Chemical and Molecular Genetics Approach to Study ROP1 Signaling Pathway in Pollen Tubes

Polarized growth in pollen tube requires complex signaling events including ROP1 GTPase pathway and calcium signaling. The role of tip calcium in negative feedback regulation of ROP1 remains a question and potentially involves ROP1 negative regulator, REN1GAP, whose activity seems to be regulated by calcium. Calcium-dependent protein kinases (CDPKs/CPKs) are calcium sensors with known function in regulating pollen tube tip growth. As such, we hypothesized that CPK substrate(s) in pollen tube may be component of tip growth regulator such as REN1. Here we report that REN1 is phosphorylated by pollen-expressed CPK16 with a relative EC50 value of ~4.6 µM. MS/MS analysis revealed calcium-dependent phosphorylation sites within REN1 which include Ser70 and Ser267. Functional analyses suggested that REN1 phosphorylation at Ser267 is required for its activity while at Ser70,71 affected its localization and subsequently activity. Mutation analysis of CPK16 loss-of-function, cpk16-3, revealed enhanced pollen tube growth and germination when grown in low calcium media. Treatment of cpk16-3 tubes with either brefeldin A or latrunculin B induced tip swelling phenotype similar to the same effects produced by chemical treatments of partially complemented ren1-1 Lat52::GFP-REN1, Overall, these results suggest a link between calcium and a major signaling pathway, ROP1, via calcium-dependent protein kinase and its substrate REN1. To dissect the causal and phasal relationships between oscillations of growth, active ROP1, F-actin dynamics, and tip-focused calcium, a chemical genetics approach was utilized with the goal to identify small molecules that would specifically activate ROP1. To this end, 20000 chemicals were screened in a cell-based yeast two hybrid assay to target inhibitors of active ROP and GAP. One compound, #7, inhibited ROP-GAP interaction as confirmed by in vitro assays as well as slightly enhanced pollen tube tip width. Treatment of ROP1 OX severely enhanced tip swelling suggesting that it primarily targets ROP1. Docking analyses of compound 7 to the protein interaction interface of RhoA and p190RhoGAP revealed possible binding sites within the GTP-binding pockets of Rho-GAP interface in potentially disrupting protein-protein interaction. This study leads to a potential activator of ROP1 which may be useful for future ROP1 studies