Potential involvement of proline and flavonols in plant responses to ozone

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The proline cycle as a eukaryotic redox valve

International audienceThe amino acid proline has been known for many years to be a component of proteins as well as an osmolyte. Many recent studies have demonstrated that proline has other roles such as regulating redox balance and energy status. In animals and plants, the well-described proline cycle is concomitantly responsible for the preferential accumulation of proline and shuttling of redox equivalents from the cytosol to mitochondria. The impact of the proline cycle goes beyond regulating proline levels. In this review, we focus on recent evidence of how the proline cycle regulates redox status in relation to other redox shuttles. We discuss how the interconversion of proline and glutamate shuttles reducing power between cellular compartments. Spatial aspects of the proline cycle in the entire plant are considered in terms of proline transport between organs with different metabolic regimes (photosynthesis versus respiration). Furthermore, we highlight the importance of this shuttle in the regulation of energy and redox power in plants, through a particularly intricate coordination, notably between mitochondria and cytosol

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

none5siAccumulation 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.Finanziato da FAR 2020, ma non è disponibile nel menu a tendinaopenLebreton, Sandrine; Cabassa-Hourton, Cécile; Savouré, Arnould; Funck, Dietmar; Forlani, GiuseppeLebreton, Sandrine; Cabassa-Hourton, Cécile; Savouré, Arnould; Funck, Dietmar; Forlani, Giusepp

Pyrroline-5-carboxylate metabolism protein complex detected in Arabidopsis thaliana leaf mitochondria

International audienceProline dehydrogenase (ProDH) and pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) catalyse the oxidation of proline into glutamate via the intermediates P5C and glutamate-semialdehyde (GSA), which spontaneously interconvert. P5C and GSA are also intermediates in the production of glutamate from ornithine and α-ketoglutarate catalysed by ornithine δ-aminotransferase (OAT). ProDH and P5CDH form a fused bifunctional PutA enzyme in Gram-negative bacteria and are associated in a bifunctional substrate-channelling complex in Thermus thermophilus; however, the physical proximity of ProDH and P5CDH in eukaryotes has not been described. Here, we report evidence of physical proximity and interactions between Arabidopsis PRODH, P5CDH, and OAT in the mitochondria of plants during dark-induced leaf senescence when all three enzymes are expressed. Pairwise interactions and localization of the three enzymes were investigated using bimolecular fluorescence complementation with confocal microscopy in tobacco and sub-mitochondrial fractionation in Arabidopsis. Evidence for a complex composed of PRODH, P5CDH, and OAT was revealed by co-migration of the proteins in native conditions upon gel electrophoresis. Co-immunoprecipitation coupled with mass spectrometry analysis confirmed the presence of the P5C metabolism complex in Arabidopsis. Pull-down assays further demonstrated a direct interaction between PRODH1 and P5CDH. P5C metabolism complexes might channel P5C among the constituent enzymes and directly provide electrons to the respiratory electron chain via PRODH