Flavonoids have long been recognized as playing multiple roles in the responses of higher plants to a wide range of environmental constraints (Winkel-Shirley, 2002; Roberts & Paul, 2006), and early proposed as developmental regulators (Stafford, 1991). Over the last three decades, as ozone depletion has become a major environmental problem, the particular role of flavonoids in screening short-wavelength solar UV-B radiation (over the 280–315 nm waveband) has been strongly emphasized (Rozema et al., 1997; Burchard et al., 2000).
Flavonoids have been reported to modulate auxin movement (Peer & Murphy, 2007) and hence have the potential to regulate the development of plants growing under different intensities of sunlight irradiance (Jansen, 2002; Buer & Djordjevic, 2009). Antioxidant flavonoids are the most effective inhibitors of basipetal auxin transport (Jacobs & Rubery, 1988;Brown et al., 2001; Jansen et al., 2001). Strong similarities have been found in the molecular targets of flavonoid modulation of auxin movement and signaling components vital to animal cellular functions (Taylor & Grotewold, 2005). In both cases the ability of flavonoids to bind to the ATP sites of a large number of proteins (e.g. the auxin efflux facilitator PIN proteins in plants,Peer et al., 2004) has been invoked, this ability being restricted to flavonoid structures with potentially effective ‘antioxidant’ properties (Williams et al., 2004).
Here, we offer evidence that may challenge the view that light-inducible ‘UV-absorbing flavonoids’ function primarily in attenuating short-wavelength solar UV-B radiation. We propose that UV-absorbing flavonoids serve an important antioxidant function in photoprotection. Our suggestion is based upon: the UV-screening and antioxidant properties of UV-inducible flavonoids; their accumulation in the mesophyll, not only in epidermal cells following sunlight exposure; the minor role of effective UV attenuators, such as hydroxycinnamic acid derivatives, in the response of plants to sunlight irradiance; and the high capacity of antioxidant flavonoid structures to regulate auxin movement.
Flavonoids serve multiple functions in photoprotection
UV-screening vs antioxidant functions
Most ‘UV-absorbing flavonoids’, with the exception of acylated structures, do not maximally absorb over the 280–315-nm waveband and hence do not equip the leaf with the most effective shield against UV-B irradiance, as compared with other phenylpropanoids (Harborne & Williams, 2000). Nevertheless, flavonoids accumulate to a greater extent than other phenylpropanoids in response to UV-B radiation. Most ‘flavonoid anthocyanins’ have negligible molar extinction coefficients (ε) over the 280–390-nm waveband, but increase greatly in concentration in response to UV irradiance (for review articles, see Gould, 2004; Manetas, 2006). The best candidates for UV-B attenuators are hydroxycinnamic acid derivatives (e.g. p-coumaric, ferulic and caffeic acid), with εmax in the 310–325-nm waveband (Harborne & Williams, 2000; Tattini et al., 2004). However, the ratio of flavonoids to hydroxycinnamates increases steeply upon exposure to UV-B or strong sunlight (Burchard et al., 2000; Tattini et al., 2000; Agati et al., 2002; Kotilainen et al., 2008). Flavonoids seem to ‘replace’ hydroxycinnamic acid derivatives as a leaf develops under UV irradiance (Burchard et al., 2000), and soluble hydroxycinnamates are usually confined to tissues receiving the lowest doses of UV radiation (Olsson et al., 1999; Tattini et al., 2004). Consequently, the UV-B-induced preferential biosynthesis of flavonoids, which have εmax > 335 nm (with the exception of some acylated forms), suggests that UV screening is just one of the multiple roles played by flavonoids in photoprotection (Markham et al., 1998; Harborne & Williams, 2000). This suggestion is strongly supported by: the deactivation of hydroxycinnamate in favour of the flavonoid branch pathway in the glandular trichomes of Phillyrea latifolia leaves following severe UV stress (Tattini et al., 2000; Agati et al., 2002); a decrease in the concentrations of the highly effective UV attenuators p-coumaric and chlorogenic acid derivatives coupled with a steep increase in the concentration of quercetin 3-O- glycosides (εmax > 350 nm) in plants growing under enhanced UV-B or UV-B + UV-A irradiance (Kotilainen et al., 2008); flavonoid accumulation in full-sunlight-treated leaves in the absence of UV irradiance (Kolb et al., 2001; Agati et al., 2009) or in plants treated with excess copper ions growing under photosynthetically active radiation (Babu et al., 2003).
