that polyamines and guazatine treatments may prevent membrane destabilization, at least in part, by inhibiting lipid peroxidation. Finally, the availability of a cDNA coding for oat arginine decarboxylase (ADC) and the generation of ‘site-directed’ antibodies specific for ADC have led to the establishment of the basis for a model of regulation of ADC gene expression in oat leaves.