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.


Proline levels in salt- and PEG-treated rice cells. Suspension cultured cells of rice (cv Vialone nano) were treated with different, growth inhibitory concentrations of either NaCl (A,C,E) or PEG 6000 (B,D,F). At increasing time after the treatments, free proline levels (A,B) and total amino acid concentrations (C,D) were determined on a fresh weight basis. Proline concentration was also expressed as percent of total amino acids (E,F). All treatments were carried out in triplicate, and means ± SE are depicted. Very similar results were obtained also with cell cultures of the cv Loto.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Proline levels in salt- and PEG-treated rice cells. Suspension cultured cells of rice (cv Vialone nano) were treated with different, growth inhibitory concentrations of either NaCl (A,C,E) or PEG 6000 (B,D,F). At increasing time after the treatments, free proline levels (A,B) and total amino acid concentrations (C,D) were determined on a fresh weight basis. Proline concentration was also expressed as percent of total amino acids (E,F). All treatments were carried out in triplicate, and means ± SE are depicted. Very similar results were obtained also with cell cultures of the cv Loto.

Mentions: To investigate the regulation of OsP5CDH during stress-induced proline accumulation, rice suspension cultured cells were treated with increasing concentrations of either NaCl or polyethylene glycol (PEG) to induce salt or osmotic stress conditions, respectively. Cell growth was progressively reduced by both stress treatments, but not completely suppressed (not shown). As expected, free proline content in salt-stressed cells increased with time in a dose-responsive manner (Figure 2A). However, also total amino acid content showed a similar pattern (Figure 2C), and if proline content was expressed as percentage of total amino acids, only minor variations, if any, were evident in response to salt treatment (Figure 2E). A remarkably different picture was obtained with PEG. In this case only a slight but reproducible increase of intracellular proline levels was found soon after the exposure to osmotic stress conditions, which at low PEG doses came back to control levels after a couple of days (Figure 2B). The concentration of free amino acids did not increase, but showed on the contrary a slight decrease (Figure 2D). Because the treatment caused a 20 to 35%-loss of cell viability (data not presented), such a decrease most likely depended on the presence of a significant amount of dead cells that influences the amino acid content, as expressed on a fresh weight basis. Interestingly, when proline levels were expressed as percentage of total amino acids, a significant increase was evident that was proportional to the severity of the stress. At 22.5% PEG, this increase was maintained over the entire culture cycle (Figure 2F). The effects of higher PEG concentrations were not tested because of the resulting undesirable consequence on cell viability. Because the plant response to hyperosmotic stress conditions is at least in part mediated by abscisic acid, similar experiments were performed also by treating cells with 50 μM ABA, either alone or in combination with PEG. Results were identical to those obtained for untreated cells or with cell treated with PEG alone, respectively (data not shown). To rule out the possibility that these results could be cultivar-specific, or depend on some mutation that may have occurred with time at the undifferentiated tissue level, where mutations that are lethal in planta can be maintained within the cell population, the experiments were carried out with independent cell cultures of two japonica rice cultivars (Loto and Vialone nano). Virtually overlapping patterns of amino acid profile changes were found in both cell cultures (data not shown).


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)

Proline levels in salt- and PEG-treated rice cells. Suspension cultured cells of rice (cv Vialone nano) were treated with different, growth inhibitory concentrations of either NaCl (A,C,E) or PEG 6000 (B,D,F). At increasing time after the treatments, free proline levels (A,B) and total amino acid concentrations (C,D) were determined on a fresh weight basis. Proline concentration was also expressed as percent of total amino acids (E,F). All treatments were carried out in triplicate, and means ± SE are depicted. Very similar results were obtained also with cell cultures of the cv Loto.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Proline levels in salt- and PEG-treated rice cells. Suspension cultured cells of rice (cv Vialone nano) were treated with different, growth inhibitory concentrations of either NaCl (A,C,E) or PEG 6000 (B,D,F). At increasing time after the treatments, free proline levels (A,B) and total amino acid concentrations (C,D) were determined on a fresh weight basis. Proline concentration was also expressed as percent of total amino acids (E,F). All treatments were carried out in triplicate, and means ± SE are depicted. Very similar results were obtained also with cell cultures of the cv Loto.
Mentions: To investigate the regulation of OsP5CDH during stress-induced proline accumulation, rice suspension cultured cells were treated with increasing concentrations of either NaCl or polyethylene glycol (PEG) to induce salt or osmotic stress conditions, respectively. Cell growth was progressively reduced by both stress treatments, but not completely suppressed (not shown). As expected, free proline content in salt-stressed cells increased with time in a dose-responsive manner (Figure 2A). However, also total amino acid content showed a similar pattern (Figure 2C), and if proline content was expressed as percentage of total amino acids, only minor variations, if any, were evident in response to salt treatment (Figure 2E). A remarkably different picture was obtained with PEG. In this case only a slight but reproducible increase of intracellular proline levels was found soon after the exposure to osmotic stress conditions, which at low PEG doses came back to control levels after a couple of days (Figure 2B). The concentration of free amino acids did not increase, but showed on the contrary a slight decrease (Figure 2D). Because the treatment caused a 20 to 35%-loss of cell viability (data not presented), such a decrease most likely depended on the presence of a significant amount of dead cells that influences the amino acid content, as expressed on a fresh weight basis. Interestingly, when proline levels were expressed as percentage of total amino acids, a significant increase was evident that was proportional to the severity of the stress. At 22.5% PEG, this increase was maintained over the entire culture cycle (Figure 2F). The effects of higher PEG concentrations were not tested because of the resulting undesirable consequence on cell viability. Because the plant response to hyperosmotic stress conditions is at least in part mediated by abscisic acid, similar experiments were performed also by treating cells with 50 μM ABA, either alone or in combination with PEG. Results were identical to those obtained for untreated cells or with cell treated with PEG alone, respectively (data not shown). To rule out the possibility that these results could be cultivar-specific, or depend on some mutation that may have occurred with time at the undifferentiated tissue level, where mutations that are lethal in planta can be maintained within the cell population, the experiments were carried out with independent cell cultures of two japonica rice cultivars (Loto and Vialone nano). Virtually overlapping patterns of amino acid profile changes were found in both cell cultures (data not shown).

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.