Isolation of an active pistil component responsible for LePRK2 dephosphorylation

WENGIER, DIEGO; MUSCHIETTI, JORGE

PINAMAR, ARGENTINA

Congreso; SAIB 2005; 2005

SAIB

LePRK2 is a receptor kinase located at the plasma membrane of tomato pollen grains. In mature pollen, it forms a high molecular weight complex together with LePRK1 (another pollen receptor kinase) and other still unknown proteins. In this complex, LePRK2 is phosphorylated. When incubated with exudates from pistils, LePRK2 is specifically dephosphorylated and the high molecular weight complex is disrupted completely. Previously, we showed that the active pistil component responsible for these two phenomena has a molecular weight of 3-10 kDa, is heat stable, partially resistant to protease activity, resistant to acid treatments but not to base hydrolysis. We decided to isolate that pistil component using a combination of different chromatographic methods (cationic and anionic exchange resins and reverse phase chromatography) and solvent extractions (chloroform-methanol extraction and acetone precipitation). In the last step of our designed purification protocol, LePRK2 dephosphorylation activity coelutes with a single peak from anionic exchange chromatography (MonoQ) at ~100 mM ammonium bicarbonate. Further studies of 13C-NMR and mass spectroscopy will be used to determine the structure and molecular weight of this component. For the understanding of LePRKs complex signal transduction during pollen tube growth, it will be essential to identify this pistil component.