Orthodihydroxy B-ring substitution is crucial in conferring effective antioxidant properties, but does substantially alter the UV-spectral features of these flavonoid glycosides as compared with the monohydroxy B-ring flavonoid structures. Flavonoid glycosides are usually encountered in healthy leaf cells, as glycosylation produces soluble flavonoids in the aqueous cellular milieu and protects highly reactive functional groups (e.g. the OH-group in the 3-position in flavonols) from auto-oxidation (Pearse et al., 2005). Orthodihydroxy B-ring substituted flavonoids may inhibit the generation of free radicals by both chelating metal ions (Brown et al., 1998; Smyk et al., 2008) and decreasing the activity of xanthine oxidase (which generates the superoxide anion; Nguyen et al., 2006), in addition to effectively quenching ROS once they are formed (Rice-Evans et al., 1996; Pourcel et al., 2006). Flavonoid–metal complexes may also mimic superoxide dismutase activity (Kostyuk et al., 2004). These unique properties of flavonoids with a catechol group in the B-ring may help to explain the steep increase in the ratio of dihydroxy to monohydroxy B-ring substituted flavonoids caused by UV radiation (Markham et al., 1998; Tattini et al., 2004, 2005; Gerhardt et al., 2008; Agati et al., 2009).
We hypothesize that light-induced changes in phenylpropanoid metabolism result in the synthesis of compounds capable of performing multiple functions. These ‘biochemical adjustments’ appear primarily to have the purpose of reducing the oxidative damage caused by the flux of short-wavelength solar UV radiation to sensitive leaf targets, rather than attenuating the flux of damaging solar wavelengths (Landry et al., 1995; Winkel-Shirley, 2002). This hypothesis is strongly supported by a recent finding (Gerhardt et al., 2008) of UV-B-induced accumulation of nonacylated quercetin in preference to acylated kaempferol derivatives, acylated kaempferols being effective at absorbing over the whole UV spectral region. In addition to the ability to attenuate UV wavelengths, metal-chelating and ROS-quenching activities have been suggested to have contributed greatly to the evolution of early land plants (Swain, 1986). It may not be a coincidence that flavonols, the most ancient and widespread of the flavonoids, are effective antioxidants (Cockell & Knowland, 1999; Winkel-Shirley, 2002).
Plants have long been reported to be equipped with a very efficient antioxidant system for coping with excess light-induced ROS generation (Schwanz et al., 1996; Logan et al., 1998; Peltzer & Polle, 2001;Halliwell, 2009). However, plants have not developed redundant metabolic networks to counter photo-induced alterations in the cellular redox homeostasis. We note that antioxidant flavonoids and antioxidant enzymes, whose concentrations and activities increase in parallel in response to high light (Grace & Logan, 2000; Close & McArthur, 2002), may operate in different cellular compartments, thus complicating the issue of how the various antioxidant systems actually co-operate in photoprotection (Hernández et al., 2009).
Antioxidant flavonoids as developmental regulators
It has long been documented that flavonoids control auxin movements (Jacobs & Rubery, 1988; Brown et al., 2001; Peer & Murphy, 2007), and a nuclear distribution of chalcone synthase (CHS) and chalcone isomerase (CHI) (Saslowsky et al., 2005) is consistent with control exerted by flavonoids on the transcription of genes required for growth and development, such as the auxin transport facilitator proteins (Peer et al., 2004). Stafford (1991) suggested that flavonoids probably played a primary role as internal regulators during the evolution of early land plants, with distinct functions carried out by the mono and dihydroxy B-ring structures in regulating auxin metabolism. These functions may be accomplished by relatively low flavonoid concentrations, as probably occurred in early land plants, in contrast to the relatively high concentrations (in the mM range) actually needed to effectively attenuate UV radiation (Stafford, 1991).
