• Δ1-pyrroline-5-carboxylate (P5C) reductase catalyses the final step of proline synthesis in plants. In Arabidopsis thaliana, protein levels are correlated neither to the corresponding mRNA copy numbers, nor to the intracellular proline concentrations. The occurrence of post-translational regulative mechanisms was therefore hypothesized, but never assessed. • The purification of A. thaliana P5C reductase was achieved through either a six-step protocol from cultured cells, or heterologous expression of AtP5CR in E. coli. The protein was characterized with respect to structural, kinetic, and biochemical properties. • P5C reductase was able to use either NADPH or NADH as the electron donor, with contrasting affinities and maximum reaction rates. The presence of equimolar levels of NADP+ completely suppressed the NADH-dependent activity, whereas the NADPH-dependent reaction was mildly affected. Proline inhibited only the NADH-dependent reaction. At physiological levels, increasing concentrations of salt steadily inhibited the NADH-dependent activity, but were stimulatory of the NADPH-dependent reaction. • The biochemical properties of A. thaliana P5C reductase suggest a complex regulation of enzyme activity by the redox status of the pyridine nucleotide pools, and the levels of proline and chloride in the cytosol. Data support a so far underestimated role of P5C reductase in controlling stress-induced proline accumulation.

Δ1-pyrroline-5-carboxylate reductase from Arabidopsis thaliana: stimulation or inhibition by chloride ions and feed-back regulation by proline depend on whether NADPH or NADH acts as co-substrate.

GIBERTI, Samuele;FORLANI, Giuseppe
2014

Abstract

• Δ1-pyrroline-5-carboxylate (P5C) reductase catalyses the final step of proline synthesis in plants. In Arabidopsis thaliana, protein levels are correlated neither to the corresponding mRNA copy numbers, nor to the intracellular proline concentrations. The occurrence of post-translational regulative mechanisms was therefore hypothesized, but never assessed. • The purification of A. thaliana P5C reductase was achieved through either a six-step protocol from cultured cells, or heterologous expression of AtP5CR in E. coli. The protein was characterized with respect to structural, kinetic, and biochemical properties. • P5C reductase was able to use either NADPH or NADH as the electron donor, with contrasting affinities and maximum reaction rates. The presence of equimolar levels of NADP+ completely suppressed the NADH-dependent activity, whereas the NADPH-dependent reaction was mildly affected. Proline inhibited only the NADH-dependent reaction. At physiological levels, increasing concentrations of salt steadily inhibited the NADH-dependent activity, but were stimulatory of the NADPH-dependent reaction. • The biochemical properties of A. thaliana P5C reductase suggest a complex regulation of enzyme activity by the redox status of the pyridine nucleotide pools, and the levels of proline and chloride in the cytosol. Data support a so far underestimated role of P5C reductase in controlling stress-induced proline accumulation.
2014
Giberti, Samuele; Funck, D.; Forlani, Giuseppe
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2277025
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 90
  • ???jsp.display-item.citation.isi??? 75
social impact