Cyto- and histochemical demonstration of lignins in plant cell walls: an evaluation of the chlorine water/ethanolamine-silver nitrate method of Coppick and Fowler

Abstract

Summary.The chlorine water/ethanolamine-silver nitrate method introduced by Coppick and Fowler for the detection of lignins was evaluated for cyto-and histochemical work using different reagents and fixatives for specimens embedded in epoxy resin. Fixation schedules tested included ethanol, glutaraldehyde, and glutaraldehyde followed by OsO4 as a post-fixative. Chlorine water, sodium hypochlorite, and calcium hypochlorite were the oxidising agents evaluated for their efficacy as part of the Coppick and Fowler procedure. The Coppick and Fowler method was tested against stem woody tissue of Lophomyrtus obcordata, and haustorial xylem tissue of the sucker of its attached dwarf mistletoe Korthalsella lindsayi.The presence of lignins in walls of these cells was indicated in thin sections for transmission electron microscopy by fine electron-dense deposits. Post-staining thin sections did not affect the lignin reaction, but tended to mask its effect due to increased wall contrast. In histological preparations lignified walls stained orange/brown. Counterstaining in methylene blue/azur B caused lignified walls to appear dark green/brown and non-lignified walls blue. Fixation in either ethanol or glutaraldehyde produced identical staining for lignins. Penetration by chlorine water was sometimes irregular, more so with glutaraldehyde fixation, with parts of tissues consequently not responding to the lignin reaction. Post-fixation in osmium tetroxide following primary fixation in glutaraldehyde slightly improved penetration of chlorine water. However, osmium caused greater amounts of extraneous stain deposits compared with other fixative regimes. Chlorine water was confirmed as the most effective oxidising agent for reacting with groups in lignins to produce reducing residues in the Coppick and Fowler method. Sodium hypochlorite caused no reaction. Calcium hypochlorite exhibited limited oxidative capacity resulting in slight staining for lignins. The Coppick and Fowler procedure was concluded to be a suitable method for demonstrating * Correspondence and reprints: Department of Plant and Microbial Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.lignins in cyto-and histochemical preparations using material fixed in either ethanol or glutaraldehyde, and with embedding in epoxy resin.

Lipids in xylem sap of woody plants across the angiosperm phylogeny

“…(Myrothamnaceae) and after further studies concluded that it was composed of phospholipids (Schneider et al, 2003). Dark layers on vessel walls that appeared under TEM after OsO 4 fixation were also found in a few other species (Fineran, 1997, Zimmermann et al, 2004, Westhoff et al, 2008). In fact, it had been suggested much earlier that a thin coat of lipids may remain on the lumen walls of conduits from live cell content after conduit maturation (Scott et al, 1960, Esau, 1965, Esau et al, 1966).…”

Lipids in xylem sap of woody plants across the angiosperm phylogeny

“…Dark layers on vessel walls that appeared under TEM after osmium tetroxide (OsO 4 ) fixation were also found in a few other species (Fineran, 1997;Zimmermann et al, 2004;Westhoff et al, 2008). In fact, it had been shown much earlier that a thin coat of lipids remains on the lumen walls of xylem conduits from live cell content after conduit maturation (Scott et al, 1960;Esau, 1965;Esau et al, 1966).…”

Xylem Surfactants Introduce a New Element to the Cohesion-Tension Theory

“…The latter seems most likely, as substances from the xylem sap could be filtered out and deposited onto or within the pit membrane (Côté, 1958;Schmid, 1965; Wheeler, 1981; Pesacreta et al, 2005). It would be fair to say that nanoparticles in intervessel pit membranes and on conduit walls have been hiding in plain sight for some time, although a few previous studies noted that they only appeared after OsO 4 fixation, indicating their lipid nature (Fineran, 1997;Westhoff et al, 2008). Wagner et al (2000) observed a dark layer lining xylem conduits of the resurrection plant Myrothamnus flabellifolia (Myrothamnaceae) and speculated that it was composed of phospholipids, but they did not conduct a chemical analysis.…”