Rhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide present in the walls of growing plant cells. We now report that RG-II, released by endopolygalacturonase treatment of the walls of suspension-cultured sycamore cells and etiolated pea stems, exists mainly as a dimer that is cross-linked by a borate ester. The borate ester is completely hydrolyzed at room temperature within 30 min at pH 1, partially hydrolyzed between pH 2 and 4, and stable above pH 4. The dimer is formed in vitro between pH 2.4 and 6. 2 by treating monomeric RG-II (0.5 mM) with boric acid (1.2 mM); the dimer formed after 24 h at pH 3.4 and 5.0 accounts for approximately 30 and approximately 5%, respectively, of the RG-II. In contrast, the dimer accounts for approximately 80 and approximately 54% of the RG-II when the monomer is treated for 24 h at pH 3.4 and 5.0, respectively, with boric acid and 0.5 m Sr2+, Pb2+, or Ba2+. The amount of dimer formed at pH 3.4 or 5.0 is not increased by addition of 0.5 mM Ca2+, Cd2+, Cu2+, Mg2+, Ni2+, and Zn2+. Steric considerations appear to regulate dimer formation since those divalent cations that enhance dimer formation have an ionic radius >1.1 A. Our data suggest that the borate ester is located on C-2 and C-3 of two of the four 3'-linked apiosyl residues of dimeric RG-II. Our results, taken together with the results of two previous studies (Kobayashi, M., Matoh, T., and Azuma, J.-I. (1996) Plant Physiol. 110, 1017-1020; Ishii, T., and Matsunaga, T. (1996) Carbohydr. Res. 284, 1-9) provide substantial evidence that this plant cell wall pectic polysaccharide is covalently cross-linked.

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The location of the 1:2 borate-diol ester cross-link in the dimer of the plant cell wall polysaccharide rhamnogalacturonan II (RG-II) has been determined. The ester cross-links the apiofuranosyl residue of the 2-Omethyl-D-xylose-containing side chains in each of the subunits of the dimer. The apiofuranosyl residue in each of the two aceric acid-containing side chains is not esterified. The site of borate esterification is identical in naturally occurring and in in vitro synthesized dimer. Pb 2؉ , La 3؉ , and Ca 2؉ increase dimer formation in vitro in a concentration-and pH-dependent manner. Pb 2؉ is the most effective cation. The dimer accounts for 55% of the RG-II when the monomer (0.5 mM) is treated for 5 min at pH 3.5 with boric acid (1 mM) and Pb 2؉ (0.5 mM); at pH 5 the rate of conversion is somewhat slower. Hg 2؉ does not increase the rate of dimer formation. A cation's charge density and its ability to form a coordination complex with RG-II, in addition to steric factors, may regulate the rate and stability of dimer formation in vitro. Our data provide evidence that the structure of RG-II itself determines which apiofuranosyl residues are esterified with borate and that in the presence of boric acid and certain cations, two RG-II monomers selfassemble to form a dimer.Rhamnogalacturonan II (RG-II) 1 is a low molecular weight, structurally complex pectic polysaccharide released from the primary walls of plants by treatment with endo-␣-1,4-polygalacturonase (1). The results of recent studies have established that RG-II exists predominantly in the cell walls of all higher plants as a dimer (dRG-II-B) that is cross-linked by a 1:2 borate-diol ester (2-5). Borate ester cross-linking of pectin may be required for the normal growth and development of plants (4 -6) because boron deficiency results in the inhibition of plant growth and in the formation of cell walls with markedly altered physical properties (7-11).A single 1:2 borate-diol ester is believed to cross-link two RG-II molecules. 1 mol of the dimer contains 1 mol of boron (3,4,(12)(13)(14). We have proposed that two of the four 3Ј-linked apiofuranosyl (Apif) residues present in dRG-II-B are crosslinked by borate because the dimer contains approximately equimolar amounts of 3Ј-and 2,3,3Ј-linked Apif residues, whereas monomeric RG-II (mRG-II) contains only 3Ј-linked Apif residues (4, 12). However, two different Apif-containing side chains are attached to the backbone of each RG-II monomer (chains A and B in Fig. 1). It is not known whether the borate cross-link involves the Apif residue in one of each of the two types of Apif-containing side chains or if the borate is attached to the same type of Apif-containing side chains in each monomer.The ability to form dRG-II-B from mRG-II and boric acid in vitro provides a convenient model system to examine the mechanism of dimer formation (4). In addition, such studies are likely to provide information on the ability of dRG-II-B, which is present in fermented beverages such as wine, to form complexes with heavy me...