Sunlight irradiance closely controls a plant’s shape, with shade plants having long internodes and large leaves, whereas sunny plants usually display very short internodes and small, thick leaves. These morphological traits are under hormonal, particularly auxin, control (Jansen, 2002). ‘Antioxidant’ flavonoids have been reported to be the most effective regulators of auxin transport in vivo (Jacobs & Rubery, 1988;Brown et al., 2001; Taylor & Grotewold, 2005), quercetin aglycone being much more effective than quercetin 3-O-rutinoside in inhibiting the basipetal transport of auxin (Jacobs & Rubery, 1988). Therefore, the high-light-induced preferential biosynthesis of ‘antioxidant’ flavonoids may have a role in regulating whole-plant and individual-organ architecture (Lazar & Goodman, 2006; Beveridge et al., 2007; Buer & Djordjevic, 2009), and increase self-shading (Barnes et al., 1996; Jansen, 2002). Flavonoid-induced control of whole-plant development is, however, far from being fully elucidated, as flavonoids have been shown to also affect auxin catabolism (Galston, 1969; Stafford, 1991). The early finding that monohydroxy B-ring flavonoids behave as cofactors and dihydroxy B-ring flavonoids as inhibitors of the peroxidase-catalysed oxidation of auxin (Galston, 1969) may help to explain the correlation between the UV-induced increase in the quercetin to kaempferol ratio and UV tolerance recently reported by Jansen et al. (2001) in tobacco (Nicotiana tabacum).
Taylor & Grotewold (2005) have recently suggested a link between anti-proliferative, anti-tumour and pro-apoptopic activities of flavonoids and their effects on auxin movement (Geisler et al., 2005; Titapiwatanakum et al., 2009). Strong similarities have been found in the molecular targets of flavonoid modulation of auxin movement and the intracellular signalling cascades vital to animal cellular function: in both cases the ability of flavonoids to bind to the ATP sites of a large number of proteins has been invoked (Conseil et al., 1998; Williams et al., 2004). The ability to bind to ATP sites depends, obviously, on the flavonoid structure, and the orthodihydroxy substitution in the B-ring and the degree of unsaturation of the C2–C3 bonds are key determinants of this biological activity (Williams et al., 2004). Indeed, one of the most selective phospho-inositide 3-kinase (PI 3-kinase) inhibitors currently available, LY294002, has been modelled on the structure of quercetin (Matter et al., 1992; Vlahos et al., 1994).
We conclude, therefore, that light-inducible ‘antioxidant flavonoid structures’ may reduce the risk of photo-oxidative damage, not only through their UV-screening features, but also as a consequence of their ability to regulate the development of the whole plant and individual shoot organs.
Flavonoids as antioxidants in photoprotection
The issue of the antioxidant functions of flavonoids in plant–environment interactions, not only in photoprotection, is still a matter of debate (Halliwell, 2009; Hernández et al., 2009). The oxidation of polyphenols may be inferred from the occurrence of enzymes responsible for this oxidation (Pourcel et al., 2006), but the relevance of flavonoids in the complex antioxidant machinery that allows higher plants to cope with photo-oxidative damage is far from clear (Hernández et al., 2009).
Yamasaki et al. (1997) proposed a model to address major criticisms regarding the antioxidant functions of flavonoids compartmentalized in epidermal cell vacuoles, and at the same time to explain the light-induced preferential biosynthesis of flavonoids with effective antioxidant properties in vitro. It was proposed that orthodihydroxy B-ring-substituted flavonoids, not their monohydroxy B-ring-substituted counterparts, are effective substrates for class III peroxidases, which quench H2O2 freely diffusing from mesophyll cellular organelles and entering the vacuoles of epidermal cells. The model was remarkable in calling out question whether vacuolar flavonoids could be effective in protecting underlying tissues from damaging solar wavelengths (actually, UV radiation may freely pass through the anticlinal cell walls of epidermal cells; Strack et al., 1988; Day, 1993; Day et al., 1994), while not protecting the epidermal cells from oxidation (Stafford, 1991). The exclusive accumulation of flavonoids at the expense of hydroxycinnamates in glandular trichomes (Tattini et al., 2000; Agati et al., 2002) and in epidermal cells upon UV irradiance (Burchard et al., 2000) led to the hypothesis that flavonoids may function as antioxidants in epidermal cells.
