- Open Access
A high-resolution method for the localization of proanthocyanidins in plant tissues
© Abeynayake et al; licensee BioMed Central Ltd. 2011
Received: 14 January 2011
Accepted: 20 May 2011
Published: 20 May 2011
Histochemical staining of plant tissues with 4-dimethylaminocinnamaldehyde (DMACA) or vanillin-HCl is widely used to characterize spatial patterns of proanthocyanidin accumulation in plant tissues. These methods are limited in their ability to allow high-resolution imaging of proanthocyanidin deposits.
Tissue embedding techniques were used in combination with DMACA staining to analyze the accumulation of proanthocyanidins in Lotus corniculatus (L.) and Trifolium repens (L.) tissues. Embedding of plant tissues in LR White or paraffin matrices, with or without DMACA staining, preserved the physical integrity of the plant tissues, allowing high-resolution imaging that facilitated cell-specific localization of proanthocyanidins. A brown coloration was seen in proanthocyanidin-producing cells when plant tissues were embedded without DMACA staining and this was likely to have been due to non-enzymatic oxidation of proanthocyanidins and the formation of colored semiquinones and quinones.
This paper presents a simple, high-resolution method for analysis of proanthocyanidin accumulation in organs, tissues and cells of two plant species with different patterns of proanthocyanidin accumulation, namely Lotus corniculatus (birdsfoot trefoil) and Trifolium repens (white clover). This technique was used to characterize cell type-specific patterns of proanthocyanidin accumulation in white clover flowers at different stages of development.
Proanthocyanidins, or condensed tannins, are polymers of flavan-3-ol subunits, which are produced by the flavonoid secondary pathway in many plants. Proanthocyanidins are best known for their protein-binding ability and are commercially significant because of their antioxidant properties and their potential health benefits when included at a low level in the diets of humans and livestock [1–5]. Proanthocyanidins are produced naturally in the leaves, flowers, fruit, seeds, bark and roots of many plant species [6–11]. A number of quantitative methods have been developed to analyze the level and subunit composition of proanthocyanidins in bulk tissue samples [5, 7, 12–14]. These methods can provide information about the degree of polymerisation and the hydroxylation pattern and stereochemistry of flavan-3-ol subunits.
Vanillin and 4-dimethylaminocinnamaldehyde (DMACA) are commonly used for histochemical staining of proanthocyanidins and their immediate precursor molecules, namely, flavan-3,4-diols and flavan-3-ols, in fresh plant material [15–20]. DMACA reagent stains proanthocyanidins a blue color by binding to meta-oriented dihydroxy- or trihydroxy-substituted benzene rings [14, 19, 21]. The main disadvantage of histochemical staining of fresh plant tissues is that cellular integrity is compromised during the sectioning process.
A range of methods have been used to localize proanthocyanidins, but each has limitations that need to be considered when planning an experiment. Established methods for localization of proanthocyanidins using electron microscopy [22–25] require a high level of technical expertise and are expensive. Epoxy and glycolmethacrylate resins have been used as embedding media for plant tissues prior to sectioning and staining to visualize proanthocyanidins [16, 24, 26]. Staining of plant tissues embedded in epoxy resin with Sudan Black to detect proanthocyanidin deposits also stained lipid bodies . Staining of sections from samples embedded in glycolmethacrylate resin has been used in combination with the more specific DMACA staining reagent to preserve the fine structure of plant tissues . This method involved heating of glycolmethacrylate-embedded semithin sections in a microwave oven in the presence of a staining solution containing DMACA to enhance the staining process. However, not all cells in sections were fixed equally well, suggesting that the glutaraldehyde fixative had not penetrated the tissue sufficiently. The heating time also needed to be carefully controlled to avoid discoloration and each section had to be treated with fresh staining solution, which was deactivated upon heating. Proanthocyanidins have also been visualized by fixing fresh samples in a solution of formalin and ferrous sulphate, but this method also stains other phenolic substances .
