Hydrogen peroxide produced by NADPH oxidases increases proline accumulation during salt or mannitol stress in Arabidopsis thaliana.

Many plants accumulate proline, a compatible osmolyte, in response to various environmental stresses such as water deficit and salinity. In some stress responses, plants generate hydrogen peroxide (H2 O2 ) that mediates numerous physiological and biochemical processes. The aim was to study the relationship between stress-induced proline accumulation and H2 O2 production. Using pharmacological and reverse genetic approaches in Arabidopsis thaliana, we investigated the role of NADPH oxidases, Respiratory burst oxidase homologues (Rboh), in the induction of proline accumulation was investigated in response to stress induced by either 200 mM NaCl or 400 mM mannitol. Stress from NaCl or mannitol resulted in a transient increase in H2 O2 content accompanied by accumulation of proline. Dimethylthiourea, a scavenger of H2 O2 , and diphenylene iodonium (DPI), an inhibitor of H2 O2 production by NADPH oxidase, were found to significantly inhibit proline accumulation in these stress conditions. DPI also reduced the expression level of Δ(1) -pyrroline-5-carboxylate synthetase, the key enzyme involved in the biosynthesis of proline. Similarly, less proline accumulated in knockout mutants lacking either AtRbohD or AtRbohF than in wild-type plants in response to the same stresses. Our data demonstrate that AtRbohs (A. thaliana Rbohs) contribute to H2 O2 production in response to NaCl or mannitol stress to increase proline accumulation in this plant.

Phospholipases C and D modulate proline accumulation in Thellungiella halophila/salsuginea differently according to the severity of salt or hyperosmotic stress.

Proline accumulation is one of the most common responses of plants to environmental constraints. Thellungiella halophila/salsuginea, a model halophyte, accumulates high levels of proline in response to abiotic stress and in the absence of stress. Recently, lipid signaling pathways have been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. Here we investigated the relationship between lipid signaling enzymes and the level of proline in T. salsuginea. Inhibition of phospholipase C (PLC) enzymes by the specific inhibitor U73122 demonstrated that proline accumulation is negatively controlled by PLCs in the absence of stress and under moderate salt stress (200 mM NaCl). The use of 1-butanol to divert some of the phospholipase D (PLD)-derived phosphatidic acid by transphosphatidylation revealed that PLDs exert a positive control on proline accumulation under severe stress (400 mM NaCl or 400 mM mannitol) but have no effect on its accumulation in non-stress conditions. This experimental evidence shows that positive and negative lipid regulatory components are involved in the fine regulation of proline metabolism. These signaling pathways in T. salsuginea are regulated in the opposite sense to those previously described in A. thaliana, revealing that common signaling components affect the physiology of closely related glycophyte and salt-tolerant plants differently.

Effects of exogenous nitric oxide on growth, proline accumulation and antioxidant capacity in Cakile maritima seedlings subjected to water deficit stress.

Nitric oxide (NO) - an endogenous signalling molecule in plants and animals - mediates responses to biotic and abiotic stresses. In the present study, we examined the role of exogenous application of NO in mediating stress responses in Cakile maritima Scop. seedlings under water deficit stress using sodium nitroprusside (SNP) as NO donor and as a pre-treatment before the application of stress. Water deficit stress was applied by withholding water for 14 days. Growth, leaf water content (LWC), osmotic potential (ψs), chlorophyll, malondialdehyde (MDA), electrolyte leakage (EL), proline and Δ1-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) protein levels were determined. Enzyme activities involved in antioxidant activities (superoxide dismutase (SOD) and catalase (CAT)) were measured upon withholding water. The results showed that shoot biomass production was significantly decreased in plants subjected to water deficit stress alone. However, in water deficit stressed plants pre-treated with SNP, growth activity was improved and proline accumulation was significantly increased. Proline accumulation was concomitant with the stimulation of its biosynthesis as shown by the accumulation of P5CS proteins. Nevertheless, no significant change in ProDH protein levels was observed. Besides plants showed lower water deficit-induced lipid membrane degradation and oxidative stress after the pretreatment with 100µM SNP. This behaviour was related to the increased activity of SOD and CAT. Thus, we concluded that NO increased C. maritima drought tolerance and mitigated damage associated with water deficit stress by the regulation of proline metabolism and the reduction of oxidative damage.

Biochemical characterization of proline dehydrogenase in Arabidopsis mitochondria.

Proline has multiple functions in plants. Besides being a building block for protein biosynthesis proline plays a central role in the plant stress response and in further cellular processes. Here, we report an analysis on the integration of proline dehydrogenase (ProDH) into mitochondrial metabolism in Arabidopsis thaliana. An experimental system to induce ProDH activity was established using cell cultures. Induction of ProDH was measured by novel photometric activity assays and by a ProDH in gel activity assay. Effects of increased ProDH activity on other mitochondrial enzymes were systematically investigated. Activities of the protein complexes of the respiratory chain were not significantly altered. In contrast, some mitochondrial dehydrogenases had markedly changed activities. Activity of glutamate dehydrogenase substantially increased, indicating upregulation of the entire proline catabolic pathway, which was confirmed by co-expression analyses of the corresponding genes. Furthermore, activity of d-lactate dehydrogenase was increased. d-lactate was identified to be a competitive inhibitor of ProDH in plants. We suggest that induction of d-lactate dehydrogenase activity allows rapid upregulation of ProDH activity during the short-term stress response in plants.

