Abstract
Mouse monoclonal antibodies were generated against dehydrodiconiferyl alcohol- or pinoresinol-p-aminohippuric acid (pAHA)-bovine serum albumin (BSA) conjugate as probes that specifically react with 8-5′ or 8-8′ linked structure of lignin in plant cell walls. Hybridoma clones were selected that produced antibodies that positively reacted with dehydrodiconiferyl alcohol- or pinoresinol-pAHA–BSA and negatively reacted with pAHA–BSA and guaiacylglycerol-beta-guaiacyl ether-pAHA–BSA conjugates containing 8-O-4′ linkage. Eight clones were established for each antigen and one of each clone that positively reacted with wood sections was selected. The specificity of these antibodies was examined by competitive ELISA tests using various lignin dimers with different linkages. The anti-dehydrodiconiferyl alcohol antibody reacted specifically with dehydrodiconiferyl alcohol and did not react with other model compounds containing 8-O-4′, 8-8′, or 5-5′ linkages. The anti-pinoresinol antibody reacted specifically with pinoresinol and syringaresinol and did not react with the other model compounds containing 8-O-4′, 8-5′, or 5-5′ linkages. The antibodies also did not react with dehydrodiconiferyl alcohol acetate or pinoresinol acetate, indicating that the presence of free phenolic or aliphatic hydroxyl group was an important factor in their reactivity. In sections of Japanese cypress (Chamaecyparis obtusa), labeling by the anti-dehydrodiconiferyl alcohol antibody was found in the secondary walls of phloem fibers and in the compound middle lamellae, and secondary walls of tracheids. Weak labeling by the anti-pinoresinol antibody was found in secondary walls of phloem fibers and secondary walls and compound middle lamellae of developed tracheids. These labelings show the localization of 8-5′ and 8-8′ linked structure of lignin in the cell walls.
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Abbreviations
- BSA:
-
Bovine serum albumin
- pAHA:
-
p-Aminohippuric acid
- ELISA:
-
Enzyme-linked immunosorbent assay
- MALDI-TOF:
-
MS Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
- PBS:
-
Phosphate buffered saline
- TBS:
-
Tris(hydroxymethyl) aminomethane buffered saline
- TEM:
-
Transmission electron microscopy
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Acknowledgments
This work was supported by a Grant in Aid for Scientific Research (C) (No. 17580143) from the Japan Society for the Promotion of Science (JSPS). The authors are indebted to Professor Emeritus Noritsugu Terashima of Nagoya University, Japan, for kindly donating dehydrodiconiferyl alcohol, dehydrodiisoeugenol. The authors are also indebted to Dr. John Ralph and Dr. Hoon Kim in Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, USA for kindly donating syringaresinol.
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Kiyoto, S., Yoshinaga, A., Tanaka, N. et al. Immunolocalization of 8-5′ and 8-8′ linked structures of lignin in cell walls of Chamaecyparis obtusa using monoclonal antibodies. Planta 237, 705–715 (2013). https://doi.org/10.1007/s00425-012-1784-x
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DOI: https://doi.org/10.1007/s00425-012-1784-x