Multiple functional roles of flavonoids in photoprotection

Abstract

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). It is now well documented that UV-B stress greatly enhances the production of reactive oxygen species (ROS), as occurs in response to a plethora of environmental stresses (Apel & Hirt, 2004; Jenkins, 2009). UV-B-responsive flavonoids in general have the potential to reduce the oxidative damage caused by short solar wavelengths, in addition to reducing the risk of ROS generation by attenuating the penetration of UV-B radiation to sensitive leaf targets (Kytridis & Manetas, 2006; Kotilainen et al., 2008; Owens et al., 2008; Agati et al., 2009). An increased ratio of the ‘effective antioxidant’ quercetin or luteolin glycosides to the ‘poor antioxidant’ kaempferol or apigenin glycosides has been reported for plants exposed to high levels of UV-B or sunlight irradiance (Markham et al., 1998; Ryan et al., 1998; Gerhardt et al., 2008; Agati et al., 2009). Flavonoids occur not only in the vacuoles and cell walls of epidermal cells and in nonsecretory and glandular trichomes (Wollenweber & Dietz, 1981; Strack et al., 1988; Hutzler et al., 1996; Tattini et al., 2007), but also in the vacuoles of mesophyll cells (Kytridis & Manetas, 2006; Agati et al., 2009) and in chloroplasts (Saunders & McClure, 1976; Takahama, 1982; Agati et al., 2007). As a consequence, they are optimally located to reduce light-induced oxidative damage near or within the sites of ROS production. Recently, a nuclear distribution of orthodihydroxylated B-ring flavonoids has been suggested to protect DNA from oxidative damage (Feucht et al., 2004). 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. 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). The UV-B-induced biosynthesis of phenolics with maximum absorbance in the UV-A region of the solar spectrum (i.e. the action spectrum for induction does not overlap with the phenolic absorbance profile, Caldwell et al., 1983; Cockell & Knowland, 1999) may be explained in part in the light of the several potential functions of flavonoids in photoprotection, as most UV-inducible flavonoids have a catechol group in the B-ring of the flavonoid skeleton (e.g. quercetin and luteolin derivatives; Markham et al., 1998; Ryan et al., 1998; Tattini et al., 2004, 2005; Agati et al., 2007; Gerhardt et al., 2008). 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). 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. 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). Flavonoids have long been reported to occur in the vacuoles and cell walls of epidermal cells and in nonsecretory and glandular trichomes and hence have been assumed primarily to have the function of attenuating short solar wavelengths (Wollenweber & Dietz, 1981; Strack et al., 1988; Schnitzler et al., 1996; Yamasaki et al., 1997; Hutzler et al., 1998;Burchard et al., 2000). Short-term experiments, and microscopy techniques inappropriate for visualizing flavonoid distribution at the level of the whole leaf, may have been responsible for such ‘superficial’ conclusions. The ‘localization–functional relationship’ of flavonoids (Olsson et al., 1999) is still a largely unexplored issue. 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 leaves exposed to full sunlight (Tattini et al., 2005), as also found in by Schmitz-Hoerner & Agati et al. quercetin and luteolin glycosides in chloroplasts in with the in P. latifolia leaves, microscopy coupled with distribution of flavonoids was reported (Saunders & McClure, and chloroplasts have been shown to be capable of flavonoid biosynthesis & 2003). and luteolin glycosides effectively oxygen by excess light in vivo (Agati et al., 2007). It is that flavonoids on the of the quench ROS formed the & 2002). We that the concentration of hydroxycinnamates and apigenin derivatives not in leaves of growing at or full sunlight irradiance, that is, receiving UV irradiance at or the concentration of quercetin and luteolin greatly on by in response to UV radiation the increase being in the mesophyll, and not in epidermal cells leaves to full sunlight in the absence of UV irradiance a much higher concentration of quercetin and luteolin glycosides than leaves, and these ‘antioxidant’ flavonoids accumulated to a greater extent in cells than in epidermal cells These that UV screening is just and not the most role played by flavonoids in photoprotection note that which are very effective UV attenuators, to the light and 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). The concentrations of hydroxycinnamic acid derivatives, quercetin luteolin and apigenin in leaves of Ligustrum at different sunlight over a Plants under & and in the and sunlight and under