Limits...
Functional characterization and expression analysis of rice δ(1)-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism.

Forlani G, Bertazzini M, Zarattini M, Funck D - Front Plant Sci (2015)

Bottom Line: Cations were found to modulate enzyme activity, whereas anion effects were negligible.This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate.Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Biotechnology, University of Ferrara Ferrara, Italy.

ABSTRACT
While intracellular proline accumulation in response to various stress conditions has been investigated in great detail, the biochemistry and physiological relevance of proline degradation in plants is much less understood. Moreover, the second and last step in proline catabolism, the oxidation of δ(1)-pyrroline-5-carboxylic acid (P5C) to glutamate, is shared with arginine catabolism. Little information is available to date concerning the regulatory mechanisms coordinating these two pathways. Expression of the gene coding for P5C dehydrogenase was analyzed in rice by real-time PCR either following the exogenous supply of amino acids of the glutamate family, or under hyperosmotic stress conditions. The rice enzyme was heterologously expressed in E. coli, and the affinity-purified protein was thoroughly characterized with respect to structural and functional properties. A tetrameric oligomerization state was observed in size exclusion chromatography, which suggests a structure of the plant enzyme different from that shown for the bacterial P5C dehydrogenases structurally characterized to date. Kinetic analysis accounted for a preferential use of NAD(+) as the electron acceptor. Cations were found to modulate enzyme activity, whereas anion effects were negligible. Several metal ions were inhibitory in the micromolar range. Interestingly, arginine also inhibited the enzyme at higher concentrations, with a mechanism of uncompetitive type with respect to P5C. This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate. Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism.

No MeSH data available.


Transcript levels of the genes involved in proline synthesis from glutamate and its re-oxidation in PEG-treated rice cells. The expression levels were quantified in suspension cultured cells of rice (cv Vialone nano) by real-time PCR following reverse transcription. Levels were normalized with respect to Actin 1 (Os03t0718100-01) cDNA (A). The same experiment was performed with cells treated with 22.5% PEG 6000, harvested at increasing time after the treatment. Expression levels were in this case normalized with respect to those in untreated controls (B). All treatments were carried out in triplicate, and means ± SE are reported. Results were subjected to analysis of variance, taking into account the Bonferroni correction for multiple testing; *, ***: significantly different at the 10% and 1% level, respectively, with respect to time 0 (A) or untreated controls (B).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4525382&req=5

Figure 3: Transcript levels of the genes involved in proline synthesis from glutamate and its re-oxidation in PEG-treated rice cells. The expression levels were quantified in suspension cultured cells of rice (cv Vialone nano) by real-time PCR following reverse transcription. Levels were normalized with respect to Actin 1 (Os03t0718100-01) cDNA (A). The same experiment was performed with cells treated with 22.5% PEG 6000, harvested at increasing time after the treatment. Expression levels were in this case normalized with respect to those in untreated controls (B). All treatments were carried out in triplicate, and means ± SE are reported. Results were subjected to analysis of variance, taking into account the Bonferroni correction for multiple testing; *, ***: significantly different at the 10% and 1% level, respectively, with respect to time 0 (A) or untreated controls (B).

Mentions: Based on these pieces of evidence, the effect of the treatment with 22.5% PEG on the expression level of the genes coding for the enzymes involved in proline synthesis and catabolism was investigated. In unstressed cells, P5C synthetase 2 and P5C reductase mRNAs were present at significantly higher levels than that of OsP5CDH, whereas P5C synthetase 1 and Proline dehydrogenase transcripts were almost undetectable (Figure 3A). In PEG-stressed cells, despite the rapid and relevant increase of free proline, mRNA levels of P5C reductase did not change at all, and only minor variations were found for the two P5C synthetases, with a two-fold increase 48 h after the start of the treatment. Concerning the mRNA for P5C dehydrogenase, a two-fold increase was also evident, and the same trend was shared by the transcript of Proline dehydrogenase (Figure 3B).


