Root border-like cells of Arabidopsis. Microscopical characterization and role in the interaction with rhizobacteria

Abstract

Plant roots of many species produce thousands of cells that are released daily into the rhizosphere. These cells are commonly termed border cells because of their major role in constituting a biotic boundary layer between the root surface and the soil. In this study, we investigated the occurrence and ultrastructure of such cells in Arabidopsis (Arabidopsis thaliana) using light and electron microscopy coupled to high-pressure freezing. The secretion of cell wall molecules including pectic polysaccharides and arabinogalactan-proteins (AGPs) was examined also using immunofluorescence microscopy and a set of anticarbohydrate antibodies. We show that root tips of Arabidopsis seedlings released cell layers in an organized pattern that differs from the rather randomly dispersed release observed in other plant species studied to date. Therefore, we termed such cells border-like cells (BLC). Electron microscopical results revealed that BLC are rich in mitochondria, Golgi stacks, and Golgi-derived vesicles, suggesting that these cells are actively engaged in secretion of materials to their cell walls. Immunocytochemical data demonstrated that pectins as well as AGPs are among secreted material as revealed by the high level of expression of AGP-epitopes. In particular, the JIM13-AGP epitope was found exclusively associated with BLC and peripheral cells in the root cap region. In addition, we investigated the function of BLC and root cap cell AGPs in the interaction with rhizobacteria using AGP-disrupting agents and a strain of Rhizobium sp. expressing a green fluorescent protein. Our findings demonstrate that alteration of AGPs significantly inhibits the attachment of the bacteria to the surface of BLC and root tip.

Figure 1.

Microscopical characterization of root BLC from Arabidopsis. A, A root tip of 2-week-old seedlings showing BLC release. Arrowheads indicate the cell layers where border cell files came from. B, Micrograph illustrating the general morphology of a BLC from a root prepared by HPF and FS. Note the abundance of Golgi-derived vesicles filled with opaque electron material. C, High magnification view of cytoplasmic content of a BLC prepared HPF/FS. CW, Cell wall; ER, endoplasmic reticulum; G, Golgi stack; M, mitochondria; mvb, multi-vesicular bodies; TGN, trans golgi network; V, vacuole; Vs, secretory vesicles. Bars = 100 μm (A); 1 μm (B); and 300 nm (C).

Figure 2.

Micrographs of Arabidopsis BLC stained with calcein-AM. The fluorescence of the probe is indicative of the cell viability. BLC from 8-d-old seedlings stained with calcein-AM. A, At the time of root cap jettisoning. B, After 4 h in water. C, After 6 h in water. D, BLC from 13-d-old seedlings stained with calcein-AM 24 h after their release from the root apex. E, Intrinsic fluorescence of BLC from 13-d-old seedlings. Bars = 50 μm.

Figure 3.

Immunofluorescence-staining of homogalacturonans. A, Immersion immunofluorescence showing labeling at the surface of the root apex with the mAb JIM5. B, Sloughing off BLC stained with JIM5. Bars = 20 μm.

Figure 4.

Immunofluorescence staining of AGPs on longitudinal section of Arabidopsis root tip. A, Staining with the mAb Mac207 antibody. B, Staining with the mAb JIM14. C, Staining with the mAb JIM13. Labeling was exclusively associated with peripheral and root BLCs. BLC, Arrowheads; M, meristem; P, peripheral cells. Bars = 20 μm.

Figure 5.

Bar graphs showing the effects of Yariv reagents and DHP on root length from 7-d-old (gray bars) and 15-d-old (dark bars) Arabidopsis seedlings. A, Treatments with 10 μm β-GlcY and 10 μm α-ManY. B, Treatments with 5 μm and 10 μm DHP. n = 30 to 100 and confidence intervals at 95% (lsd).

Figure 6.

Micrographs of Arabidopsis root tips treated with AGP-disrupting agents. A, Control. B, 10 μm β-GlcY. C, 5 μm DHP. Bars = 100 μm (A) and 50 μm (B and C).

Figure 7.

Double localization showing the association between BLC and bacteria YAS34-GFP. A, BLC, labeled with JIM 5 antibodies, present a red fluorescence. The GFP-expressing rhizobacteria appear green. B, Higher magnification showing the association between BLC and rhizobacteria. Bars = 50 μm (A) and 20 μm (B).

Figure 8.

Effect of Yariv reagents and DHP on the association between root tip cells and Rhizobium sp. YAS34GFP. A, Effect of β-GlcY 10 μm and α-ManY 10 μm; 7-d-old plants. B, Effect of DHP 5 μm; 15-d-old plants. n = 3 to 15. Average and confidence intervals are at 95.0% (lsd).

Figure 9.

Effects of AGP-disrupting agents on the association between Arabidopsis root tips and Rhizobium sp. YAS34GFP. Images represent the projection of Z-sections over 15-μm depth (1-μm step). Root tips are stained in red with Nile red and the expressing-GFP bacteria appear green. A, Control. B, 10 μm Yariv. C, 5 μm DHP. Bars = 50 μm (A and C) and 30 μm (B).