Phenolic profile of a Parma violet unveiled by chemical and fluorescence imaging

Abstract

The ability of phenolic compounds to autofluoresce upon illumination by UV or blue light was exploited to explore the nature and distribution of these metabolites within the flower petals, leaves and roots of the violet, Viola alba subsp. dehnhardtii. This was achieved through a dual complementary approach that combined fluorescence microscopy imaging of living intact tissues and chemical extraction of pulverized material. The blue to red fluorescence displayed by living tissues upon illumination was indicative of their richness in phenolic compounds. Phenolic acids were found in all tissues, while flavonoids characterized the aerial part of the plant, anthocyanidins being restricted to the petals. The chemical quantification of phenolics in plant extracts confirmed their tissue-specific distribution and abundance. A key finding was that the spectral signatures obtained through confocal microscopy of endogenous fluorophores in living tissues and their counterpart extracts share the same fluorescence patterns, pointing out the potential of fluorescence imaging of intact organs for a proper estimation of their phenolic content. In addition, this study highlighted a few distinct morphology cell types, in particular foliar-glandular-like structures, and jagged petal cell walls. Altogether, these data provide a comprehensive histochemical localization of phenolics in living tissues of a violet. Converting fluorescence imaging into a chemical imprint indicated that one can rely on fluorescence microscopy of intact living tissues as a rapid, non-destructive means to follow their phenolic imprint under various environmental conditions.

Keywords: Autofluorescence; Parma violet; Viola alba subsp. dehnhardtii; chemical quantification; confocal microscopy; phenolic profile.

Figure 1.

Chasmogamous flowers of Viola alba subsp. dehnhardtii harbouring 20–40 petals and petal-like stamens.

Figure 2.

Overall views and transverse sections of leaf blade (A–I), petal (J–P) and root (Q). (A–I) Observation in epifluorescence wide-field microscopy of the adaxial face of the leaf blade excited at the UV range (A). Note the small blue spots at the end of the midrib (open arrow), and at each tooth of the dentate blade (arrows); the latter are shown at higher magnification in bright field (B) and fluorescence microscopy upon excitation in the UV range (C) and blue range (D), respectively. Scale bars: 1 mm (A), 100 µm (B–D). (E–G) Observation by epifluorescence wide-field microscopy of the abaxial face of the leaf blade excited in the UV range reveals the numerous stomata and blue patches of fluorescence (E); detail of the latter showing the epidermal cells surrounding the stomata strongly emitting in bright blue (F) and the area at the tip of the midrib (G). Scale bars: 200 µm (E), 50 µm (F), 400 µm (G). (H, I) Transverse sections of the leaf blade observed in wide-field (H) and confocal (I) fluorescence microscopy. Details from adaxial (ad) to abaxial (ab) face: cuticle of the epidermal cell layer, epidermis (ep), palisadic parenchyma (pp), spongy parenchyma (sp), pocket filled with fluorescent compounds (open star). Scale bars: 200 µm (H), 300 µm (I). (J–P) Observation of the adaxial (J–M) and abaxial (N) faces of a petal in bright field (J) and fluorescence microscopy (K–N), with a focus on cells which fluoresce both in the red (K) and blue range (L), and image overlay (M); transverse section of the petal (ad, adaxial epidermis) in bright field (O) and fluorescence (P) microscopy upon excitation at the UV range. Scale bars: 30 µm (L, M, O, P), 50 µm (J, K), 100 µm (N). (Q) Transverse section of the root showing the rhizoderm, xylem vessels and the pith. Scale bars: 300 µm.

Figure 3.

Phenolic acids (A), flavonoids (B) and anthocyanidins (C) content (µg g⁻¹ FW) of flowers, leaves and root extracts. Whiskers represent max and min, the box edges are the first and third quartiles and the middle line represents the median of at least three independent biological repeats.

Figure 4.

Fluorescence emission spectra (mean intensity in arbitrary units ± standard error vs. wavelength in nanometers) of living plant parts (A–C), extracts from the corresponding tissues (D–F) and reference compounds (G–I). (A–C) Spectra from: 15 petal epidermal cells (A); 20 different cells of palisadic parenchyma (dotted green), spongiform parenchyma (green) and glandular structure (yellow) (B); and 20 root cortical cells (C). (D–F) Spectra of extracts from 20 mg FW of: petals (D); leaves (green) and leaf borders (yellow) (E); and roots (F). (G–I) Spectra of reference compounds: the anthocyanidin-cyanidin chloride (G); the rutin (yellow) and quercetin (dotted yellow) flavonoids (H); the ferulic-hydroxycinnamic acid (brown); and the 4-methyl umbelliferone coumarin (dotted brown) (I).