Recent evidence suggests that flavonoids may scavenge ROS within or near the sites of their generation (Gould et al., 2002; Schmitz-Hoerner & Weissenböck, 2003; Kytridis & Manetas, 2006; Agati et al., 2007, 2009). Anthocyanins have been shown to accumulate in the vacuoles of mesophyll cells in several species, and ex vivo experiments strongly support a function as scavengers of superoxide anions and hydrogen peroxide (Gould et al., 2000; Neill & Gould, 2003; Kytridis & Manetas, 2006). Similarly, the effective antioxidant quercetin and luteolin glycosides accumulated in the vacuoles of mesophyll cells in Ligustrum vulgareleaves exposed to full sunlight, in the presence or absence of UV irradiance (Agati et al., 2009). This finding, which is consistent with previous reports that UV irradiance is not a prerequisite for the biosynthesis of flavonoids (for a review, see Jenkins, 2009), leads to the hypothesis that excess light-induced oxidative damage may regulate the biosynthesis of flavonoids, irrespective of the proportion of solar wavelengths reaching and penetrating a leaf. The accumulation of mesophyll flavonoids and the ratio of orthodihydroxy to monohydroxy B-ring-substituted flavonoids correlated with the extent of oxidative damage in P. latifolia and L. vulgare leaves exposed to full sunlight (Tattini et al., 2005), as also found in barley (Hordeum vulgare) lines by Schmitz-Hoerner & Weissenböck (2003).
Agati et al. (2007) detected quercetin and luteolin glycosides in chloroplasts (probably in association with the chloroplast envelope) in P. latifolia leaves, using three-dimensional deconvolution microscopy coupled with multispectral fluorescence micro-imaging. A chloroplast distribution of flavonoids was reported earlier (Saunders & McClure, 1976;Takahama, 1982), and chloroplasts have been shown to be capable of flavonoid biosynthesis (Zaprometov & Nikolaeva, 2003). Quercetin and luteolin glycosides effectively quenched singlet oxygen generated by excess blue light in vivo (Agati et al., 2007). It is noteworthy that flavonoids on the outer surface of the chloroplast envelope might additionally quench ROS formed outside the chloroplast (Mullineaux & Karpinski, 2002).
We report here that the concentration of hydroxycinnamates and apigenin derivatives did not vary in leaves of L. vulgare growing at 20 or 100% full sunlight irradiance, that is, receiving UV irradiance at 12.4 or 28.9 μmol m−2 s−1, respectively. By contrast, the concentration of quercetin 3-O-glycosides and luteolin 7-O-glycosides greatly increased, on average by 95%, in response to UV radiation (Fig. 1), the increase mainly being observed in the mesophyll, and not in epidermal cells (Fig. 2b,c). Furthermore, leaves acclimated to full sunlight in the absence of UV irradiance had a much higher concentration of quercetin and luteolin glycosides than UV-treated leaves, and these ‘antioxidant’ flavonoids accumulated to a greater extent in adaxial palisade parenchyma cells than in adaxial epidermal cells (Fig. 2a). These data confirm that UV screening is just one, and perhaps not the most important, role played by flavonoids in photoprotection (please note that hydroxycinnamates, which are very effective UV attenuators, were unresponsive to the light treatments; Fig. 1), and suggest the hypothesis that the light-induced biosynthesis of flavonoids has the primary purpose of reducing photo-oxidative damage (Landry et al., 1995; Agati et al., 2009).
Nevertheless, the questions raised by Hernández et al. (2009) regarding the ‘spatio-temporal correlations of flavonoids and oxidative damage’ merit close consideration, and highlight serious technical problems, both analytical and histochemical. The oxidation products of ‘UV-absorbing flavonoids’ are difficult to detect, as they are unstable at the pHs of various cell compartments (oxidation products of rutin were detected in vitro at pH 4.0; Takahama, 1986). The oxidized quinone and semiquinone forms of flavonoids are still highly reactive and undergo further nonenzymatic reactions to produce polymeric species (Pourcel et al., 2006). In healthy leaves, particularly those exposed to full sunlight, the recycling of phenoxyl radicals to their reduced forms may occur unless ascorbic acid and glutathione-S-transferase are depleted (Edwards et al., 2000; Sakihama et al., 2000). Oxidation products of flavan-3-ols have been identified in pea (Pisum sativum) plants suffering from severe drought stress (Hernández et al., 2006), although the possibility could not be excluded that the ascorbic acid content and the integrity of the cell membranes were greatly altered by the severity of the stress. However, this issue merits detailed investigation, for example by exposing species that differ in constitutive ascorbate content or mutants defective in ascorbate biosynthesis to high sunlight irradiance and increasing water stress. Drought stress has been widely reported to increase the concentration of phenylpropanoids, particularly the dihydroxy substituted structures of hydroxycinnamates and flavonoids (Grace & Logan, 2000; Hernández et al., 2004; Tattini et al., 2004; Turtola et al., 2005).
