Biomedical subjects

Melinda J Mayer

Publications and source records attributed to Melinda J Mayer.

6 recordsLinked to original sources

PubMed2004

Effects of spermidine synthase overexpression on polyamine biosynthetic pathway in tobacco plants.

Transgenic tobacco plants overexpressing the Datura stramonium spermidine synthase (EC 2.5.1.16) cDNA were produced in order to understand the role of this gene in the polyamine metabolism and in particular in affecting spermidine endogenous levels. All the analysed transgenic clones displayed a high Level of overexpression of the exogenous cDNA with respect to the endogenous spermidine synthase. No relationship was detected between the mRNA expression level of S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50), which did not change between the negative segregant control and the transgenic plants, and spermidine synthase, suggesting the existence of an independent regulatory mechanism for transcription of the two genes. The determination of enzyme activities indicated an increased spermidine synthase and S-adenosylmethionine decarboxylase activity, with the last being mainly recovered in the particulate fraction. ODC (ODC, EC 4.1.1.17) was the most active enzyme and its activity was equally distributed between the soluble and the particulate fraction, while ADC (ADC, EC 4.1.1.19) activity in the transgenic plants did not particularly change with respect to the controls. In comparison to the controls, the transformed plants displayed an increased spermidine to putrescine ratio in the majority of the clones assayed, white the total polyamine content remained almost unchanged. These findings suggest a high capacity of the transformed plants to tightly regulate polyamine endogenous levels and provide evidence that spermidine synthase is not a limiting step in the biosynthesis of polyamines.

Adenosylmethionine Decarboxylase↗

PubMed2003

The diverse bacterial origins of the Arabidopsis polyamine biosynthetic pathway.

We functionally identified the last remaining step in the plant polyamine biosynthetic pathway by expressing an Arabidopsis thaliana agmatine iminohydrolase cDNA in yeast. Inspection of the whole pathway suggests that the arginine decarboxylase, agmatine iminohydrolase, N-carbamoylputrescine amidohydrolase route to putrescine in plants was inherited from the cyanobacterial ancestor of the chloroplast. However, the rest of the pathway including ornithine decarboxylase and spermidine synthase was probably inherited from bacterial genes present in the original host cell, common ancestor of plants and animals, that acquired the cyanobacterial endosymbiont. An exception is S-adenosylmethionine decarboxylase, which may represent a eukaryote-specific enzyme form.

Amino Acid Sequence↗

PubMed2003

Vanillin.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is an important flavour and aroma molecule, but is also of interest because of its biogenetic relationship to the phenylpropanoid pathway and to other molecules of physiological significance, notably salicylate. Recent progress towards characterisation of the biosynthesis of vanillin is reviewed. In Vanilla, there is some evidence that the route to vanillin-beta-D-glucoside may proceed from 4-coumaric acid via 4-hydroxybenzaldehyde, with glucoside formation occurring not necessarily as the final step, and possibly with the involvement of 4-hydroxybenzyl alcohol beta-D-glucoside tartrate bis-esters as "shunt" metabolites. This appears to be given tentative support by the recent partial characterisation of a 4-hydroxybenzaldehyde synthase from Vanilla. On the other hand, a well-characterised, CoA-dependent, non-oxidative chain-shortening mechanism to produce vanillin from ferulic acid, occurring as part of a pathway of hydroxycinnamate degradation in Pseudomonas, may not be representative of hydroxycinnamate chain-shortening mechanism(s) occurring in Vanilla and other plants. Nevertheless, by expression of the Pseudomonas enzyme 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL), attempts have been made to introduce a direct capacity for vanillin formation into model plants by diversion of the phenylpropanoid pathway. The results obtained have emphasised the obstacles to achieving the desired oxidation level (aldehyde) and ring substitution (4-hydroxy-3-methoxyphenyl), even when a substantial metabolic diversion is successfully achieved. Finally, the significance of the latest biosynthetic and biotechnological developments is reviewed briefly in relation to authentication of vanillin.

