Immunolocalization of 8-5′ and 8-8′ linked structures of lignin in cell walls of Chamaecyparis obtusa using monoclonal antibodies

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.

A rapid and quantitative safranin‐based fluorescent microscopy method to evaluate cell wall lignification

“…The described safranin method is not only easy to use, but also provides high‐resolution spatial information on cell wall lignin that can usually only be obtained by using more time‐consuming and fastidious techniques, such as light/SEM immunolocalization or Raman spectroscopy (Gierlinger, ; Kiyoto et al , ; Day et al , ; Simon et al , ).…”

Visualization of plant cell wall lignification using fluorescence‐tagged monolignols

“…The direct monitoring of cell wall lignification processes in live plants and cells is a promising strategy to gain insight into the elusive aspects of lignin biosynthesis. Unlike proteins, and similarly to polysaccharides or lipids, lignins are not amenable to being genetically tagged for (Lewis and Yamamoto, 1990;Donaldson, 2001;Fromm et al, 2003), direct microspectrophotometric detection (Singh et al, 2009;Sun et al, 2011;Ding et al, 2012;Gierlinger et al, 2012;Donaldson, 2013), mass spectrometry-based chemical imaging (Saito et al, 2005(Saito et al, , 2012Jung et al, 2012), and immunochemical labeling using lignin-specific antibodies (Ruel et al, 2009;Tranquet et al, 2009;Kiyoto et al, 2013), have been developed and used to visualize lignins in plant tissues. However, these methods typically involve several disadvantages either in sensitivity, specificity, sample-preparation times, artifacts from sample fixation, and/or applicability to living plant systems.…”

Ectopic Lignification in the Flax lignified bast fiber1 Mutant Stem Is Associated with Tissue-Specific Modifications in Gene Expression and Cell Wall Composition

“…In contrast to outer-stem tissues, the S/G ratio (0.12) estimated from lbf12D NMR spectra (Supplemental Figure 1) was very similar to that estimated by thioacidolysis (0.13). Further information on lbf1 lignin was obtained using the KM1 antibody targeted against lignin phenylcoumaran linkages (b-5) (Kiyoto et al, 2013). TEM observation showed that labeling was present in the whole cell wall (primary cell wall, secondary cell wall) of lbf1 fibers, but was conspicuously absent in wild-type fibers (Figure 6).…”