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.

• 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.