Requirement of Borate Cross-Linking of Cell Wall Rhamnogalacturonan II for Arabidopsis Growth M. A. O'Neill, S. Eberhard, P. Albersheim, A. G. Darvill

Supplementary Material

Supplemental Figure 1. A. The size-exclusion chromatography elution profiles of the material released by EPG treatment of WT, mur1-1, and mur1-2 cell walls. Solutions of the EPG-soluble material (~5 mg) in 50 mM ammonium formate, pH 5 (200 L), were filtered (0.2 m Nylon 66 microcentrifuge filter) and then fractionated on a Superdex-75 HR10/30 column by elution at 0.6 ml/min with 50 mM ammonium formate, pH 5. Material eluting from the column was detected using a Hewlett Packard 1037A refractive index detector. The elution positions of rhamnogalacturonan I (RG-I), dimeric and monomeric RG-II, and oligogalacturonides (OGAs) are indicated as are the column excluded (V₀) and included (V_i) volumes. B. The elution profiles of monomeric RG-II generated from WT and mur1-2 RG-II. WT and mur1-2 RG-II was treated for 30 min at 20°C with 0.1M HCl. The solutions were then dialyzed and freeze dried. Portions (500 g) of each RG-II were then analyzed using a Superdex Peptide HR10/30 column eluted at 0.6 ml/min with 50 mM ammonium formate, pH 5. The elution position of dimeric RG-II and the column void (V₀) and included (V_i) volumes are shown.

Medium version | Full size version

---

Supplemental Figure 2. Wild-type, and mur2 plants are visibly indistinguishable. Wild-type and mur2 plants have comparable morphologies, in contrast to mur1-2 plants which are dwarfed, when grown under the same growth conditions. The photographs were taken five weeks after planting. Scale bar = 2.5 cm. The digital images were manipulated with Photoshop (Adobe, Mountain View, CA) to prepare the figure.

Medium version | Full size version

---

Supplementary methods. RG-IIs released by EPG-treatment of WT and mur1-2 cell walls was purified by SEC on a Sephadex G-75 SEC column and by ion-exchange chromatography on a Q-sepharose fast-flow anion-exchange column that was eluted with 1 M imidazole-HCl, pH 7. Mur1-2 and WT RG-IIs were converted to their respective monomers by treatment for 30 min at 20°C with 0.1 M HCl. Separate solutions of each monomer (500 g) in 50 mM phthalate-HCl, pH 3.6 (250 L), were reacted for between 0.1 h and 6 h at 20°C with 1 mM boric acid and 1 mM SrCl₂. In a second series of experiments the SrCl₂ was omitted. The amount of RG-II dimer that formed was determined by SEC (6). In a third series of experiments each monomer (500 g) was reacted for 1 h at 20°C in 50 mM phthalate-HCl, pH 3.6 (250 L), containing boric acid (0 - 5 mM) and either 1 mM SrCl₂ or 10 mM CaCl₂. These cations were used because Sr⁺² promotes rapid dimer formation in vitro (8) whereas Ca⁺² promotes and stabilizes dimer formation in muro (4). The amount of RG-II dimer formed was determined by SEC.

The RG-IIs purified from mur1-2 and WT plants were reacted separately for 24 h at 20°C in 50 mM phthalate-HCl, pH 3.6, containing 5 mM boric acid and 5mM SrCl₂. This treatment ensured that at least 80% of the RG-II existed as the dimer (6). Samples of the mur1-2 and WT RG-II dimers (500 g) in 50 mM Na acetate (pH 4 and 5), 50 mM phthalate-HCl (pH 3.5, 3.0, and 2.5), and KCl-HCl (pH 2.0, 1.5, and 1.0) (250 L), were allowed to react for 1 h. The amount of monomeric RG-II that formed under each reaction condition was determined by SEC. In a second series of experiments the material released by EPG from the walls of boric acid or L-Fuc-treated WT and mur1-2 plants and from the leaf walls of mur1-3 and mur2-1 plants were treated for 24 h at 20°C in 50 mM phthalate-HCl, pH 3.6 containing 5 mM boric acid and 5mM SrCl₂. Separate aliquots of each sample (2.5 mg) in 50 mM phthalate-HCl, pH 2.5 (250 L), were kept at 20°C for 1 h and the amount of RG-II monomer that had formed was determined by SEC.