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

Abstract

In plant cells, linkage between the cytoskeleton, plasma membrane and cell wall is crucial to maintain 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 Pro-rich extracellular domain (ECD) and are candidate coordination factors. Here, 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 ECD of both is necessary for their function and identify distinct O-glycans attached to these sequences, AtFH5 being Hyp-arabinosylated and AtFH3 carrying arabinogalactan chains. Loss of Hyp-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 where AtFH5 and AtFH3, restricted to specific plasma membrane domains by their ECDs and the glycans attached to them, organize distinct subarrays of actin during pollen tube elongation.

The hydroxyproline O-arabinosyltransferase FIN4 is required for tomato pollen intine development

“…Leucine-rich repeat EXT-like proteins (LRXs), Proline-rich extensin-like receptor kinases (PERKs) and Class I Formin Homology proteins (FHs); Ding et al, 2020). Several LRX and FH genes have been implicated in pollen tube growth in Arabidopsis (Borassi et al, 2016;Sede et al, 2018;Lara-Mondragón et al, 2022;Mecchia et al, 2017;Wang et al, 2018). A role in intine formation has also been briefly described for some Arabidopsis LRX members.…”

“…Full-length FIN4 mRNA was amplified using forward and reverse primers designed based on the maximum extent of available mRNA sequence reads mapping to the FIN4 region (table 1; Kudo et al, 2017;Lara-Mondragón et al, 2022). The resulting 1291 bp product was cloned into PCR II TOPO vector using TOPO TA cloning kit (ThermoFisher Scientific, catalog number 461020) and sequenced with T7 and SP6 primers to confirm the sequence of FIN4 mRNA.…”

“…This EXT network is hypothesized to function as a scaffold for pectin assembly, forming extensin pectate (Cannon et al, 2008;Lamport et al, 2011). In addition to the heavily Hyp-Ara modified EXTs, other targets of this modification include EXT chimeras and secreted signaling peptides of the CLE (CLAVATA3/Embryo Surrounding Region-Related) family (Lara-Mondragón et al, 2022;Ohyama et al, 2009;Petersen et al, 2021). The HPAT family is strongly conserved across plant genomes and in the absence of HPAT activity, no detectable Hyp-Ara is formed (MacAlister et al, 2016).…”

Transmembrane formins as active cargoes of membrane trafficking

“…While both AtFH3 and AtFH5 localize to the plasma membrane and endomembranes of the pollen tube tip, participate in actin nucleation at the plasmalemma, and contribute to actin fringe organization, they exhibit distinct plasmalemma localization patterns; AtFH5 is restricted to the tip, while AtFH3 also decorates the tube shank ( Lan et al , 2018 ). This difference appears to be due to distinct glycosylation patterns of the extracytoplasmic part of these formins and restriction of their lateral mobility by binding to specific cell wall components, as documented by effects of mutations affecting the glycosylation pathway or target glycosylation sites on localization of these formins ( Lara-Mondragón et al , 2022 ). AtFH5 localizes to exocytotic vesicles before pollen tube emergence, participating directly in profilin-aided actin nucleation on them ( C. Liu et al , 2018 , 2021 ).…”