Characterization of Metal Binding Properties of Rhamnogalacturonan II from Plant Cell Walls by Size-Exclusion HPLC/ICP-MS

Abstract

IntroductionRhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide isolated from vascular plant cell walls, and is composed of a backbone of galacturonic acid (GalA) residues and four side chains of diverse glycosyl residues (Fig. 1). 1,2 RG-II is one of the most complex polysaccharides identified so far; however, its function remained unclear for many years. Recently, a dimeric RG-II-borate complex (dRG-II-B) in which a borate-diol ester (1:2) cross-links two monomeric RG-II (mRG-II) was isolated from pectinase digests of plant cell walls. [3][4][5] It has been shown that RG-II is covalently linked to homogalacturonan (HG), a major component of pectin in plant cell walls, 6 and that dRG-II-B formation contributes to stabilization of the cell walls.7 These studies have demonstrated that the primary function of boron, an essential microelement for plants, is to covalently cross-link wall pectin by dRG-II-B formation, and thereby to contribute to the strength and integrity of the cell walls. 7,8 The dRG-II-B complex contains specific metals, such as Ca, Sr, Ba, and Pb. 5,9-11 The divalent cations (Sr 2+ , Pb 2+ , and Ba 2+ ) with an ionic radius >1.10 Å and trivalent cations (Eu 3+ , Pr 3+ , La 3+ , and Ce 3+ ) with an ionic radius >0.90 Å significantly increased in vitro dRG-II-B formation from mRG-II and boric acid.12 The dRG-II-B with Ca 2+ was likely to stabilize the pectic polysaccharides in the cell walls. 13 Moreover, most of Pb in wine existed as a complex with dRG-II-B;10 Pb bound to the dimer was less available than Pb acetate in rats. 14 However, the function of metal ions bound to dRG-II-B has not been established. In the present study, we characterized the metal binding properties of RG-II using size-exclusion highperformance liquid chromatography/inductively coupled plasma mass spectrometry (SE-HPLC/ICP-MS). 15,16 The metal contents of RG-II and its partial hydrolysates that were treated with metal ions were determined by the SE-HPLC/ICP-MS to understand the RG-II's affinity and binding site for metals. Materials and Methods Preparation of dRG-II-B and mRG-IIdRG-II-B was isolated from sugar beet pulp and red wine, as described previously.4,17 mRG-II was prepared by a 0.1 M HCl treatment of dRG-II-B for 30 min at room temperature. The solution was dialyzed against deionized water and then freezedried. The mineral contents of dRG-II-B were determined by SE-HPLC/ICP-MS using a Diol-120 (8 × 300 mm, YMC Co., Kyoto, Japan) column. Treatment of dRG-II-B and mRG-II with metal ionsSeparate solutions of sugar beet dRG-II-B (0.025 mM) and mRG-II (0.05 mM) in 40 mM ammonium acetate, pH 5.0, containing either CaCl2, SrCl2, BaCl2, Pb(NO3)2, or LaCl3 (0.25 mM) were kept for several hours or more. In a second experiment, solutions of mRG-II (0.025 mM) in 40 mM ammonium acetate, pH 5.0, containing either LaCl3, EuCl3, or LuCl3 (0.25 mM) were prepared. The pH condition (pH 5.0) was selected based on the fact that dRG-II-B is stable above pH 4, 5 and that the apoplastic pH in plants is 5 -6. 18...

Effect of boric acid on the ability of plants to accumulate heavy metals

“…One of probable mechanisms accounting for the activating effect of boric acid on HM removal from the soil by plants is that boron is involved in complexation with carbohydrate derivatives, pectin and rhamnoga lacturonan II, in the cell wall (Kobayashi, Matoh, and Junichi, 1996;O'Neill et al, 1996), with the resulting complexes also including metal ions such as Ca 2+ , Sr 2+ , Ba 2+ , Pb 2+ , and La 3+ (Matsunaga and Ishii, 2004). These complexes reinforce transversely inter laced structures of the cell wall, thereby stabilizing them.…”

“…The role of boron in plants is being actively studied. It has been shown that it is involved in structural organization of the cell wall (Kobayashi, Matoh, and Junichi, 1996;O'Neill et al, 1996;Matsunaga and Ishii, 2004) and has an effect on ion transport through membranes (Blaser Grill et al 1989;Ferrol and Donaire, 1992;Cakmak, Kurz, and Marschner, 1995;Wang et al, 1999).…”

Borate Cross-Linked/Total Rhamnogalacturonan II Ratio in Cell Walls for the Biochemical Diagnosis of Boron Deficiency in Hydroponically Grown Pumpkin

“…The flow-injection conditions were as follows: carrier, deionized water; flow rate, 1.0 mL/min; and injection volume, 20 μL. 15,19 The residue extracted with deionized water, as described above, for the determination of water-soluble B was suspended in 800 μL of aqueous 80% (v/v) ethanol, mixed by a homogenizer POLYTRON PT1300D (KINEMATICA, Lucerne, Switzerland), shaken for 20 min and centrifuged. The residue was then washed with 80% (v/v) ethanol, 99.5% (v/v) ethanol, chloroform:methanol (1:1, v/v), acetone and deionized water, freeze-dried, and used as a cell-wall sample.…”

“…8,11 Waterinsoluble B has been revealed to exist in cell walls as a dimeric rhamnogalacturonan II-borate (dRG-II-B) complex, in which a borate-apiose (1:2) ester cross-links two monomeric RG-IIs. [12][13][14][15][16] The pectic polysaccharide RG-II is covalently bound to homogalacturonan, which is a major component of pectin. 17 The borate cross-linked RG-II or dRG-II-B/total RG-II ratio in cell walls of B-sufficient and deficient plants is reported to be 0.8 -0.9 and 0 -0.2, respectively.…”