Improved chemical fixation of lipid-secreting plant cells for transmission electron microscopy.

Cultured Lithospermum erythrorhizon cells were fixed with a new fixation method to visualize the metabolism of shikonin derivatives, the lipophilic naphthoquinone pigments in Boraginaceae. The new fixation method combined glutaraldehyde containing malachite green, imidazole-osmium and p-phenylenediamine treatments, and cells were then observed with a transmission electron microscope. The method prevented the extraction of lipids, including shikonin derivatives, and improved the visualization of subcellular structures, especially the membrane system, when compared with that of conventional fixation. The improved quality of the transmission electron micrographs is because malachite green ionically binds to the plasma membrane, organelles and lipids and acts as a mordant for electron staining with osmium tetroxide. Imidazole promotes the reaction of osmium tetroxide, leading to enhanced electron staining. p-Phenylenediamine reduces osmium tetroxide bound to cellular materials and increases the electron density. This protocol requires only three additional reagents over conventional chemical fixation using glutaraldehyde and osmium tetroxide.

Distribution of Lignin, Hemicellulose, and Arabinogalactan Protein in Hemp Phloem Fibers.

The distribution of lignin, 8-5' and 8-8' linked lignin substructure, and noncellulosic polysaccharides in hemp (Cannabis sativa L.) phloem fibers were explored based on histochemical and immunological methods. Ultraviolet absorption and potassium permanganate staining were observed mainly in the compound middle lamella (CML) and S1 layers, and rarely in the G-layer of phloem fibers, suggesting that lignin concentration is high at the CML and S1 layers, and very low at the G-layer of hemp fibers. Acriflavine staining, uniform KM1 labeling (8-5' linked lignin substructure), and no KM2 labeling (8-8' linked structure) were observed in the G-layer, suggesting that there is a small amount of lignin-like compound with 8-5' linked structure in the G-layer. In addition, some fiber cells showed a multilayered structure. Uniform arabinogalactan protein (AGP) labeling was observed on the S1 layers and G-layers using JIM14, but little appeared in the CML of hemp fibers, indicating that these layers of the phloem fibers contain AGP. Immunogold labeling of xylan (LM11) and glucomannan (LM21) showed that xylan and glucomannan were mainly present in the S1 layers and the G-layers, respectively. In some phloem fibers, LM21 immunofluorescence labeling showed multilayered structure, suggesting the heterogeneous distribution of glucomannan.

Relative deposition of xylan and 8-5'-linked lignin structure in Chamaecyparis obtusa, as revealed by double immunolabeling by using monoclonal antibodies.

MAIN CONCLUSION: Immunolabeling by using monoclonal antibodies showed that xylan deposition precedes the formation of 8-5'-linked structure of lignin in normal and compression woods of Chamaecyparis obtusa. Xylan deposition and formation of 8-5'-linked lignin structure in differentiating xylems from normal and compression woods in Chamaecyparis obtusa were examined by immunoelectron microscopy using monoclonal antibodies (LM10 or LM11) to detect xylan localization. The 8-5'-linked lignin structure was immunolocalized using KM1 antibody. Xylan and 8-5'-linked lignin double immunolabeling was performed using secondary antibodies labeled with colloidal gold particles of different diameters. In normal wood, KM1 labeling occurred in the compound middle lamella (CML) and S1 layer during S1 layer formation and increased as S2 and S3 layers formed, with labeling occurring at the outer part of the previous layer. In compression wood, mild KM1 labeling occurred in the CML and outer part of the S1 layer at the later S1 layer formation stage, with increased labeling as the S2 layer formed. Minor labeling occurred in the outer part of the S2 layer during helical cavity formation. Comparison between KM1 labeling and KMnO4 staining suggested that lignin other than 8-5'-linked structure was formed during early lignification, and the proportion of 8-5'-linked lignin structure increased at later stages of lignification in both normal and compression woods. LM10 and LM11 labeling occurred slightly earlier than KM1 labeling, suggesting that xylan deposition preceded the formation of 8-5'-linked lignin in normal and compression woods. Less labeling by KM1, LM10, and LM11 occurred in the outer part of the S2 layer in compression wood, which has abundant lignin. Thus, lignin in these parts is composed of lignin substructures other than the 8-5' linkage.

Ectopic lignification in the flax lignified bast fiber1 mutant stem is associated with tissue-specific modifications in gene expression and cell wall composition.

Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply.

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

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.