Boron-bridged RG-II and calcium are required to maintain the pectin network of the Arabidopsis seed mucilage ultrastructure

The structure of a pectin network requires both calcium (Ca2+) and boron (B). Ca2+ is involved in crosslinking pectic polysaccharides and arbitrarily induces the formation of an “egg-box” structure among pectin molecules, while B crosslinks rhamnogalacturonan II (RG-II) side chain A apiosyl residues in primary cell walls to generate a borate-dimeric-rhamnogalacturonan II (dRG-II-B) complex through a boron-bridge bond, leading to the formation of a pectin network. Based on recent studies of dRG-II-B structures, a hypothesis has been proposed suggesting that Ca2+is a common component of the dRG-II-B complex. However, no in vivo evidence has addressed whether B affects the stability of Ca2+ crosslinks. Here, we investigated the L-fucose-deficient dwarf mutant mur1, which was previously shown to require exogenous B treatment for phenotypic reversion. Imbibed Arabidopsis thaliana seeds release hydrated polysaccharides to form a halo of seed mucilage covering the seed surface, which consists of a water-soluble outer layer and an adherent inner layer. Our study of mur1 seed mucilage has revealed that the pectin in the outer layer of mucilage was relocated to the inner layer. Nevertheless, the mur1 inner mucilage was more vulnerable to rough shaking or ethylene diamine tetraacetic acid (EDTA) extraction than that of the wild type. Immunolabeling analysis suggested that dRG-II-B was severely decreased in mur1 inner mucilage. Moreover, non-methylesterified homogalacturonan (HG) exhibited obvious reassembly in the mur1 inner layer compared with the wild type, which may imply a possible connection between dRG-II-B deficiency and pectin network transformation in the seed mucilage. As expected, the concentration of B in the mur1 inner mucilage was reduced, whereas the distribution and concentration of Ca2+in the inner mucilage increased significantly, which could be the reason why pectin relocates from the outer mucilage to the inner mucilage. Consequently, the disruption of B bridges appears to result in the extreme sensitivity of the mur1 mucilage pectin complex to EDTA extraction, despite the reinforcement of the pectin network by excessive Ca2+. Therefore, we propose a hypothesis that B, in the form of dRG-II-B, works together with Ca2+to maintain pectin network crosslinks and ultimately the mucilage ultrastructure in seed mucilage. This work may serve to complement our current understanding of mucilage configuration.