Cell Wall Microstructure Analysis Implicates Hemicellulose Polysaccharides in Cell Adhesion in Tomato Fruit Pericarp Parenchyma  Ordaz-Ortiz José J. , Marcus Susan E. , Paul Knox J.   Molecular Plant  Volume 2, Issue 5, Pages 910-921 (September 2009) DOI: 10.1093/mp/ssp049 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 Indirect Immunofluorescence Micrographs of Pectic HG Rat Monoclonal Antibody LM7 Binding to the Surface of Intact Cells Isolated from Green Mature 40-dpa (40) and Red Ripe 60-dpa (60) Tomato Pericarp Parenchyma. At the surface of both green mature and red ripe cells, the LM7 epitope is abundant in continuous lines reflecting the former edge of adhesion planes (arrows). Former adhesion planes are indicated by asterisks. Pectate lyase pre-treatment abolished LM7 binding (40,PL) and Calcofluor binding shows intact cell surface of the same cell (40,PL,Calc.) with arrowhead indicating edge of cell. The pattern of LM7 HG epitope occurrence was present at the surface of red ripe 60-dpa fruit (60) and equivalent fruit from Cnr (Cnr, 60). Scale bar  =  100 μm. Molecular Plant 2009 2, 910-921DOI: (10.1093/mp/ssp049) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 Indirect Immunofluorescence Micrographs of LM6 Arabinan and LM5 Galactan Monoclonal Antibodies Binding to the Surface of Intact Cells Isolated from Green Mature 40-dpa (40) and Red Ripe 60-dpa (60) Tomato Pericarp Parenchyma. At the surface of both green mature cells (40), the epitopes are evenly distributed. At the surface of cells from red ripe fruit (60), the epitopes were less abundant in regions reflecting former intercellular space (arrows). Former adhesion planes are indicated by asterisks. In equivalent cells from 60-dpa Cnr fruit (Cnr, 60), the LM6 epitope is weakly present in regions formerly lining intercellular space and the LM5 epitope is abundant in these regions (arrows). Scale bar  =  100 μm. Molecular Plant 2009 2, 910-921DOI: (10.1093/mp/ssp049) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 Indirect Immunofluorescence Micrographs of Antibodies Directed to Hemicellulosic Polysaccharides, LM11 Xylan, LM15 Xyloglucan, and LM21 Mannan, Binding to the Surface of Intact Cells Isolated from Green Mature 40-dpa (40) and Red Ripe 60-dpa (60) Tomato Pericarp Parenchyma. In a section of mature green pericarp, the LM11 epitope was detected in cell walls lining intercellular space (blue shows Calcofluor staining of all cell walls) and also some middle lamellae regions (arrowheads) but was absent from the surface of separated cells. The LM15 xyloglucan epitope was detected at the surface of green mature cell regions reflecting the edge of former adhesion planes (arrows) but with some gaps (double-headed arrows). An equivalent pattern was not detected at the surface of red ripe 60-dpa cells (60) nor in Cnr cells at either 40 or 60 dpa stage. The LM21 epitope was weakly present evenly across the cell surface of 40-dpa cells, punctuate at 60-dpa cells. In Cnr cells from both 40 and 60-dpa fruit, the mannan epitope was in an ordered pattern reflecting the cell walls formerly lining the intercellular space (arrows). Former adhesion planes are indicated by asterisks. Scale bar  =  100 μm. Molecular Plant 2009 2, 910-921DOI: (10.1093/mp/ssp049) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 Recovery of Intact Separated Cells after Incubation of Slices of Tomato Pericarp from Wild-Type Tomato Ailsa Craig at the Green Ripe Stage 40-dpa with Arabinanase (A), Galactanase (G), Xylanase (X), Xyloglucanase (XG), Mannanase (M), Endo-Polygalacturonase (PG), Pectate Lyase (PL), or Without Enzyme (c). Arrows indicate the top of sedimented cells in tubes. Numbers beneath tubes indicate volumes (mL) of sedimented cells obtained for each enzyme treatment. Result shown is representative of three separate experiments. Molecular Plant 2009 2, 910-921DOI: (10.1093/mp/ssp049) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 5 Indirect Immunofluorescence Micrographs of Monoclonal Antibodies LM15-Xyloglucan and LM7-Pectic-HG Binding to the Surface of Intact Cells Isolated from Green Mature 40-dpa (40) Pericarp Parenchyma by the Action of Pectate Lyase, Mannanase, and Galactanase. Scale bar  =  100 μm. Molecular Plant 2009 2, 910-921DOI: (10.1093/mp/ssp049) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 6 Polysaccharide Epitope Detection in Pooled Fractions of Cell Wall Polymers Isolated by the Sequential Extraction of an Alcohol-Insoluble Residue of Green Mature (40-dpa) Material with Water, Calcium Chelator CDTA, Na2CO3, and KOH. Epitope levels determined by ELISA. Microtitre plates were coated with 50 μg mL−1 polymers and antibody hybridoma supernatants used at five-fold dilution. Results shown are representative of three separate experiments and the error bars indicate ±standard deviation for absorbances obtained from one experiment. (A) Pectin-directed monoclonal antibodies. (B) Hemicellulose-directed monoclonal antibodies. (C) Assay of sensitivity of LM15 (xyloglucan-directed) binding to water extract (*) when subjected to pre-treatment with xyloglucanase. Molecular Plant 2009 2, 910-921DOI: (10.1093/mp/ssp049) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions