Phenotypic variation in <i>frb1</i> mutant seedlings.

<p>A. Wild-type <i>FRB1</i> seedling after 5 days growth on MS media. B. A 3-day old <i>frb1–1</i> seedling where the hypocotyl has spontaneously broken during germination. C. An example of a <i>frb1–1</i> seedling where the cotyledons have become fused as the seedling germinated. D. A <i>frb1–2</i> seedling showing severe cell dissociation. E. A two week old <i>FRB1</i> seedling grown on MS media. F. and G. At the same stage of growth as seedlings in E, <i>frb1–1</i> and <i>frb1–2</i> seedlings have little cell separation but many do have extensive fusions between leaves and other aerial organs. All scale bars equal 0.5. mm.</p

Xyloglucan oligosaccharide mass profile (OLIMP) of crude cell wall material from shoots (A, B, C) and its respective 4 M KOH fractions (D, E, F).

<p>A. and D. Representative spectra derived from <i>FRB1</i>. B. and E. Representative spectra derived from <i>frb1</i>. C. OLIMP analysis of shoots. D. OLIMP analysis of 4 M KOH fraction derived from shoots. Nomenclature of xyloglucan oligosaccharides are taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042914#pone.0042914-Neumetzler1" target="_blank">[38]</a> and references within. Stars indicate significant alterations with p<0.05.</p

Comparison of pectin methylesterase (PME) activity in <i>FRB1</i> and <i>frb1</i> alleles and analysis of esterified and non-esterified cell wall regions using FTIR.

<p>A. PME activity in <i>frb1</i> alleles is from 45–70% higher than in wild-type seedlings. Degree of methylesterification of the pectin used as substrate in PME assays is indicated as percentage. Each column is the average of three separate assays; error bars indicate ± standard deviation. All levels are significantly different with p<0.05 for all three <i>frb1</i> lines compared to wild-type B. Comparison of <i>FRB1</i> and <i>frb1</i> of FTIR scans. C. Close-up of region III from A. showing particular differences in the degree of methylesterification between <i>FRB1</i> and <i>frb1</i>.</p

Sugar composition and sugar incorporation in membrane preparations from <i>FRB1</i> and <i>frb1</i> plants.

<p>A. Sugar composition derived from microsomal preparations of wild-type and <i>frb1</i>–<i>2</i>. Monosaccharides and uronic acids were identified based on retention time and comparison to pure standards and values are expressed in % of total area. Significant changes (two-tailed Student's t-test, n = 3, *: p<0.05; **: p<0.01) are marked with stars. Rha: rhamnose, Ara: arabinose, Gal: galacatose, Glc: glucose, Xyl-Man: unseparated peak containing xylose and mannose, GalA: galacturonic acid, GlcA: glucuronic acid. B. The total radioactivity in the chloroform insoluble material. C. The total radioactivity in the chloroform soluble material. See the experimental procedures for details.</p

GFP-FRB1 fusion protein accumulates in subcellular compartments.

<p>A. Hypocotyl cells of transgenic plants expressing GFP-FRB1 fusion protein under the control of a constitutive promoter. Note that the GFP accumulates in subcellular compartments (green). Red fluorescence is from chloroplasts. B. GFP-FRB1 fluorescent particles in the cortical cytoplasm. C. Higher magnification image showing the ring morphology of the GFP-tagged compartments. D. Hypocotyl cells of GFP-FRB1 plants treated with 100 µg/ml Brefeldin A for 15 min. Note the redistribution of the GFP-labeled compartments to aggregates, especially around nuclei. Some of the GFP signal has also become soluble. E. Transient over-expression of GFP-FRB1 fusion protein or F. mCherry (CD3–967) fusion protein in an epidermal tobacco cell. G. Micrograph showing overlap of E and F. Scale bars equal 10 µm in A–D and 20 µm in E–G</p

Annotation of cell wall genes upregulated in <i>frb1</i> and immunolabeling of transverse hypocotyl sections using antibodies against arabinosylated cell wall epitopes.

<p>A. Differentially expressed genes involved in cell wall metabolism. Increase of expression of in <i>frb1</i> upregulated cell wall genes with n = 3. Error bars are ± standard deviation. B. to K. Immunolabeling of transverse hypocotyl sections (10 µm) using antibodies against arabinosyl epitopes predominantly present in extensins (LM1; D to G) or in RGI side chains (LM6; H to K). B. <i>FRB1</i> anti-rat control. C. <i>frb1–1</i> anti-rat control. D. and E. <i>FRB1</i> staining using LM1. F. and G. <i>frb1–1</i> staining using LM1. H. and I. <i>FRB1</i> staining using LM6. J. and K. <i>frb1–1</i> staining using LM6. Scale bars in B, C, D, F, H, J equal 50 µm, scale bars in E, G, I, K equal 25 µm.</p

Comparison of embryo morphology of <i>FRB1</i> and <i>frb1–1.</i>

<p>A. Optical section through a <i>FRB1</i> seed. B. Optical section through a <i>frb1–1</i> seed. C. <i>FRB1</i> embryo that has been dissected out of the seed coat. D. <i>frb1–1</i> embryo that has been dissected out of the seed coat. E. Optical section through hypocotyl of a <i>FRB1</i> embryo where tissue layers are visible. F. Optical section through hypocotyl of a <i>frb1–1</i> embryo showing partly disrupted cell files. Scale bars are 20 µm.</p