Functional characterization and expression analysis of rice δ(1)-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism.

Forlani G, Bertazzini M, Zarattini M, Funck D - Front Plant Sci (2015)

Transcript levels of the genes involved in proline synthesis from glutamate and its re-oxidation in PEG-treated rice cells. The expression levels were quantified in suspension cultured cells of rice (cv Vialone nano) by real-time PCR following reverse transcription. Levels were normalized with respect to Actin 1 (Os03t0718100-01) cDNA (A). The same experiment was performed with cells treated with 22.5% PEG 6000, harvested at increasing time after the treatment. Expression levels were in this case normalized with respect to those in untreated controls (B). All treatments were carried out in triplicate, and means ± SE are reported. Results were subjected to analysis of variance, taking into account the Bonferroni correction for multiple testing; *, ***: significantly different at the 10% and 1% level, respectively, with respect to time 0 (A) or untreated controls (B).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525382&req=5

Figure 3: Transcript levels of the genes involved in proline synthesis from glutamate and its re-oxidation in PEG-treated rice cells. The expression levels were quantified in suspension cultured cells of rice (cv Vialone nano) by real-time PCR following reverse transcription. Levels were normalized with respect to Actin 1 (Os03t0718100-01) cDNA (A). The same experiment was performed with cells treated with 22.5% PEG 6000, harvested at increasing time after the treatment. Expression levels were in this case normalized with respect to those in untreated controls (B). All treatments were carried out in triplicate, and means ± SE are reported. Results were subjected to analysis of variance, taking into account the Bonferroni correction for multiple testing; *, ***: significantly different at the 10% and 1% level, respectively, with respect to time 0 (A) or untreated controls (B).
Mentions: Based on these pieces of evidence, the effect of the treatment with 22.5% PEG on the expression level of the genes coding for the enzymes involved in proline synthesis and catabolism was investigated. In unstressed cells, P5C synthetase 2 and P5C reductase mRNAs were present at significantly higher levels than that of OsP5CDH, whereas P5C synthetase 1 and Proline dehydrogenase transcripts were almost undetectable (Figure 3A). In PEG-stressed cells, despite the rapid and relevant increase of free proline, mRNA levels of P5C reductase did not change at all, and only minor variations were found for the two P5C synthetases, with a two-fold increase 48 h after the start of the treatment. Concerning the mRNA for P5C dehydrogenase, a two-fold increase was also evident, and the same trend was shared by the transcript of Proline dehydrogenase (Figure 3B).

Bottom Line: Cations were found to modulate enzyme activity, whereas anion effects were negligible.This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate.Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Biotechnology, University of Ferrara Ferrara, Italy.

ABSTRACT
While intracellular proline accumulation in response to various stress conditions has been investigated in great detail, the biochemistry and physiological relevance of proline degradation in plants is much less understood. Moreover, the second and last step in proline catabolism, the oxidation of δ(1)-pyrroline-5-carboxylic acid (P5C) to glutamate, is shared with arginine catabolism. Little information is available to date concerning the regulatory mechanisms coordinating these two pathways. Expression of the gene coding for P5C dehydrogenase was analyzed in rice by real-time PCR either following the exogenous supply of amino acids of the glutamate family, or under hyperosmotic stress conditions. The rice enzyme was heterologously expressed in E. coli, and the affinity-purified protein was thoroughly characterized with respect to structural and functional properties. A tetrameric oligomerization state was observed in size exclusion chromatography, which suggests a structure of the plant enzyme different from that shown for the bacterial P5C dehydrogenases structurally characterized to date. Kinetic analysis accounted for a preferential use of NAD(+) as the electron acceptor. Cations were found to modulate enzyme activity, whereas anion effects were negligible. Several metal ions were inhibitory in the micromolar range. Interestingly, arginine also inhibited the enzyme at higher concentrations, with a mechanism of uncompetitive type with respect to P5C. This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate. Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism.

No MeSH data available.