Preventing False Negatives for Histochemical Detection of Phenolics and Lignins in PEG-Embedded Plant Tissues

Abstract

SummaryPolyethylene glycol (PEG) is a low-cost and advantageous embedding medium, which maintains the majority of cell contents unaltered during the embedding process. Some hard or complex plant materials are better embedded in PEG than in other usual embedding media. However, the histochemical tests for phenolics and lignins in PEG-embedded plant tissues commonly result in false negatives. We hypothesize that these false negatives should be prevented by the use of distinct fixatives, which should avoid the bonds between PEG and phenols. Novel protocols for phenolics and flavanols detection are efficiently tested, with fixation of the samples in ferrous sulfate and formalin or in caffeine and sodium benzoate, respectively. The differentiation of lignin types is possible in safranin-stained sections observed under fluorescence. The Maule's test faultlessly distinguishes syringyl-rich from guaiacyl-and hydroxyphenyl-rich lignins in PEG-embedded material under light microscopy. Current hypothesis is corroborated, that is, the adequate fixation solves the false-negative results, and the new proposed protocols fill up some gaps on the detection of phenolics and lignins. (J Histochem Cytochem 65:105-116, 2017)

Increased Gibberellins and Light Levels Promotes Cell Wall Thickness and Enhance Lignin Deposition in Xylem Fibers

“…When half of the initial solution volume was reached, 75% (w/v), the PEG 6000 solution was again added. When the solution reached half the total volume again, the fragments were incorporated to a 90% PEG 6000 and gum arabic solution ( Ferreira et al, 2017 ). The stem fragments were placed in cassettes mounted on a wood base with adhesive tape and immediately frozen at −16°C.…”

“…The stem fragments were placed in cassettes mounted on a wood base with adhesive tape and immediately frozen at −16°C. Samples were then unformed, cut using a hand-rotated microtome (thickness 25–35 μm) and plated with water between 35 and 50°C ( Souza et al, 2016 ; Ferreira et al, 2017 ). Staining was performed using astra blue and safranin/basic fuchsin 1% (w/v) ( Kraus et al, 1998 ) and slides were then mounted between the slide and a cover slip with 50% glycerin.…”

“…Staining was performed using astra blue and safranin/basic fuchsin 1% (w/v) ( Kraus et al, 1998 ) and slides were then mounted between the slide and a cover slip with 50% glycerin. Digital images were obtained on an EKB-2F epifluorescence microscope (Eikonal Ind., São Paulo, Brazil) at the violet excitation wavelength (400 nm) ( Ferreira et al, 2017 ). The images were processed with the Bel Eurisko software (Bel Photonics, Piracicaba, Brazil) and analyzed qualitatively and quantitatively for changes in vascular cylinder morphology with the Image-Pro-Plus ® v.4.5 software.…”

Galls on Smilax campestris Griseb. (Smilacaceae) protect the insects against restinga constraints, but do not provide enriched nutrition

“…The lignification of some cortical cell walls, when compared to non-galled leaves, guarantees structural stability and provides a favorable microenvironment for the development of the larvae (Motta et al 2005). Lignification of cell walls within gall tissues may be the result of increased oxidative stress caused by the midges, which seems to initiate a chemical signaling cascade that culminate in the accumulation of phenols, and trigger gall development (Del Rio and Puppo 2009;Oliveira et al , 2011Oliveira et al , 2016Ferreira et al 2017b). The residues of lignins may capture free radical molecules and polymerize in cell walls, being a chemical adaptation toward the control of oxidative stress in several plants (Boerjan et al 2003;Oliveira et al 2016).…”

Anatomy, histochemistry, and comparative analysis of hydroxycinnamic derivatives in healthy leaves and galls induced by Baccharopelma spp. (Hemiptera: Psyllidae) in Baccharis spicata (Lam) Baill (Asteraceae)

“…The material was fixed with FAA and included in polyethylene glycol 6000 according to (Ferreira et al, 2014(Ferreira et al, , 2017. The sections were obtained with a rotating microtome (70 mm) using the technique described in (Argüeso, 1986).…”