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.

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.

Pectin and the role of the physical properties of the cell wall in pollen tube growth of Solanum chacoense.

The cell wall is one of the structural key players regulating pollen tube growth, since plant cell expansion depends on an interplay between intracellular driving forces and the controlled yielding of the cell wall. Pectin is the main cell wall component at the growing pollen tube apex. We therefore assessed its role in pollen tube growth and cytomechanics using the enzymes pectinase and pectin methyl esterase (PME). Pectinase activity was able to stimulate pollen germination and tube growth at moderate concentrations whereas higher concentrations caused apical swelling or bursting in Solanum chacoense Bitt. pollen tubes. This is consistent with a modification of the physical properties of the cell wall affecting its extensibility and thus the growth rate, as well as its capacity to withstand turgor. To prove that the enzyme-induced effects were due to the altered cell wall mechanics, we subjected pollen tubes to micro-indentation experiments. We observed that cellular stiffness was reduced and visco-elasticity increased in the presence of pectinase. These are the first mechanical data that confirm the influence of the amount of pectins in the pollen tube cell wall on the physical parameters characterizing overall cellular architecture. Cytomechanical data were also obtained to analyze the role of the degree of pectin methyl-esterification, which is known to exhibit a gradient along the pollen tube axis. This feature has frequently been suggested to result in a gradient of the physical properties characterizing the cell wall and our data provide, for the first time, mechanical support for this concept. The gradient in cell wall composition from apical esterified to distal de-esterified pectins seems to be correlated with an increase in the degree of cell wall rigidity and a decrease of visco-elasticity. Our mechanical approach provides new insights concerning the mechanics of pollen tube growth and the architecture of living plant cells.

More than a leak sealant. The mechanical properties of callose in pollen tubes.

While callose is a well-known permeability barrier and leak sealant in plant cells, it is largely unknown whether this cell wall polymer can also serve as a load-bearing structure. Since callose occurs in exceptionally large amounts in pollen, we assessed its role for resisting tension and compression stress in this cell. The effect of callose digestion in Solanum chacoense and Lilium orientalis pollen grains demonstrated that, depending on the species, this cell wall polymer represents a major stress-bearing structure at the aperture area of germinating grains. In the pollen tube, it is involved in cell wall resistance to circumferential tension stress, and despite its absence at the growing apex, callose is indirectly involved in the establishment of tension stress resistance in this area. To investigate whether or not callose is able to provide mechanical resistance against compression stress, we subjected pollen tubes to local deformation by microindentation. The data revealed that lowering the amount of callose resulted in reduced cellular stiffness and increased viscoelasticity, thus indicating clearly that callose is able to resist compression stress. Whether this function is relevant for pollen tube mechanics, however, is unclear, as stiffened growth medium caused a decrease in callose deposition. Together, our data provide clear evidence for the capacity of cell wall callose to resist tension and compression stress, thus demonstrating that this amorphous cell wall substance can have a mechanical role in growing plant cells.