Abstract text: The AtFLA4 locus in Arabidopsis thaliana encodes a fasciclin-like arabinogalactan protein involved in root expansion, root hydropatterning, and seed coat mucilage adhesion. Here we show that the stem cell niche and auxin polar transport are additional cellular processes influenced by AtFLA4. Orthologues of AtFLA4 occur across most land plants, including liverworts and lycophytes, although their mode of action remains unclear. To address this, we investigated the cellular trafficking and functional mechanism of AtFLA4 and examined the role of FLA4-related genes across the plant kingdom.

AtFLA4 contains two proline-rich domains proposed to anchor type II arabinogalactans. Proteomic analysis of recombinant AtFLA4 identified potential glycosylation sites within clustered proline residues in the C-terminal domain. Site-directed proline-to-alanine substitutions in these clusters caused proteolytic shedding of FLA4 from the plasma membrane. Plasma membrane stability was restored by introducing ectopic O- and N-linked glycosylation sites, as well as by intrinsic disorder-promoting amino acids that cannot be glycosylated. These results suggest that intrinsic disorder together with O-linked glycosylation is essential for proper localization and stability of FLA proteins.

Genetic evidence indicates that AtFLA4 acts in a linear pathway with the receptor-like kinases FEI1 and FEI2, which regulate ethylene biosynthesis, root hydrotropism, and cell wall deposition. Proteomics and interaction assays suggest complex interactions between FLA4, other FLAs, and extracellular signaling components at the plasma membrane.