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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.


Arginine and proline utilization in amino acid-fed rice cells. Suspension cultured cells of rice (cv Loto) were supplied with different concentrations of either proline (A,C,E) or arginine (B,D,F). At increasing time after the addition of the exogenous amino acid, its residual level in the medium was determined (A,B). Cells from the same samples were harvested and washed thoroughly before intracellular levels of total amino acid (C,D), free proline (E) and free arginine (F) were determined on a fresh weight basis. All treatments were carried out in triplicate, and means ± SE are reported. Virtually overlapping patterns were obtained with cell cultures of the cv Vialone nano.
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Figure 4: Arginine and proline utilization in amino acid-fed rice cells. Suspension cultured cells of rice (cv Loto) were supplied with different concentrations of either proline (A,C,E) or arginine (B,D,F). At increasing time after the addition of the exogenous amino acid, its residual level in the medium was determined (A,B). Cells from the same samples were harvested and washed thoroughly before intracellular levels of total amino acid (C,D), free proline (E) and free arginine (F) were determined on a fresh weight basis. All treatments were carried out in triplicate, and means ± SE are reported. Virtually overlapping patterns were obtained with cell cultures of the cv Vialone nano.

Mentions: Even under the best conditions found, the increase of proline concentration in the cells appeared too slight to allow the investigation of its utilization during a subsequent relief from the stress. As an alternative, the fate of exogenously-supplied proline was studied. Rice cells were treated with millimolar concentrations of proline. The addition did not significantly affect cell viability up to 4 days after the treatment (results not shown). Under the experimental conditions adopted, exogenous proline was rapidly and actively taken up by the cells, whereas no significant decrease of proline concentration was found in the absence of cells during the time of the experiment (Figure 4A). Intracellular proline levels reached their maximum 24 h after addition of proline to the medium (Figure 4E). Thereafter, intracellular concentrations slowly came back to control levels. On the contrary, no variations were found with respect to total free amino acid content (Figure 4C). Because the absolute amount of proline utilized was higher in cells treated with 5 mM proline than in cells treated with 2 mM proline (Figure 4E), it is likely that proline is not simply used for protein synthesis for growth. Indeed, transcript level analysis of the genes involved in P5C metabolism in proline-fed cells revealed that 24 h after proline supply Proline dehydrogenase transcript levels were 5-fold higher than those of OsP5CDH and Ornithine-δ-aminotransferase (OAT), the gene encoding for the enzyme catalyzing the conversion of ornithine into P5C. This corresponds to a 60-fold increase of Proline dehydrogenase transcripts over basal levels (Figure 5A). At 48 h after proline treatment the Proline dehydrogenase transcript came back to control levels. Notwithstanding this, the mRNA levels of OsP5CDH were unaffected at both analyzed time points (Figure 5B).


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)

Arginine and proline utilization in amino acid-fed rice cells. Suspension cultured cells of rice (cv Loto) were supplied with different concentrations of either proline (A,C,E) or arginine (B,D,F). At increasing time after the addition of the exogenous amino acid, its residual level in the medium was determined (A,B). Cells from the same samples were harvested and washed thoroughly before intracellular levels of total amino acid (C,D), free proline (E) and free arginine (F) were determined on a fresh weight basis. All treatments were carried out in triplicate, and means ± SE are reported. Virtually overlapping patterns were obtained with cell cultures of the cv Vialone nano.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Arginine and proline utilization in amino acid-fed rice cells. Suspension cultured cells of rice (cv Loto) were supplied with different concentrations of either proline (A,C,E) or arginine (B,D,F). At increasing time after the addition of the exogenous amino acid, its residual level in the medium was determined (A,B). Cells from the same samples were harvested and washed thoroughly before intracellular levels of total amino acid (C,D), free proline (E) and free arginine (F) were determined on a fresh weight basis. All treatments were carried out in triplicate, and means ± SE are reported. Virtually overlapping patterns were obtained with cell cultures of the cv Vialone nano.
Mentions: Even under the best conditions found, the increase of proline concentration in the cells appeared too slight to allow the investigation of its utilization during a subsequent relief from the stress. As an alternative, the fate of exogenously-supplied proline was studied. Rice cells were treated with millimolar concentrations of proline. The addition did not significantly affect cell viability up to 4 days after the treatment (results not shown). Under the experimental conditions adopted, exogenous proline was rapidly and actively taken up by the cells, whereas no significant decrease of proline concentration was found in the absence of cells during the time of the experiment (Figure 4A). Intracellular proline levels reached their maximum 24 h after addition of proline to the medium (Figure 4E). Thereafter, intracellular concentrations slowly came back to control levels. On the contrary, no variations were found with respect to total free amino acid content (Figure 4C). Because the absolute amount of proline utilized was higher in cells treated with 5 mM proline than in cells treated with 2 mM proline (Figure 4E), it is likely that proline is not simply used for protein synthesis for growth. Indeed, transcript level analysis of the genes involved in P5C metabolism in proline-fed cells revealed that 24 h after proline supply Proline dehydrogenase transcript levels were 5-fold higher than those of OsP5CDH and Ornithine-δ-aminotransferase (OAT), the gene encoding for the enzyme catalyzing the conversion of ornithine into P5C. This corresponds to a 60-fold increase of Proline dehydrogenase transcripts over basal levels (Figure 5A). At 48 h after proline treatment the Proline dehydrogenase transcript came back to control levels. Notwithstanding this, the mRNA levels of OsP5CDH were unaffected at both analyzed time points (Figure 5B).

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.