O-glycosylation of the extracellular domain of pollen class I formins modulates their plasma membrane mobility.

In plant cells, linkage between the cytoskeleton, plasma membrane, and cell wall is crucial for maintaining cell shape. In highly polarized pollen tubes, this coordination is especially important to allow rapid tip growth and successful fertilization. Class I formins contain cytoplasmic actin-nucleating formin homology domains as well as a proline-rich extracellular domain and are candidate coordination factors. Here, using Arabidopsis, we investigated the functional significance of the extracellular domain of two pollen-expressed class I formins: AtFH3, which does not have a polar localization, and AtFH5, which is limited to the growing tip region. We show that the extracellular domain of both is necessary for their function, and identify distinct O-glycans attached to these sequences, AtFH5 being hydroxyproline-arabinosylated and AtFH3 carrying arabinogalactan chains. Loss of hydroxyproline arabinosylation altered the plasma membrane localization of AtFH5 and disrupted actin cytoskeleton organization. Moreover, we show that O-glycans differentially affect lateral mobility in the plasma membrane. Together, our results support a model of protein sub-functionalization in which AtFH5 and AtFH3, restricted to specific plasma membrane domains by their extracellular domains and the glycans attached to them, organize distinct subarrays of actin during pollen tube elongation.

Partial purification and immunodetection of cell surface glycoproteins from plants.

Cell surface glycoproteins in plants were first described more than 50 years ago, and yet, the precise mechanisms by which they operate remain elusive to this day. Studying glycoproteins is often challenging due to their subcellular localization (many secreted or membrane associated) and the extent of glycosylation present on the protein backbone, which can have profound effects on protein structure and behavior. In plants, additional layers of complexity exist as cell surface glycoproteins are in close contact, and in some cases, establish direct linkages with the polysaccharide networks present in the cell wall. In this chapter, we guide the reader through a protocol aimed to address the glycosylation status of a presumed cell surface glycoprotein. First, we discuss the advantages and disadvantages of using plants as homologous expression systems for recombinant glycoprotein production. Next, we describe a protocol for microsomal enrichment, followed by partial purification by affinity chromatography and finally glycodetection by immunoblotting using monoclonal antibodies targeting cell wall glycans. We particularly focus on the hydroxyproline-rich glycoprotein (HRGP) family, the most abundant family of glycoproteins in the plant cell wall. We provide examples of two putative HRGP chimeric proteins, one akin to extensins and the second an arabinogalactan protein (AGP)-like protein. For the latter, we provide an AGP-specific protocol to ensure enrichment of members of this group, which can be used independently or in conjunction with the described protocol. Throughout the chapter, we provide recommendations for the handling of plant glycoproteins and highlight special considerations for experimental design, along with troubleshooting suggestions.