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

Proline 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.

Pyrroline-5-carboxylate dehydrogenase is an essential enzyme for proline dehydrogenase function during dark-induced senescence in Arabidopsis thaliana.

During leaf senescence, nitrogen is remobilized and carbon backbones are replenished by amino acid catabolism, with many of the key reactions occurring in mitochondria. The intermediate Δ1 -pyrroline-5-carboxylate (P5C) is common to some catabolic pathways, thus linking the metabolism of several amino acids, including proline and arginine. Specifically, mitochondrial proline catabolism involves sequential action of proline dehydrogenase (ProDH) and P5C dehydrogenase (P5CDH) to produce P5C and then glutamate. Arginine catabolism produces urea and ornithine, the latter in the presence of α-ketoglutarate being converted by ornithine δ-aminotransferase (OAT) into P5C and glutamate. Metabolic changes during dark-induced leaf senescence (DIS) were studied in Arabidopsis thaliana leaves of Col-0 and in prodh1prodh2, p5cdh and oat mutants. Progression of DIS was followed by measuring chlorophyll and proline contents for 5 days. Metabolomic profiling of 116 compounds revealed similar profiles of Col-0 and oat metabolism, distinct from prodh1prodh2 and p5cdh metabolism. Metabolic dynamics were accelerated in p5cdh by 1 day. Notably, more P5C and proline accumulated in p5cdh than in prodh1prodh2. ProDH1 enzymatic activity and protein amount were significantly down-regulated in p5cdh mutant at Day 4 of DIS. Mitochondrial P5C levels appeared critical in determining the flow through interconnected amino acid remobilization pathways to sustain senescence.

Potential involvement of proline and flavonols in plant responses to ozone.

Ozone is considered to be a major phytotoxic pollutant. It is an oxidizing molecule with harmful effects that can affect human health and vegetation. Due to its phytotoxicity, it constitutes a threat to food security in a context of climate change. Proline accumulation is induced in response to numerous stresses and is assumed to be involved in plant antioxidant defense. We therefore addressed the question of the putative involvement of proline in plant ozone responses by analyzing the responses of two Arabidopsis mutants (obtained in the Col-0 genetic background) altered in proline metabolism and different ecotypes with various degrees of ozone sensitivity, to controlled ozone treatments. Among the mutants, the p5cs1 mutant plants accumulated less proline than the double prodh1xprodh2 (p1p2) mutants. Ozone treatments did not induce accumulation of proline in Col-0 nor in the mutant plants. However, the variation of the photosynthetic parameter Fv/Fm in the p1p2 mutant suggests a positive effect of proline. Proline accumulation induced by ozone was only observed in the most ozone-sensitive ecotypes, Cvi-0 and Ler. Contrary to our expectations, proline accumulation could not be correlated with variations in protein oxidation (carbonylation). On the other hand, flavonols content, measured here, using non-destructive methods, reflected exactly the genotypes ranking according to ozone sensitivity.

The proline cycle as an eukaryotic redox valve.

The 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.

How Does Proline Treatment Promote Salt Stress Tolerance During Crop Plant Development?

Soil salinity is one of the major abiotic stresses restricting the use of land for agriculture because it limits the growth and development of most crop plants. Improving productivity under these physiologically stressful conditions is a major scientific challenge because salinity has different effects at different developmental stages in different crops. When supplied exogenously, proline has improved salt stress tolerance in various plant species. Under high-salt conditions, proline application enhances plant growth with increases in seed germination, biomass, photosynthesis, gas exchange, and grain yield. These positive effects are mainly driven by better nutrient acquisition, water uptake, and biological nitrogen fixation. Exogenous proline also alleviates salt stress by improving antioxidant activities and reducing Na+ and Cl- uptake and translocation while enhancing K+ assimilation by plants. However, which of these mechanisms operate at any one time varies according to the proline concentration, how it is applied, the plant species, and the specific stress conditions as well as the developmental stage. To position salt stress tolerance studies in the context of a crop plant growing in the field, here we discuss the beneficial effects of exogenous proline on plants exposed to salt stress through well-known and more recently described examples in more than twenty crop species in order to appreciate both the diversity and commonality of the responses. Proposed mechanisms by which exogenous proline mitigates the detrimental effects of salt stress during crop plant growth are thus highlighted and critically assessed.

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

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.

Proline oxidation fuels mitochondrial respiration during dark-induced leaf senescence in Arabidopsis thaliana.

Leaf senescence is a form of developmentally programmed cell death that allows the remobilization of nutrients and cellular materials from leaves to sink tissues and organs. Among the catabolic reactions that occur upon senescence, little is known about the role of proline catabolism. In this study, the involvement in dark-induced senescence of proline dehydrogenases (ProDHs), which catalyse the first and rate-limiting step of proline oxidation in mitochondria, was investigated using prodh single- and double-mutants with the help of biochemical, proteomic, and metabolomic approaches. The presence of ProDH2 in mitochondria was confirmed by mass spectrometry and immunogold labelling in dark-induced leaves of Arabidopsis. The prodh1 prodh2 mutant exhibited enhanced levels of most tricarboxylic acid cycle intermediates and free amino acids, demonstrating a role of ProDH in mitochondrial metabolism. We also found evidence of the involvement and the importance of ProDH in respiration, with proline as an alternative substrate, and in remobilization of proline during senescence to generate glutamate and energy that can then be exported to sink tissues and organs.

Proteomic and functional analysis of proline dehydrogenase 1 link proline catabolism to mitochondrial electron transport in Arabidopsis thaliana.

Proline accumulates in many plant species in response to environmental stresses. Upon relief from stress, proline is rapidly oxidized in mitochondria by proline dehydrogenase (ProDH) and then by pyrroline-5-carboxylate dehydrogenase (P5CDH). Two ProDH genes have been identified in the genome of the model plant Arabidopsis thaliana To gain a better understanding of ProDH1 functions in mitochondria, proteomic analysis was performed. ProDH1 polypeptides were identified in Arabidopsis mitochondria by immunoblotting gels after 2D blue native (BN)-SDS/PAGE, probing them with an anti-ProDH antibody and analysing protein spots by MS. The 2D gels showed that ProDH1 forms part of a low-molecular-mass (70-140 kDa) complex in the mitochondrial membrane. To evaluate the contribution of each isoform to proline oxidation, mitochondria were isolated from wild-type (WT) and prodh1, prodh2, prodh1prodh2 and p5cdh mutants. ProDH activity was high for genotypes in which ProDH, most likely ProDH1, was strongly induced by proline. Respiratory measurements indicate that ProDH1 has a role in oxidizing excess proline and transferring electrons to the respiratory chain.