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.

Organ-dependent oxylipin signature in leaves and roots of salinized tomato plants (Solanum lycopersicum).

Oxylipins have been extensively studied in plant defense mechanisms or as signal molecules. Depending on the stress origin (e.g. wounding, insect, pathogen), and also on the plant species or organ, a specific oxylipin signature can be generated. Salt stress is frequently associated with secondary stress such as oxidative damage. Little is known about the damage caused to lipids under salt stress conditions, especially with respect to oxylipins. In order to determine if an organ-specific oxylipin signature could be observed during salt stress, tomato (Solanum lycopersicum cv. Money Maker) plants were submitted to salt stress (100 mM of NaCl) for a 30-d period. A complete oxylipin profiling and LOX related-gene expression measurement were achieved in leaves and roots. As expected, salt stress provoked premature senescence in leaves, as revealed by a decrease in photosystem II efficiency (F(v)/F(m) ratio) and sodium accumulation in leaves. In roots, a significant decrease in several oxylipins (9- and 13-hydro(pero)xy linole(n)ic acids, keto and divinyl ether derivatives) was initiated at day 5 and intensified at day 21 after salt treatment, whereas jasmonic acid content increased. In leaves, the main changes in oxylipins were observed later (at day 30), with an increase in some 9- and 13-hydro(pero)xy linole(n)ic acids and a decrease in some keto-derivatives and in jasmonic acid. Oxylipin enantiomeric characterization revealed that almost all compounds were formed enzymatically, and therefore a massive auto-oxidation of lipids that can be encountered in abscission processes can be excluded here.

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.

Phospholipase D is a negative regulator of proline biosynthesis in Arabidopsis thaliana.

Accumulation of proline has been observed in a large number of plant species in response to drought and salt stresses, suggesting a key role of this amino acid in plant stress adaptation. Upstream components of the proline biosynthesis signal transduction pathways are still poorly defined. We provide experimental evidence that phospholipase D (PLD) is involved in the regulation of proline metabolism in Arabidopsis thaliana. The application of primary butyl alcohols, which divert part of PLD-derived phosphatidic acid by transphosphatidylation, stimulated proline biosynthesis even without hyperosmotic constraints. Moreover, application of primary butyl alcohols enhanced the proline responsiveness of seedlings to mild hyperosmotic stress. These data indicate that some PLDs are negative regulators of proline biosynthesis and that plants present a higher proline responsiveness to hyperosmotic stress when this regulator is abolished. We clearly demonstrate that PLD signaling for proline biosynthesis is similar to RD29A gene expression and different from the abscisic acid-dependent RAB18 gene expression. Our data reveal that PLDs play positive and negative roles in hyperosmotic stress signal transduction in plants, contributing to a precise regulation of ion homeostasis and plant salt tolerance.