A picture emerges in which excess light, irrespective of the proportions of different solar wavelengths reaching and penetrating the leaf, enhances the biosynthesis of flavonoids capable of performing multiple functions, at the expense of constitutive ‘poor antioxidant’ flavonoids and hydroxycinnamates. Dihydroxy B-ring-substituted flavonoids, particularly those with flavonol structures such as quercetin, are virtually absent in leaves growing under shade conditions and hence may be termed ‘light-inducible’ pigments. Other flavonoids and hydroxycinnamates are present at constitutively higher concentrations than flavonols, and are mainly distributed in the epidermal layers. It is noted that a concentration of few micromoles g−1 leaf dry weight results into a mM concentration in the epidermal layers: monohydroxy B-ring-substituted flavonoids may effectively attenuate UV irradiance and at the same time protect leaf tissues from pathogens (both functions being fully accomplished at a flavonoid concentration in the mM range; Edwards et al., 2008). Then the preferential accumulation of dihydroxy over monohydroxy flavonoids in response to high light is likely to reduce photo-oxidative damage. In the other side, antioxidant mesophyll flavonoids, in the micromolar range, may effectively avoid the generation (e.g. by chelating transition metal ions) and reduce reactive oxygen forms.
Simplified systems, such as stomatal guard cells and the highly specialized glandular trichomes, may be conveniently investigated to address the localization–functional relationship of flavonoids in photoprotection. The detection of reactive species (ROS and nitric oxide (NO)) and analysis of the expression of genes involved in the biosynthesis of antioxidant flavonoid structures in response to various stresses may be carried out simultaneously, at any scale, using histochemical analyses. Studies incorporating fusion of the promoters of flavonol synthase (FLS; Owens et al., 2008) and flavonoid 3′-hydroxylase (F3′H; which mediates the addition of an OH group to the B-ring) to the GUS reporter gene, coupled with analysis of the intracellular distribution of antioxidant flavonoids, may help to answer some basic questions regarding the oxidative-mediated up-regulation of antioxidant flavonoid biosynthesis in vivo. Indeed, recent findings of regulation of flavonol biosynthesis by R2R3MYB transcription factors, which are reduction-oxidation controlled and involved in the cross-talk between abiotic and biotic stress responses (Fujita et al., 2006; Quattrocchio et al., 2006), may link the REDOX potential of the cell to the control of flavonoid accumulation (Taylor & Grotewold, 2005).
It may be hypothesized that common oxidative signal components may up-regulate flavonoid biosynthesis, irrespective of their origins. This hypothesis is consistent with recent suggestions that plant phenolics perform the primary function of protecting leaves from photo-oxidative damage, rather than herbivore damage, by acting as antioxidants (Close & McArthur, 2002); that light-induced ROS generation activates defences against pathogens (Karpinski et al., 2003); that flavonols are mainly involved in the response mechanisms to abiotic and biotic stresses (Roberts & Paul, 2006; Kilian et al., 2007;Mellway et al., 2009); and that a link may exist between the allelopathic and antitumour activities of flavonoids (Taylor & Grotewold, 2005). These antioxidant-related functions appear to be important as they are conserved across the kingdoms (and, apparently, are free of scale; Sweetlove & Fernie, 2005), although the in vivo bioactive forms of flavonoids in animals (Williams et al., 2004) differ from the glycosylated forms found in plants. Future research should explore in depth how flavonoids may reduce oxidative stress generated in animal and plant cells, which differ greatly in the complexity of their subcellular structures. It may not be a mere coincidence that UV-screening compounds similar to those found in plants play a role in protecting human cells from photo-oxidative damage.