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

Abstract

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.

Extracellular hydrogen peroxide, produced through a respiratory burst oxidase/superoxide dismutase pathway, directs ingrowth wall formation in epidermal transfer cells of Vicia faba cotyledons

“…The H 2 O 2 signal activates cell wall biosynthesis and provides a positional cue directing polarized deposition of the uniform wall, the attenuation of which comprises WI formation. 10 Findings presented in this Addendum confirm that extra-rather than intracellular produced H 2 O 2 is the principal reactive oxygen species (ROS) driving TC transdifferentiation and that this ROS signal is generated by respiratory burst oxidase homologs (rbohs). Cotyledons were cultured on mS medium for 1, 3 or 15 h. thereafter, cotyledons were rinsed in several changes of distilled water before transferring each cotyledon, adaxial surface down, into a well of a 24 well plate containing amplex red solution with or without 5 mm sodium azide.…”

“…Extracellular H 2 O 2 production by adaxial epidermal cells of cultured V. faba cotyledons is characterized temporally by two sequential oxidative bursts, the first peaking at 1 h followed by a second slower rise commencing at 3 h from the onset of cotyledon culture. 10 Based on comparable inhibitions of H 2 O 2 production by DPI and DDC, H 2 O 2 production and expression of epidermal-specific Vfrbohs sharing similar temporal profiles and both being subject to ethylene regulation led us to conclude that the extracellular H 2 O 2 signal was generated by a rboh-SOD pathway. 10 However, DPI is known to inhibit H 2 O 2 synthesis by not only rbohs, but also by haem-containing cell wall peroxidases.…”

“…The signaling cascade is instigated Our conclusion that extracellular H 2 O 2 is a component of a signaling pathway regulating ingrowth wall formation relied on its inhibition by diphenyleneiodonium (DPI), a potent inhibitor of NADPH oxidases and other flavin-containing enzymes, 11 and rescue from DPI inhibition by H 2 O 2 supplementation. 10 To remove any ambiguity introduced by possible dual DPI action on extra-and intracellular H 2 O 2 synthesis, 12 we explored the impact of selectively depleting extracellular H 2 O 2 levels (see ref. 10) on uniform wall formation by culturing cotyledons on the extracellular Cu/ Zn superoxide dismutase (SOD) inhibitor, diethyldithiocarbamate (DDC 13 ), and ROS scavenger, ascorbic acid (AA 14 ).…”

“…These pharmacological treatments strongly suppressed extracellular H 2 O 2 production. 10 We repeated these experiments and monitored extra-and intracellular H 2 O 2 production using acid loaded 3',3'-diaminobenzidine (DAB) that forms a brown insoluble polymer on reacting with H 2 O 2, 15 Unfortunately AA interfered with DAB polymerization (data not shown). However, AA is unlikely to enter cotyledon epidermal cells as it will be ionized at pH 5.8 (pK a 4.2 and 11.6) in the MS medium.…”

Spatio-Temporal Dynamics of Fructan Metabolism in Developing Barley Grains

“…Although they are associated with a number of detrimental effects, ROS are also important in plant development, e.g., they have a regulatory function in establishing cell wall ingrowths in the V. faba cotyledon (Zhou et al, 2010;Andriunas et al, 2012). In suberin-and lignin-rich cell walls, the cross-linking of phenolics is mediated by H 2 O 2 (Fry, 1998;Schweikert et al, 2000), a process that occurs in chalazal cells during the final stage of barley grain development (Cochrane, 2000;Asthir et al, 2002).…”