Specific staining for proanthocyanidins, flavan-3-ols and flavan-3,4-diols without damage to the fine structure of plant tissues is a challenging task, due to the acidity of the DMACA staining solution [27, 28]. In this study, DMACA staining was used in combination with two commonly-used embedding techniques to analyze the accumulation of proanthocyanidins in Lotus corniculatus and Trifolium repens tissues. Physical integrity of plant tissues was retained during the staining, fixing, embedding and sectioning steps. Embedding of the tissues in LR White and paraffin matrices lead to the appearance of brown coloration in proanthocyanidin-accumulating cells. This is likely to have been the result of non-enzymatic oxidation of proanthocyanidins and the formation of colored semiquinones and quinones. This method is very simple and has the potential to provide high-resolution images showing cell-specific localization of proanthocyanidins in a range of plant tissues.
Results and discussion
Proanthocyanidin accumulation in Lotus corniculatus and Trifolium repens
When immature flowers were stained with DMACA and embedded in LR White resin prior to sectioning, brown/red coloration was most pronounced in the epidermal cell layer on the abaxial and adaxial surfaces of stamen filaments and on the abaxial surface of carpels (Figure 3D). A longitudinal section of an immature flower showed the presence of brown/red coloration in a standard petal and a trichome (Figure 3E). Brown coloration was seen in vacuoles of multiple cells within a trichome when viewed under higher magnification (Figure 3F).
The brown metabolite is likely to be oxidized proanthocyanidins for two main reasons. The spatio-temporal pattern of the metabolite correlated well with DMACA staining of cells in developing white clover flowers. Non-enzymatic oxidation of proanthocyanidins could lead to the formation of quinoidal compounds. Semiquinones and quinones are highly reactive species that undergo further non-enzymatic reactions, reacting spontaneously with phenols, amino acids or proteins, yielding a complex mixture of brown products .
A method has been demonstrated for localizing proanthocyanidin deposits in cells of two proanthocyanidin-rich legume species, namely, Lotus corniculatus and Trifolium repens. Sectioning and microscopic analysis of DMACA-stained tissues embedded in LR White or paraffin matrices allowed high-resolution imaging. The embedding procedure alone allowed the spatial pattern of proanthocyanidin accumulation to be visualized, probably due to the oxidation of proanthocyanidins and flavan-3-ols. This method is simple and allows proanthocyanidins to be visualized in specific tissues and cell types when combined with high resolution microscopic analysis.
4-Dimethylaminocinnamaldehyde (DMACA) staining and fixing of samples
White clover flowers at different stages of maturity as well as leaves from Lotus corniculatus plants were decolorized in absolute ethanol for 3 h and stained for the presence of proanthocyanidins and flavan-3-ols using 0.01% (w/v) 4-dimethylaminocinnamaldehyde (DMACA) in absolute ethanol containing 0.8% w/v hydrochloric acid . Flowers were stained for 20 min and the remaining organs were stained for 2 h before being transferred to 100% ethanol. After DMACA staining, samples were vacuum-infiltrated for 1 min with fixative (6% w/v glutaraldehyde, 4% w/v paraformaldehyde in 50 mM sodium phosphate buffer, pH 7.4) in 1.5 mL microcentrifuge tubes and were incubated for 2 h at 4°C. Samples were then washed three times for 5 min in 50 mM sodium phosphate buffer, pH 7.4.
Embedding of samples in LR white resin
Samples were dehydrated using 5 sequential 15 min washes in an ethanol series (30%, 60%, 70%, 90% and 100% ethanol). The ethanol was replaced with a 3:7 mixture of LR White resin (ProSciTech, Australia) and absolute ethanol. After 1 h of incubation at ambient temperature, this mixture was replaced with a 7:3 mixture of LR White resin and absolute ethanol and incubated for 1 h at ambient temperature. The samples were subsequently incubated for approximately 14 h at ambient temperature in 100% LR White resin. Finally, samples were placed in plastic capsules containing 100% LR White resin and incubated for approximately 14 h under vacuum at 60°C.
Embedding of samples in paraffin
Sectioning and imaging of embedded samples
Transverse and longitudinal sections of embedded samples in LR White resin (6 - 10 μm) and paraffin (8 μm) were generated using a microtome. Cell walls in some of the sections were stained with 0.05% Toluidine Blue (ProSciTech, Cat. # C078) so that the fine structure of plant tissues could be seen. Images were captured using a Leica MZFLIII light microscope (Leica, Germany) fitted with a CCD camera.
The authors would like to thank Bruce Abaloz at the University of Melbourne and Edgar Sakers at La Trobe University for help with embedding of samples and preparation of slides. The Molecular Plant Breeding Co-operative Research Centre is gratefully acknowledged for funding the research.
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