Mesophyll distribution of 'antioxidant' flavonoid glycosides in Ligustrum vulgare leaves under contrasting sunlight irradiance

Abstract

Background and aims: Flavonoids have the potential to serve as antioxidants in addition to their function of UV screening in photoprotective mechanisms. However, flavonoids have long been reported to accumulate mostly in epidermal cells and surface organs in response to high sunlight. Therefore, how leaf flavonoids actually carry out their antioxidant functions is still a matter of debate. Here, the distribution of flavonoids with effective antioxidant properties, i.e. the orthodihydroxy B-ring-substituted quercetin and luteolin glycosides, was investigated in the mesophyll of Ligustrum vulgare leaves acclimated to contrasting sunlight irradiance.

Methods: In the first experiment, plants were grown at 20 % (shade) or 100% (sun) natural sunlight. Plants were exposed to 100 % sunlight irradiance in the presence or absence of UV wavelengths, in a second experiment. Fluorescence microspectroscopy and multispectral fluorescence microimaging were used in both cross sections and intact leaf pieces to visualize orthodihydroxy B-ring-substituted flavonoids at inter- and intracellular levels. Identification and quantification of individual hydroxycinnamates and flavonoid glycosides were performed via HPLC-DAD.

Key results: Quercetin and luteolin derivatives accumulated to a great extent in both the epidermal and mesophyll cells in response to high sunlight. Tissue fluorescence signatures and leaf flavonoid concentrations were strongly related. Monohydroxyflavone glycosides, namely luteolin 4'-O-glucoside and two apigenin 7-O-glycosides were unresponsive to changes in sunlight irradiance. Quercetin and luteolin derivatives accumulated in the vacuoles of mesophyll cells in leaves growing under 100 % natural sunlight in the absence of UV wavelengths.

Conclusions: The above findings lead to the hypothesis that flavonoids play a key role in countering light-induced oxidative stress, and not only in avoiding the penetration of short solar wavelengths in the leaf.

Fig. 1.

Extinction coefficient spectra of 40 µm solutions (phosphate buffer, pH 6·8) of hydroxycinnamates (A) and flavonoid glycosides (B) with the addition (continuous lines) or without the addition (dotted lines) of 200 µm 2-amino ethyl diphenyl boric acid (NR). api 7-O-rut, Apigenin 7-O-rutinoside; lut 7-O-glc, luteolin 7-O-glucoside; que 3-O-rut, quercetin 3-O-rutinoside.

Fig. 2.

Fluorescence emission spectra of horizontal cross-sections of the epidermis (A) and the mesophyll (B) of Ligustrum vulgare leaves exposed to 20 % (blue lines) or 100 % solar radiation (red lines). Cross-sections (100 µm thick) were stained with 0·1 % (w/v) 2-amino ethyl diphenyl boric acid in phosphate buffer (NR) and excited at 365 ± 5 nm. Spectra have been normalized to fluorescence intensity at 570 nm of adaxial epidermis (A) or palisade tissue (B) of sun leaves, respectively.

Fig. 3.

Fluorescence spectra of 10 µm solutions (phosphate buffer, pH 6·8) of echinacoside, luteolin 7-O-glucoside (lut 7-O-glc) and quercetin 3-O-rutinoside (que 3-O-rut) with the addition of 50 µm NR under (A) UV- (λ_exc = 365 ± 5 nm) and (B) blue-light excitation (λ_exc = 488 ± 5 nm).

Fig. 4.

Distribution of the fluorescence at 580 nm, F₅₈₀, as false-colour surface plots, in leaves of Ligustrum vulgare acclimated to full-sunlight irradiance. Cross-sections were stained with NR and excited with (A) far blue- (λ_exc = 488 ± 5 nm) or (B) UV light (λ_exc = 365 ± 5 nm).

Fig. 5.

Images of the green and red fluorescence at a depth of 20 µm from the adaxial leaf surface of Ligustrum vulgare growing at different sunlight irradiance: (A) leaves exposed to 100 % natural sunlight over the 300–1100-nm waveband (PAR100 + UV); (B) leaves exposed to 100 % natural sunlight over the 400–1100-nm waveband (PAR100); (C) leaves exposed to 25 % natural sunlight over the 400–1100 nm waveband (PAR25). Measurements were performed on leaf pieces (approx. 25 mm²) infiltrated with 100 µL of NR. CLSM analysis performed in two-channel sequential mode: flavonoid fluorescence was recorded in the green channel (λ_exc = 488 nm, acquisition in the 560–600-nm band), and the chlorophyll fluorescence in the red channel (λ_exc = 514 nm, detection in the 670–750-nm band), respectively. (D) In vivo emission spectrum of blue-light excited (λ_exc = 488 nm) palisade cells of leaves in the PAR100 treatment. Fluorescence signal was integrated, in the λ-scan mode at 10-nm spectral resolution, over a 246 × 246 µm area. Scale bars = 50 µm.