Characterization of the Putative A. thaliana Protein O-fucosyltransferase OFT1 which is Essential for Plant Reproduction

Abstract

AbstractIn flowering plants, pollen tubes grow from the pollen grain in a polarized mannerin order to deliver two haploid non-motile sperm cells to the female gamete. Thefollowing fertilization event results in the formation of a seed, which allows for thedispersal of the organism’s genetic material and thereby, propagation of the species.Signaling events throughout the female gynoecium guide the pollen tube, while a tipfocusedCa2+ gradient establishes the direction of cytoplasmic streaming, which deliversvesicles containing plasma membrane (PM) and cell wall material to the apical tip of thegrowing tube. At3g05320 (OFT1) was identified as a putative protein Ofucosyltransferasein the A. thaliana genome in a large scale transcriptome study whichdefined transcriptional changes that occur in pollen tubes growing through the stigma.The protein O-fucosyltransferase POFUT1 plays a critical role in the metazoan Notchsignaling pathway where it O-fucosylates serine and threonine resuidues in EGF repeatson the Notch extracellular domain. Furthermore, two other putative protein Ofucosyltransfreasesin the A. thaliana genome have been demonstrated to play a role incell adhesion. To date, however, no protein O-fucosyltransferase has been fullycharacterized in the A. thaliana genome. In silico analysis reveals that OFT1 sharesconserved catalytic residues with known metazoan protein O-fucosyltransferase.Phylogenetic analysis also revealed that OFT1 is more closely related to known metazoanptotein O-fucosyltransferases than it is to any of the other 38 putative A. thaliana proteinO-fucosyltransferases. The results of segregation distortion studies indicate that OFT1 isa protein involved in male sided fertility in A. thaliana. Further phenotypiccharacterization of three different OFT1 T-DNA insertional mutant alleles revealedvarious reproductive phenotypes, including reduced silique elongation and reduced seedset. In vitro characterization of oft1 mutant pollen revealed no overt defect in pollen tubeelongation or germination, however, in vivo staining of pollen tubes showed that fewsuccessfully fertilize an ovule. Semi-in vivo germination assays reveal that oft1 mutantpollen tubes have a difficult time penetrating the female stigmatic tissue. Additionally,visualization of GFP in pollen tubes driven under the OFT1 native promotor reveal thatOFT1 is expressed throughout the pollen tube. These results suggest that OFT1 is aputative protein O-fucoslytransferase involved in stigma penetration.