Plasma membrane H+-ATPase is involved in auxin-mediated cell elongation during wheat embryo development

Abstract

Previous investigations suggested that specific auxin spatial distribution due to auxin movements to particular embryonic regions was important for normal embryonic pattern formation. To gain information on the molecular mechanism(s) by which auxin acts to direct pattern formation in specific embryonic regions, the role of a plasma membrane (PM) ATPase was evaluated as downstream target of auxin in the present study. Western-blot analysis revealed that the PM H(+)-ATPase expression level was significantly increased by auxin in wheat (Triticum aestivum) embryos (two-three times increase). In bilaterally symmetrical embryos, the spatial expression pattern of the PM H(+)-ATPase correlates with the distribution pattern of the auxin analog, tritiated 5-azidoindole-3-acetic acid. A strong immunosignal was observed in the abaxial epidermis of the scutellum and in the epidermal cells at the distal tip of this organ. Pseudoratiometric analysis using a fluorescent pH indicator showed that the pH in the apoplast of the cells expressing the PM H(+)-ATPase was in average more acidic than the apoplastic pH of nonexpressing cells. Cellulose staining of living embryos revealed that cells of the scutellum abaxial epidermis expressing the ATPase were longer than the scutellum adaxial epidermal cells, where the protein was not expressed. Our data indicate that auxin activates the proton pump resulting in apoplastic acidification, a process contributing to cell wall loosening and elongation of the scutellum. Therefore, we suggest that the PM H(+)-ATPase is a component of the auxin-signaling cascade that may direct pattern formation in embryos.

Figure 1

Effect of IAA on the level of PM H⁺-ATPase antigen in wheat embryos. A, PM-enriched microsomal preparations of Arabidopsis (A), maize (M), and wheat (W) probed with the monoclonal antibody 46 E5B11. B, Left panel, Gel blot of protein extracts from IAA-treated and untreated embryos. Equal amounts of total protein extracts were separated by electrophoresis on SDS-PAGE gels and stained by Ponceau Red. Right panel, Western-blot analysis of total protein extracts from embryos incubated for 2 h either on Murashige and Skoog medium supplemented with 30 μm IAA or on auxin-free Murashige and Skoog medium. Detection of the PM H⁺-ATPase was performed using the specific monoclonal antibody 46E5B11.

Figure 2

Immunolocalization of PM H⁺-ATPase in wheat embryos at different developmental stages. A, Globular embryo; B, late transition stage; C, early coleoptilar stage; D, late coleoptilar stage/early “stage 1”; E, fully differentiated immature embryo. F, Immunogold staining of the H⁺-ATPase in epidermal cells of the scutellum (walls in between epidermal and subepidermal cell layers). ab, Abaxial; ad, adaxial; cr, coleoptilar ring; sc, scutellum; sm, shoot meristem; sp, suspensor; w, cell wall; bars in A through E = 100 μm; magnification in F, 25,000×.

Figure 3

Immunolocalization of PM H⁺-ATPase in wheat embryos treated with auxin and different auxin transport inhibitors in vitro. A, Normal in vitro grown embryo; B, embryo isolated at the globular stage and grown on media supplemented with N-1-naphthylphthalamic acid; C, abnormal embryo induced by treatment on media supplemented with 2,3,5-triiodobenzoic acid (TIBA); D and E, radial growth phenotypes observed on media supplemented with 2,4,5-T; F, arrested growth phenotype observed on media supplemented with 2,4,5-T. ced, Area of cell elongation and division; cr, coleoptilar ring; rep, rounded embryo proper; sc, scutellum; sm, shoot meristem; sp, suspensor; bars = 100 μm.

Figure 4

Ratiometric measurements of cell wall pH. A, Calibration of the OG to TR ratio to wall pH. For each pH, 10 to 13 measurements on two to three embryos have been performed. Embryonic stages used for calibration are the same as those used for measurements. B, Areas in which the ratiometric measurements were conducted on the scutellum adaxial epidermis. In area 1, ratios were measured in walls of cells expressing PM H⁺-ATPase at the tip of the scutellum. In area 2, ratios were measured in walls of cells not expressing the PM-H⁺ATPase near the coleoptilar ring. C, Wall pH has been pseudocolor coded according to the inset scale. D, Representative ratio image of adaxial epidermal cell walls at the tip of the scutellum. E, Representative ratio image of adaxial scutellar epidermal cell walls near the coleoptilar ring. Ratio images in D and E have been processed to subtract the background and to eliminate the fluorescence of the underlying cell walls. Only the cell walls used for evaluation are visible on the photographs. cr, Coleoptilar ring; sc, scutellum; sm, shoot meristem. Bars in D and E = to 10 μm.

Figure 5

Plant material used to perform cell length measurement. Embryonic stages and localization of the epidermal cells used for measurements. A, Middle stage embryo whose first leaf primordium approximately covers one-half of the shoot meristem. B, Older embryo whose first leaf primordium covers the shoot meristem. ab, Abaxial scutellum epidermis; ad, adaxial scutellum epidermis; cr, coleoptilar ring; lp, first leaf primordium; sc, scutellum; sm, shoot meristem. Arrows indicate cell length measurement areas.