Benzaldehydes↗

PubMed2003

Polyamine homeostasis in transgenic plants overexpressing ornithine decarboxylase includes ornithine limitation.

It was reported recently that overexpression of human ornithine decarboxylase (ODC) cDNA in transgenic rice plants resulted in increased steady-state concentration of polyamines, i.e., enough biosynthetic control is invested at this step to enable adjustment of polyamine levels. To investigate critically whether constitutive overexpression of ODC is sufficient to control steady-state polyamine levels, we expressed an ODC cDNA from Datura stramonium in transgenic tobacco plants. Transgenic progeny of self-fertilised primary transformants exhibited increases in ODC activity of 25-fold in leaves and 5-fold in flower buds. However, the increase in putrescine levels was only 1.5- to 2.1-fold in leaves and 1.1- to 1.3-fold in flower buds. Emphatically, no changes to spermidine or spermine steady-state levels or to soluble or insoluble hydroxycinnamic acid-conjugated polyamines were observed. Ornithine feeding to cell suspension cultures derived from the transgenic plants indicated that putrescine accumulation was limited in part by ornithine availability. These results demonstrate that a large increase in the capacity of the tobacco plants to decarboxylate ornithine does not result in a comparable increase in the level of free or conjugated polyamines. Plant polyamine homeostatic mechanisms efficiently accommodate increased ODC activity, suggesting that polyamine biosynthetic control is invested at multiple interdependent steps.

Cells, Cultured↗

PubMed2002

Abrogation of upstream open reading frame-mediated translational control of a plant S-adenosylmethionine decarboxylase results in polyamine disruption and growth perturbations.

S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in polyamine biosynthesis. We show that the plant AdoMetDC activity is subject to post-transcriptional control by polyamines. A highly conserved small upstream open reading frame (uORF) in the AdoMetDC mRNA 5' leader is responsible for translational repression of a downstream beta-glucuronidase reporter cistron in transgenic tobacco plants. Elimination of the small uORF from an AdoMetDC cDNA led to increased relative translational efficiency of the AdoMetDC proenzyme in transgenic plants. The resulting increased activity of AdoMetDC caused disruption to polyamine levels with depletion of putrescine, reduction of spermine levels, and a more than 400-fold increase in the level of decarboxylated S-adenosylmethionine. These changes were associated with severe growth and developmental defects. The high level of decarboxylated S-adenosylmethionine was not associated with any change in 5'-methylcytosine content in genomic DNA and S-adenosylmethionine levels were more or less normal, indicating a highly efficient system for maintenance of S-adenosylmethionine levels in plants. This work demonstrates that uORF-mediated translational control of AdoMetDC is essential for polyamine homeostasis and for normal growth and development.

Adenosylmethionine Decarboxylase↗

PubMed2002

4-Hydroxycinnamoyl-CoA hydratase/lyase, an enzyme of phenylpropanoid cleavage from Pseudomonas, causes formation of C(6)-C(1) acid and alcohol glucose conjugates when expressed in hairy roots of Datura stramonium L.

4-Hydroxycinnamoyl-CoA hydratase/lyase (HCHL), a crotonase homologue of phenylpropanoid catabolism from Pseudomonas fluorescens strain AN103, led to the formation of 4-hydroxybenzaldehyde metabolites when expressed in hairy root cultures of Datura stramonium L. established by transformation with Agrobacterium rhizogenes. The principal new compounds observed were the glucoside and glucose ester of 4-hydroxybenzoic acid, together with 4-hydroxybenzyl alcohol- O-beta- D-glucoside. In lines actively expressing HCHL, these together amounted to around 0.5% of tissue fresh mass. No protocatechuic derivatives were found, although a trace of vanillic acid-beta- D-glucoside was detected. There was no accumulation of 4-hydroxybenzaldehydes, whether free or in the form of their glucose conjugates. There was some evidence suggesting a diminished availability of feruloyl-CoA for the production of feruloyl putrescine and coniferyl alcohol. The findings are discussed in the context of a diversion of phenylpropanoid metabolism, and the ability of plants and plant cultures to conjugate phenolic compounds.

Benzaldehydes↗