Abstract
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.
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Acknowledgements
We are grateful to Professor Toru Matoh and Dr. Masaru Kobayashi (Kyoto University, Japan) for providing us with the RG-II antibody. We also thank Professor Michael G. Hahn (Complex Carbohydrate Research Center, University of Georgia, USA) for providing us with the CCRC-M36 and CCRC-M38 antibodies, and we thank Professor Paul Knox (University of Leeds, UK) for providing us with the JIM5 antibody. This work was supported by the National Natural Science Foundation of China (Grant No. 31470291 and Grant No. 31670302 to YK), the National Key Technology R & D Program (Grant No. 2015BAD15B03-05, to YK), the Taishan Scholar Program of Shandong (to GZ), the Elite Youth Program of CAAS (to YK), the Grants from Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the US Department of Energy (Grant No. DE-FG02-12ER16324 and DE-FG02-96ER20220 to MAO) and the China Postdoctoral Science Foundation (Grant No. 2015M571173, to DS).
Author contributions
DS, GZ and YK designed the study. DS and JW performed the majority of the experiments. RH performed the ICP-MS analysis. MAO performed the GC-MS analysis. DS, YK and MAO wrote the paper. All of the authors discussed the results, edited the manuscript, and approved the final manuscript.
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Supplementary Table 2—Composition of extracted mucilage from wild-type, mur1-1 and mur1-1 + B Arabidopsis seeds (μg/mg intact seeds) (DOCX 16 KB)
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Supplementary Table 3—Composition of EDTA-extracted mucilage from wild-type, mur1-1 and mur1-1 + B Arabidopsis seeds (μg/mg intact seeds) (DOCX 16 KB)
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Supplementary Figure 5—Seed coat mucilage phenotypes of wild-type, mur1-1, mur1-2, mur3-3 and mur1 seeds pretreated with boric acid under rough shaking (TIF 735 KB)
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Supplementary Figure 6—Sections of developing wild-type and mur1-1 seeds. Sections were stained with toluidine blue O (TBO) (TIF 2339 KB)
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Supplementary Figure 7—Expression profiles of MUR1 based on online data. Charts of MUR1 expression levels in the developing seed coat was developed based on the dataset generated Schmid et al., <link rid="bib45">2005</link> (TIF 1368 KB)
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Supplementary Figure 8—Structural change of mur1 mucilage. A–F, immunolabeling of pectin in adherent mucilage released from wild-type and mur1-1 seeds using JIM5, JIM7 and CCRC-M36. Bar = 50 μm. G, Comparison of methanol release of wild-type, mur1-1 and mur1-2 mucilage under oxidation with an alcohol oxidase enzyme (TIF 2494 KB)
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Shi, Dc., Wang, J., Hu, Rb. et al. Boron-bridged RG-II and calcium are required to maintain the pectin network of the Arabidopsis seed mucilage ultrastructure. Plant Mol Biol 94, 267–280 (2017). https://doi.org/10.1007/s11103-017-0606-8
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DOI: https://doi.org/10.1007/s11103-017-0606-8