Fig. 4

Differential interference contrast (DIC) (a, c), immunofluorescence (b, d) and transmission electron microscopy (TEM) (e–j) images of a Penium cell wall before and after treatment with 20 mM EDTA for 20 min. a General appearance of a cell, where the outer layer (OL) of the cell wall has a rough, punctate appearance (arrows). b JIM5 mAb immunolabeling of a regular cell shows the homogalacturonan (HG)-rich OL that forms a lattice with a punctate shape. c After treatment with EDTA, the surface of the cell appears smooth, devoid of the OL. d JIM5 mAb immunolabeling of a treated cell shows the absence of labeling of the cell wall. The red color is the autofluorescence signal emitted by the chloroplast. e Ultrastructure of the cell wall of an untreated cell showing the external adhesive layer (AL), the HG-rich OL that forms the lattice, and the rhamnogalacturonan I (RGI)-rich middle layer (ML) that is embedded in a cellulose-rich inner layer (IL) (arrows). f JIM5 mAb immunogold labeling of the cell wall of an untreated cell shows the gold particles distribute to both OL and ML (arrows). g INRA-RU1 mAb immunogold labeling of the cell wall of an untreated cell shows the gold particles located in the ML (arrow). h TEM of a treated cell shows the OL completely removed (white arrow), while AL, ML and IL remain intact (black arrows). i JIM5 mAb immunogold labeling of a treated cell shows the lack of the OL (white arrow), and consequently no labeling, and the presence of gold particles in ML (black arrows). j INRA-RU1 mAb immunogold labeling of a treated cell shows the lack of the OL (white arrow) and the labeling of the ML (black arrows). IS, intracellular space. Scale bars: a–d = 10 µm; e, g, h, j = 500 nm; f = 350 nm; i = 450 nm