Ethylene and hydrogen peroxide regulate formation of a sterol-enriched domain essential for wall labyrinth assembly in transfer cells

Transfer cells (TCs) facilitate high rates of nutrient transport into, and within, the plant body. Their transport function is conferred by polarized wall ingrowth papillae, deposited upon a specialized uniform wall layer, that form a scaffold supporting an amplified area of plasma membrane enriched in nutrient transporters. We explored the question of whether lipid-enriched domains of the TC plasma membrane could serve as organizational platforms for proteins regulating the construction of the intricate TC wall labyrinth using developing Vicia faba cotyledons. When these cotyledons are placed in culture, their adaxial epidermal cells trans-differentiate to a TC phenotype regulated by auxin, ethylene, extracellular hydrogen peroxide (_apoH₂O₂), and cytosolic Ca²⁺ ([Ca²⁺]_cyt) arranged in series. Staining cultured cotyledons with the sterol-specific dye, Filipin III, detected a polarized sterol-enriched domain in the plasma membrane of their trans-differentiating epidermal transfer cells (ETCs). Ethylene activated sterol biosynthesis while extracellular _apoH₂O₂ directed sterol-enriched vesicles to fuse with the outer periclinal region of the ETC plasma membrane. The sterol-enriched domain was essential for generating the [Ca²⁺]_cyt signal and orchestrating construction of both the uniform wall layer and wall ingrowth papillae. A model is presented outlining how the sterol-enriched plasma membrane domain forms and functions to regulate wall labyrinth assembly.