in the photosynthetically active radiation UV irradiance, as a was on and in the full sunlight full sunlight and and of individual compounds carried out with a following the of Agati et al. are with different at a the p-coumaric acid and of caffeic acid (i.e. and both and The of changes in sunlight irradiance on the of at nm through the whole of Ligustrum leaves. Plants under & and in the and sunlight and under in the photosynthetically active radiation UV irradiance, as a was on and in the full sunlight, full sunlight and through the leaves the and the leaf with a with and with light at was over a a following the in Tattini et al. the soluble phenylpropanoids in leaves, dihydroxy B-ring-substituted flavonoid glycosides contributed to under The in the molar extinction coefficients of hydroxycinnamates (εmax in from to nm upon with and apigenin derivatives 335 to nm) not the luteolin glycosides (εmax from to nm upon with and quercetin glycosides to nm) absorbance over wavelengths of light-induced changes in leaf may to some extent have the (Agati et al., 2002); which are by glandular trichomes and are with may have contributed to the of the et al., dihydroxy B-ring flavonoids in the epidermal cells are to have contributed to in the leaves, of the steep leaf The are for Nevertheless, the by Hernández et al. regarding the of flavonoids and oxidative and both and The oxidation of ‘UV-absorbing flavonoids’ are to as they are at the of various cell of in at Takahama, 1986). The and of flavonoids are still highly reactive and to species (Pourcel et al., 2006). In healthy leaves, particularly exposed to full sunlight, the of radicals to their may occur acid and are et al., 2000; et al., 2000). of have been in plants from severe stress (Hernández et al., 2006), the could not be that the acid and the of the cell greatly by the of the However, this issue for by species that in or in biosynthesis to high sunlight irradiance and stress has been reported to increase the concentration of particularly the dihydroxy substituted structures of hydroxycinnamates and flavonoids (Grace & Logan, 2000; Hernández et al., 2004; Tattini et al., 2004; et al., 2005). in which excess irrespective of the of different solar wavelengths reaching and penetrating the leaf, enhances the biosynthesis of flavonoids capable of performing multiple at the expense of ‘poor antioxidant’ flavonoids and B-ring-substituted flavonoids, particularly with structures such as are in leaves growing under shade and hence may be flavonoids and hydroxycinnamates are at higher concentrations than flavonols, and are in the epidermal It is that a concentration of leaf a mM concentration in the epidermal monohydroxy B-ring-substituted flavonoids may effectively attenuate UV irradiance and at the same time protect leaf tissues from functions being fully accomplished at a flavonoid concentration in the mM et al., 2008). the preferential accumulation of dihydroxy over monohydroxy flavonoids in response to high light is to reduce photo-oxidative In the other antioxidant mesophyll flavonoids, in the may effectively the generation (e.g. by chelating metal and reduce reactive oxygen such as cells and the highly glandular may be to address the of flavonoids in photoprotection. The of reactive species and and of the of genes in the biosynthesis of antioxidant flavonoid structures in response to various stresses may be carried out at of the of synthase Owens et al., 2008) and flavonoid which the addition of an group to the to the coupled with of the intracellular distribution of antioxidant flavonoids, may help to some regarding the of antioxidant flavonoid biosynthesis in Indeed, recent of of biosynthesis by transcription which are and in the between and stress responses et al., 2006; et al., 2006), may link the potential of the cell to the control of flavonoid accumulation (Taylor & Grotewold, 2005). It may be that oxidative components may flavonoid irrespective of their This hypothesis is consistent with recent that plant phenolics the primary function of protecting leaves from photo-oxidative damage, rather than damage, by as antioxidants & McArthur, 2002); that light-induced ROS generation against et al., that are in the response to and stresses & Paul, 2006; et al., et al., and that a link may between the and activities of flavonoids (Taylor & Grotewold, 2005). These functions appear to be important as they are the are free of & 2005), the in vivo of flavonoids in (Williams et al., 2004) from the found in in how flavonoids may reduce oxidative stress in animal and plant cells, which greatly in the of their structures. It may not be a coincidence that UV-screening compounds to found in plants a role in protecting cells from photo-oxidative