Reactive oxygen species form part of a regulatory pathway initiating trans-differentiation of epidermal transfer cells in Vicia faba cotyledons

Various cell types can trans-differentiate to a transfer cell (TC) morphology characterized by deposition of polarized ingrowth walls comprised of a uniform layer on which wall ingrowths (WIs) develop. WIs form scaffolds supporting amplified plasma membrane areas enriched in transporters conferring a cellular capacity for high rates of nutrient exchange across apo- and symplasmic interfaces. The hypothesis that reactive oxygen species (ROS) are a component of the regulatory pathway inducing ingrowth wall formation was tested using Vicia faba cotyledons. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, on being placed into culture, their adaxial epidermal cells rapidly (hours) form ingrowth walls on their outer periclinal walls. These are readily visualized by electron microscopy, and epidermal peels of their trans-differentiating cells allow measures of cell-specific gene expression. Ingrowth wall formation responded inversely to pharmacological manipulation of ROS levels, indicating that a flavin-containing enzyme (NADPH oxidase) and superoxide dismutase cooperatively generate a regulatory H2O2 signature. Extracellular H2O2 fluxes peaked prior to the appearance of WIs and were followed by a slower rise in H2O2 flux that occurred concomitantly, and co-localized, with ingrowth wall formation. De-localizing the H2O2 signature caused a corresponding de-localization of cell wall deposition. Temporal and epidermal cell-specific expression profiles of VfrbohA and VfrbohC coincided with those of extracellular H2O2 production and were regulated by cross-talk with ethylene. It is concluded that H2O2 functions, downstream of ethylene, to activate cell wall biosynthesis and direct polarized deposition of a uniform wall on which WIs form