Enzymes contributing to the hydrogen peroxide signal dynamics that regulate wall labyrinth formation in transfer cells

Transfer cells are characterized by an amplified plasma membrane area supported on a wall labyrinth composed of a uniform wall layer (UWL) from which wall ingrowth (WI) papillae arise. Adaxial epidermal cells of developing Vicia faba cotyledons, when placed in culture, undergo a rapid (hours) trans-differentiation to a functional epidermal transfer cell (ETC) phenotype. The trans-differentiation event is controlled by a signalling cascade comprising auxin, ethylene, apoplasmic reactive oxygen species ((apo)ROS), and cytosolic Ca(2+). Apoplasmic hydrogen peroxide ((apo)H(2)O(2)) was confirmed as the (apo)ROS regulating UWL and WI papillae formation. Informed by an ETC-specific transcriptome, a pharmacological approach identified a temporally changing cohort of H(2)O(2) biosynthetic enzymes. The cohort contained a respiratory burst oxidase homologue, polyamine oxidase, copper amine oxidase, and a suite of class III peroxidases. Collectively these generated two consecutive bursts in (apo)H(2)O(2) production. Spatial organization of biosynthetic/catabolic enzymes was deduced from responses to pharmacologically blocking their activities on the cellular and subcellular distribution of (apo)H(2)O(2). The findings were consistent with catalase activity constraining the (apo)H(2)O(2) signal to the outer periclinal wall of the ETCs. Strategic positioning of class III peroxidases in this outer domain shaped subcellular (apo)H(2)O(2) signatures that differed during assembly of the UWL and WI papillae.