Appropriate Activity Assays Are Crucial for the Specific Determination of Proline Dehydrogenase and Pyrroline-5-Carboxylate Reductase Activities.

Sandrine Lebreton,Arnould Savouré,Giuseppe Forlani,Dietmar Funck,Cécile Cabassa-Hourton

doi:10.3389/fpls.2020.602939

Abstract

Accumulation of proline is a widespread plant response to a broad range of environmental stress conditions including salt and osmotic stress. Proline accumulation is achieved mainly by upregulation of proline biosynthesis in the cytosol and by inhibition of proline degradation in mitochondria. Changes in gene expression or activity levels of the two enzymes catalyzing the first reactions in these two pathways, namely pyrroline-5-carboxylate (P5C) synthetase and proline dehydrogenase (ProDH), are often used to assess the stress response of plants. The difficulty to isolate ProDH in active form has led several researchers to erroneously report proline-dependent NAD+ reduction at pH 10 as ProDH activity. We demonstrate that this activity is due to P5C reductase (P5CR), the second and last enzyme in proline biosynthesis, which works in the reverse direction at unphysiologically high pH. ProDH does not use NAD+ as electron acceptor but can be assayed with the artificial electron acceptor 2,6-dichlorophenolindophenol (DCPIP) after detergent-mediated solubilization or enrichment of mitochondria. Seemingly counter-intuitive results from previous publications can be explained in this way and our data highlight the importance of appropriate and specific assays for the detection of ProDH and P5CR activities in crude plant extracts.

Highlights

Many prokaryotes and eukaryotes accumulate free proline as a compatible solute in response to abiotic or biotic stress (Forlani et al, 2019; Trovato et al, 2019)
When the deduced mature polypeptides (ProDH1 N12 and ProDH2 N13) or the pre-proteins of Arabidopsis ProDH1 and ProDH2 were expressed as GSTfusion proteins in E. coli, very little soluble protein of the expected size was obtained by expression over night at 10– 18◦C (Figure 2A)
The rates of P5C-dependent NADPHoxidation at pH 7.5 and of proline-dependent NAD+ reduction at pH 10 were very similar in cells carrying the empty vector or expressing ProDH1 or ProDH2, but increased more than 180-fold in cells expressing P5C reductase (P5CR) (Figure 2B)

Summary

Introduction

Many prokaryotes and eukaryotes accumulate free proline as a compatible solute in response to abiotic or biotic stress (Forlani et al, 2019; Trovato et al, 2019). In order to improve crop performance under stress conditions, a lot of research has been dedicated to understand the molecular basis of proline accumulation and the role of this amino acid in plant acclimation and stress tolerance (Szabados and Savoure, 2010; Forlani et al, 2019; ProDH or P5CR Activity NAD(P)H NAD(P)+ H2O GSA P5C spontaneous P5CR ProDH FAD(H2) Proline QH2 Q. Proline production is catalyzed by the sequential action of P5C synthetase and P5C reductase (P5CR), the latter being the only known enzyme to synthesize proline in plants (Trovato et al, 2019). Proline degradation occurs in mitochondria by the sequential action of proline dehydrogenase (ProDH) and P5C dehydrogenase (P5CDH; Trovato et al, 2019). To account for the frequent observation of oxygen consumption in conjunction with proline oxidation, ProDH was and is occasionally referred to as proline oxidase, but good evidence for direct electron transfer to molecular oxygen has not been obtained so far

Methods

Results

Conclusion