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

Abstract

The intricate, and often polarized, ingrowth walls of transfer cells (TCs) amplify their plasma membrane surface areas to confer a transport function of supporting high rates of nutrient exchange across apo-/symplasmic interfaces. The TC ingrowth wall comprises a uniform wall layer on which wall ingrowths are deposited. Signals and signal cascades inducing trans-differentiation events leading to formation of TC ingrowth walls are poorly understood. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, when placed into culture, their adaxial epidermal cells rapidly (h) and synchronously form polarized ingrowth walls accessible for experimental observations. Using this model, we recently reported findings consistent with extracellular hydrogen peroxide, produced through a respiratory burst oxidase homolog/superoxide dismutase pathway, initiating cell wall biosynthetic activity and providing directional information guiding deposition of the polarized uniform wall. Our conclusions rested on observations derived from pharmacological manipulations of hydrogen peroxide production and correlative gene expression data sets. A series of additional studies were undertaken, the results of which verify that extracellular hydrogen peroxide contributes to regulating ingrowth wall formation and is generated by a respiratory burst oxidase homolog/superoxide dismutase pathway.

Figure 1. Cellular sources of (A - G), and biosynthetic pathway producing (H), H₂O₂ regulating ingrowth wall formation in adaxial epidermal cells of cultured cotyledons of Vicia faba. (A-G). Role of extra- (A - C) and intracellular (D - G) H₂O₂. Cotyledons were cultured for 15 h on MS medium without (A, D) or with (B) 1 mM DDC, (E) 500 µM BHA or (F) 1.3 mM flavone and thereafter processed as follows. (A - B; D - F) H₂O₂ was detected in outer periclinal walls and protoplasts of adaxial epidermal cells in 100 - 120 µm thick transverse sections of freshly-harvested cotyledons acid loaded (pH 3.5) with 10 mg/mL 3′,3′-diaminobenzidine (DAB), contained within the MS medium. Upon reacting with H₂O₂, DAB forms a brown insoluble polymer. Sections were cut on a vibrotome. (C, G) Estimates of extra- and intracellular H₂O₂ levels derived from pixel densities of the cell wall (region outlined in A with white dashed line) and protoplasm (region outlined in A with black dashed line) scanned with ImageJ software (http://rsbweb.nih.gov/ij/) and corrected for background. Cotyledons cultured on MS medium plus pharmacological reagents, but without DAB, were processed for (C) TEM to estimate thickness of the uniform wall or for (G) SEM to determine the percentages of adaxial epidermal cells containing WIs. Mean ± SE of four replicate cotyledons per treatment. ep, epidermal cell; sp, storage parenchyma cell. Bar for A, B = 30 µm, for D-f = 20 µm (H). Impact of 5 mM azide, a cell wall peroxidase inhibitor, on net efflux of H₂O₂. 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. After 15 min, the cotyledons were removed, re-rinsed in several changes of distilled water and the adaxial face of each cotyledon scanned for surface area determination by ImageJ software. Optical densities of the well solutions were determined at 560 nm spectrophometrically and amounts of H₂O₂ released into each solution determined from standard curves run concurrently. Net fluxes of H₂O₂ were estimated from the measurements described above. Mean ± SE of four replicate cotyledons per treatment (* p < 0.05 for treatment against control). For further procedural information, see ref. .

Figure 2. Model of signals and signaling pathways regulating uniform wall formation in adaxial epidermal cells of Vicia faba cotyledons. Auxin induces ethylene biosynthesis and lowering of intracellular glucose levels ([glu]_L) removes the glu block on EIN3 allowing ethylene signaling to proceed. This leads to upregulated expression of VfrbohA that catalyzes a burst in extracellular reactive oxygen species (ROS). This induces VfrbohC expression through an unidentified signal transduction pathway (black broken arrow) to mediate a second burst in extracellular ROS production. The latter extracellular ROS signal acts through two unidentified signal transduction pathways. One induces expression of cell wall biosynthetic machinery (black broken arrow) and the other (gray broken arrow) provides the positional cue to direct polarized trafficking of wall building machinery to catalyze assembly of the polarized uniform wall.