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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SummaryThe metabolism by suspension-cultured tobacco cells of oligogalacturonides was investigated. Dodecagalacturonic acid-

The biological activity of reducing-end-modified oligogalacturonides was quantified in four tobacco (Nicotiana tabacum) tissue culture bioassays. The derivatives used were oligogalacturonides with the C-1 of their reducing end (a) covalently linked to a biotin hydrazide, (b) covalently linked to tyramine, (c) chemically reduced to a primary alcohol, or (d) enzymatically oxidized to a carboxylic acid. These derivatives were tested for their ability to (a) alter morphogenesis of N. tabacum cv Samsun thin cell-layer explants, (b) elicit extracellular alkalinization by suspension-cultured cv Samsun cells, (c) elicit extracellular alkalinization by suspensioncultured N. tabacum cv Xanthi cells, and (d) elicit H 2 O 2 accumulation in the cv Xanthi cells. In all four bioassays, each of the derivatives had reduced biological activity compared with the corresponding underivatized oligogalacturonides, demonstrating that the reducing end is a key element for the recognition of oligogalacturonides in these systems. However, the degree of reduction in biological activity depends on the tissue culture system used and on the nature of the specific reducing-end modification. These results suggest that oligogalacturonides are perceived differently in each tissue culture system.Carbohydrates that act as signal molecules in plants (oligosaccharins) have been isolated from plant and fungal cell wall polysaccharides, from the cell walls of bacterial symbionts of plants, and from fungal glycopeptides (for review, see Ryan and Farmer, 1991;Darvill et al., 1992;Cô té and Hahn, 1994). We are interested in determining the mechanism by which one type of the plant cell wallderived oligosaccharins, the oligogalacturonides, elicit biological responses in plants.The biological effects elicited in plants by oligosaccharins are diverse (for review, see Ryan and Farmer, 1991;Darvill et al., 1992;Cô té and Hahn, 1994) but can generally be placed in two groups: delayed responses and rapid responses. Delayed responses are usually observed hours or days after oligosaccharin treatment and are often directly involved in adaptation to environmental conditions, whereas rapid responses generally occur at the plant cell surface and are observed within minutes after addition of oligosaccharins. The delayed responses elicited by oligogalacturonides can be broadly divided into those in which defense responses are induced and those in which growth and development are modified. The defense-related responses, depending on the plant species, include phytoalexin accumulation (Hahn et al., 1981), lignification of cell walls (Robertsen, 1986), and proteinase inhibitor accumulation (Bishop et al., 1984). The responses involving growth and development include induction of ethylene in tomato fruit (Brecht and Huber, 1988), inhibition of auxin-induced pea stem elongation (Branca et al., 1988), and regulation of tobacco (Nicotiana tabacum) TCL explant morphogenesis (Eberhard et al., 1989). The rapid responses induced by oligogalacturonides include enhanced protein phosp...