Abstract
Under several stress conditions, such as excess salt and drought, many plants accumulate proline inside the cell, which is believed to help counteracting the adverse effects of low water potential. This increase mainly relies upon transcriptional induction of δ1-pyrroline-5-carboxylate synthetase (P5CS), the enzyme that catalyzes the first two steps in proline biosynthesis from glutamate. P5CS mediates both the phosphorylation of glutamate and the reduction of γ-glutamylphosphate to glutamate-5-semialdehyde, which spontaneously cyclizes to δ1-pyrroline-5-carboxylate (P5C). In most higher plants, two isoforms of P5CS have been found, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress-induced. Despite the number of papers to investigate the regulation of P5CS at the transcriptional level, to date, the properties of the enzyme have been only poorly studied. As a consequence, the descriptions of post-translational regulatory mechanisms have largely been limited to feedback-inhibition by proline. Here, we report cloning and heterologous expression of P5CS2 from Oryza sativa. The protein has been fully characterized from a functional point of view, using an assay method that allows following the physiological reaction of the enzyme. Kinetic analyses show that the activity is subjected to a wide array of regulatory mechanisms, ranging from product inhibition to feedback inhibition by proline and other amino acids. These findings confirm long-hypothesized influences of both, the redox status of the cell and nitrogen availability, on proline biosynthesis.
Highlights
Water stress tolerance is a major goal for plant genetic improvement, and most likely a need to secure crop productivity threatened by ongoing climate changes (Ahanger et al, 2017)
The gene coding in rice for the stress-induced pyrroline5-carboxylate synthetase (P5CS) isoenzyme 2 was cloned into the expression vector pET151 and used to transform E. coli BL21(DE3) pLysS cells
Following the treatment with IPTG, glutamate and ATP-dependent NADPH oxidation activity was detectable in crude extracts, which was not present in extracts from either non-induced cells or induced cells transformed with the empty vector
Summary
Water stress tolerance is a major goal for plant genetic improvement, and most likely a need to secure crop productivity threatened by ongoing climate changes (Ahanger et al, 2017). The first two steps were found to be catalyzed by a single bifunctional enzyme bearing both catalytic domains of glutamyl kinase and glutamyl-phosphate reductase, P5C synthetase (P5CS; Hu et al, 1992; Rai and Penna, 2013). Isolation and sequencing of P5CS led to the identification of two isogenes in several plant species and phylogenetic analyses demonstrated that P5CS duplication occurred independently in several taxonomic groups (Turchetto-Zolet et al, 2008; Rai and Penna, 2013, Fichman et al, 2015) These paralogs (named P5CS1 and P5CS2) showed non-overlapping roles, with varying temporal and spatial expression patterns. We report cloning, recombinant expression, purification, and functional characterization of P5CS2 from rice, providing a first body of evidence toward this goal
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