2. Biochemical approaches to study pollen receptor kinases.

MUSCHIETTI, JORGE

. ?Frontiers in Pollen Tube Biology Shanghai, China

Conferencia; Pollen Biology International Workshop; 2012

Shanghai Institute of Plant Physiology and Ecology, CAS

The tip-growing pollen tube is a useful model for studying polarized cell growth in plants. We previously characterized LePRK1 and LePRK2, two pollen-specific receptor-like kinases from tomato (Solanum lycopersicum). We used different biochemical and molecular techniques to demonstrate that 1) LePRK1 and LePRK2 interact in pollen membranes and when expressed in yeast; 2) LePRK1 and LePRK2 are part of a high molecular weight protein complex (LePRK complex); 3) LePRK1-LePRK2 interaction is disrupted when pollen is germinated in the presence of style extracts; 4) LePRK2 dephosphorylation is mediated by a heat-, acid-, base-, DTT- and protease resistant component from styles. Style exudates were subjected to chloroform extraction, anionic exchange, and C18 reverse-phase chromatography columns, obtaining a single ~3,550 Da compound that increased pollen tube lengths of in vitro germinated pollen in a dose dependent manner. Using comparative sequence analysis and phosphorylation site prediction programs, we identified two putative phosphorylation motifs in the cytoplasmic juxtamembrane (JM) domain of LePRK2. Site-directed mutagenesis in these motifs, followed by transient overexpression in tobacco pollen, showed that both motifs have opposite effects in regulating pollen tube length suggesting that LePRK2 may have a role in pollen tube growth through regulation of its own phosphorylation status.