Involvement of Phosphatidylinositol 3-kinase in the regulation of proline catabolism in Arabidopsis thaliana.

Plant adaptation to abiotic stresses such as drought and salinity involves complex regulatory processes. Deciphering the signaling components that are involved in stress signal transduction and cellular responses is of importance to understand how plants cope with salt stress. Accumulation of osmolytes such as proline is considered to participate in the osmotic adjustment of plant cells to salinity. Proline accumulation results from a tight regulation between its biosynthesis and catabolism. Lipid signal components such as phospholipases C and D have previously been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. In this study, we demonstrate that proline metabolism is also regulated by class-III Phosphatidylinositol 3-kinase (PI3K), VPS34, which catalyses the formation of phosphatidylinositol 3-phosphate (PI3P) from phosphatidylinositol. Using pharmacological and biochemical approaches, we show that the PI3K inhibitor, LY294002, affects PI3P levels in vivo and that it triggers a decrease in proline accumulation in response to salt treatment of A. thaliana seedlings. The lower proline accumulation is correlated with a lower transcript level of Pyrroline-5-carboxylate synthetase 1 (P5CS1) biosynthetic enzyme and higher transcript and protein levels of Proline dehydrogenase 1 (ProDH1), a key-enzyme in proline catabolism. We also found that the ProDH1 expression is induced in a pi3k-hemizygous mutant, further demonstrating that PI3K is involved in the regulation of proline catabolism through transcriptional regulation of ProDH1. A broader metabolomic analysis indicates that LY294002 also reduced other metabolites, such as hydrophobic and aromatic amino acids and sugars like raffinose.

A new method for accurately measuring Delta(1)-pyrroline-5-carboxylate synthetase activity.

Proline is a key factor in plant adaptation to environmental stresses. The Delta(1)-pyrroline-5-carboxylate synthetase catalyzes the first committed step and the rate-limiting step for proline biosynthesis in both plants and mammals. This enzyme catalyzes the reduction of glutamate to pyrroline-5-carboxylate in two sequential steps including the phosphorylation and the reduction of its precursor. Several methods were established to assay P5CS activity but however none of them are fully reliable. Therefore, we developed a new simple and reliable assay which is based on the quantification of Pi. This assay allowed us to determine the optimal pH, the apparent K(m) and V(m) of P5CS with regard to ATP and glutamate.

Comparative salt tolerance analysis between Arabidopsis thaliana and Thellungiella halophila, with special emphasis on K(+)/Na(+) selectivity and proline accumulation.

The eco-physiology of salt tolerance, with an emphasis on K(+) nutrition and proline accumulation, was investigated in the halophyte Thellungiella halophila and in both wild type and eskimo-1 mutant of the glycophyte Arabidopsis thaliana, which differ in their proline accumulation capacity. Plants cultivated in inert sand were challenged for 3 weeks with up to 500mM NaCl. Low salinity significantly decreased A. thaliana growth, whereas growth restriction was significant only at salt concentrations equal to or exceeding 300mM NaCl in T. halophila. Na(+) content generally increased with the amount of salt added in the culture medium in both species, but T. halophila showed an ability to control Na(+) accumulation in shoots. The analysis of the relationship between water and Na(+) contents suggested an apoplastic sodium accumulation in both species; this trait was more pronounced in A. thaliana than in T. halophila. The better NaCl tolerance in the latter was associated with a better K(+) supply, resulting in higher K(+)/Na(+) ratios. It was also noteworthy that, despite highly accumulating proline, the A. thaliana eskimo-1 mutant was the most salt-sensitive species. Taken together, our findings indicate that salt tolerance may be partly linked to the plants' ability to control Na(+) influx and to ensure appropriate K(+) nutrition, but is not linked to proline accumulation.

Calcium signaling via phospholipase C is essential for proline accumulation upon ionic but not nonionic hyperosmotic stresses in Arabidopsis.

Proline (Pro) accumulation occurs in various plant organisms in response to environmental stresses. To identify the signaling components involved in the regulation of Pro metabolism upon water stress in Arabidopsis (Arabidopsis thaliana), a pharmacological approach was developed. The role of phosphoinositide-specific phospholipases C (PLCs) in Pro accumulation was assessed by the use of the aminosteroid U73122, a commonly employed specific inhibitor of receptor-mediated PLCs. We found that U73122 reduced pyrroline-5-carboxylate synthetase transcript and protein as well as Pro levels in salt-treated seedlings. Inhibition of PLC activity by U73122 was quantified by measuring the decrease of inositol 1,4,5-trisphosphate (InsP(3)) levels. Moreover, the utilization of diacylglycerol kinase and InsP(3)-gated calcium release receptor inhibitors suggested that InsP(3) or its derivatives are essential for Pro accumulation upon salt stress, involving calcium as a second messenger in ionic stress signaling. This observation was further supported by a partial restoration of Pro accumulation in salt- and U73122-treated seedlings after addition of extracellular calcium, or when calcium homeostasis was perturbed by cyclopiazonic acid, a blocker of plant type IIA calcium pumps. Taken together, our data indicate that PLC-based signaling is a committed step in Pro biosynthesis upon salinity but not in the case of mannitol stress. Calcium acts as a molecular switch to trigger downstream signaling events. These results also demonstrated the specific involvement of lipid signaling pathway to discriminate between ionic and nonionic stresses.