A Chemical Genetic Analysis of Galactose Biochemistry in Growing Pollen Tubes

Background: Pollen tubes grow by tip growth rather than normal cell division. Tip growth is an extremely rapid process in which enzymes simultaneously destroy and recreate the cell wall until the cell extends to more than 1000 times its original length. In this process, pollen tubes break down dense plant cell walls composed of complex carbohydrates. Microarray data suggests that two β-galactosidases, BGAL11 and BGAL13, exhibit increased expression in hydrated pollen and tube elongation.  Preliminary studies revealed that a double knockout strain of Arabidopsis (deficient in BGAL11 and BGAL13) disrupts pollen tube elongation in vitro and in vivo. As such, we are interested in exploring the role of BGAL11 and BGAL13 in pollen tube tip growth using both chemical genetic and biochemical techniques Methods: I created a high-throughput assay that provides accurate correlation between fluorescence and pollen tube length and/or germination rates without requiring microscopy. I also optimized a procedure for a multi-step reaction to produce a library of 15 galactosyl triazoles. The final compounds were clean enough to screen “as is” without further purification. Using Pichia pastoris, I began cloning, expressing and purifying BGAL11 and BGAL13 using in order to characterize these enzymes in vitro. Previous research showed that E. coli is unable to easily express these large, highly glycosylated proteins, and yeast may provide the eukaryotic post-translational modifications necessary for the BGALs to fold into soluble proteins. Results: Using the optimized pollen growth assay, I showed that that fluorescence inversely correlated with inhibitor concentration. I then screened over 50 library compounds for reduced pollen growth. Several glycosyl triazoles consistently reduced pollen growth. These compounds were further evaluated in a β-galactosidase activity assay using total protein content extracted from pollen grains. In the next few months, I will optimize the expression and purification of both BGAL11 and BGAL13 to be used in enzyme activity assays in order to fully evaluate the biological activity of all library compounds. Conclusions: This project has taken steps towards identifying a small molecule probe selective for BGAL11 or BGAL13, two enzymes essential for pollen tube elongation.