Introduction

Flavonoids, the vast class of secondary metabolites encompassing more than 10,000 structures (Harborne and Williams 2000), have long been reported to display wide range of uses in plant-environment interactions (Winkel-Shirley 2002; D'Auria and Gershenzon 2005; Agati and Tattini 2010) . In the recent past, the idea behind functioning of flavonoids primarily as attenuators of short solar wavelengths in plants exposed to UV-B or full solar irradiance has been questioned (Gerhardt et al. 2008; Agati and Tattini 2010; Agati et al. 2011; Akhtar et al. 2010), as fla-vonoids most responsive to UV-radiation are far from being the most effective UV-B absorbers among the thousands of polyphenol structures (Cockell and Knowland 1999; Harborne and Williams 2000). These suggestions are consistent with the early authoritative views of Swain (1986) and Stafford (1991) of flavonols, the most ancient and widespread of the flavonoids (Rausher 2006; Winkel 2006), having served key antioxidant or "internal regulatory" functions during the evolution of early terrestrial plants.

The biosynthesis of flavonoids is upregulated not only as a consequence of UV-radiation but also in response to a wide range of other abiotic (and biotic) stresses, ranging from nitrogen/phosphorus depletion to cold and salinity/drought stress (Tattini et al. 2004, 2005; Lillo et al. 2008; Olsen et al. 2009; Agati et al. 2011). Since different stresses have in common the generation of reactive oxygen species (Mittler et al. 2004; Mittler 2006), it has been postulated that fla-vonoids are synthesized to effectively counter the stress-induced oxidative damage. Flavonoids may accomplish their antioxidant functions by both preventing the generation of ROS (through their ability to chelate transition metal ions such as Fe and Cu, Brown et al. 1998; Melidou et al. 2005; Hernández et al. 2009; Agati and Tattini 2010) and scavenging ROS once formed (Ryan et al. 2002; Babu et al. 2003; Tattini et al. 2004; Agati et al. 2007; Jaakola and Hohtola 2010).

How flavonoids may perform their scavenging activity in an in vivo condition is still a matter of conflict, and major criticisms regarding the localization functional relationships (Halliwell 2009; Hernández et al. 2009) have been only partially addressed (Yamasaki et al. 1997; Agati et al. 2007; Agati and Tattini 2010; Akhtar et al. 2010) . However, we note that flavonoids do not exclusively occur in the vacuoles of epidermal cells (as long been reported, Saunders and Mc Clure 1976), and hence far enough from the sites of ROS production. Relatively recent experiments have reported a large accumulation of fla-vonoids in mesophyll cells both in the vacuole (Agati et al. 2002, 2009, 2011; Gould et al. 2002; Tattini et al. 2005, 2006; Kytridis and Manetas 2006) and in the chloroplasts (Agati et al. 2007). This subcellular distribution of flavonoids is, therefore, consistent with a their putative role as ROS-quenchers. Chloroplast flavonoids (chloro-plasts have been reported to be both capable of flavonoid biosynthesis and may represent an important site of flavonoid accumulation), (Oettmeier and Heupel 1972; Saunders and Mc Clure 1976; Takahama 1982; Takahama and Oniki 1997; Zaprometov and Nikolaeva 2003) have been shown to effectively quench singlet oxygen generated upon excess blue-light irradi-ance (Agati et al. 2007) . A vacuolar distribution of mesophyll flavonoids may be of key significance in reducing hydrogen peroxide (H2O2) that may freely escape from the chloroplast under severe stress conditions, as also hypothesized to occur for flavonoids located in the vacuoles of epidermal cells (Yamasaki et al. 1997; Sakihama et al. 2000). Nevertheless, the matter is far being conclusively elucidated and poses serious concerns from an analytical and technical point of view, mostly concerning the simultaneous visualization of reactive oxygen species and flavonoid distribution at inter and intracellular levels (Hernández et al. 2009; Agati and Tattini 2010).

We highlight that even though polyphenols, particularly flavonoids have long been shown to scavenge various forms of reactive oxygen "in vitro", the flavonoids usually encountered in plants, e.g., in leaf tissues, are the glycosylated structures, as glycosylation both increases the solubility of carbon-based metabolites in an aqueous cellular milieu and preserve the most reactive

Fig. 9.1 Scheme of the main flavonoid pathway leading II flavone synthase I and II, F3'H flavonoid 3'-hydroxy-

to the most common flavanones, flavones, dihydrofla- lase, FHT flavanone 3b-hydroxylase, FLS flavonol syn-

vonols, flavonols, and flavonol glycosides. Enzymes - thase, GT glycosyl transferases CHS chalcone synthase, CHI chalcone isomerase, FNS I/

Fig. 9.1 Scheme of the main flavonoid pathway leading II flavone synthase I and II, F3'H flavonoid 3'-hydroxy-

to the most common flavanones, flavones, dihydrofla- lase, FHT flavanone 3b-hydroxylase, FLS flavonol syn-

vonols, flavonols, and flavonol glycosides. Enzymes - thase, GT glycosyl transferases CHS chalcone synthase, CHI chalcone isomerase, FNS I/

functional groups from autooxidation (Pearse et al. 2005). Actually, few glycosylated flavonoids are effective antioxidants, whereas most flavonoid aglycones (i.e., lacking the sugar moiety) are actually capable of quenching ROS (Rice-Evans et al. 1996) . Quercetin 3-O- and luteolin 7-O-glycosides that posses a catechol group (ortho-dihydroxy B-ring substitution, Fig. 9.1) in the B-ring of the flavonoid skeleton, but not kaempferol 3-O- or apigenin 7-O-glycosides, display an appreciable antioxidant activity, in the molar concentration-range likely encountered in plant cells (Tattini et al. 2004).

Noticeably, stress-responsive flavonoids have the greatest antioxidant potential, and the ratio of "effective antioxidant" to "poor antioxidant" fla-vonoids has been conclusively shown to increase steeply in response to a plethora of abiotic stresses (Kolb et al. 2001; Schmitz-Hoerner and Weissenbock 2003; Tattini et al. 2004; Lillo et al. 2008; Kotilainen et al. 2008; Jaakola and Hohtola

2010; Agati et al. 2009, 2011). The issue of "how significant are flavonoids as antioxidants in plants" has been recently explored by Hernández et al. (2009): these authors have suggested of minor significance the contribution of flavonoids to the highly integrated and constitutive antioxi-dant defense system (which includes antioxidant enzymes, ascorbic acid, and glutathione) operating in plants suffering from various abiotic stresses (as early authoritatively suggested by Halliwell 2009) .

Nevertheless, the first line of defense against stress-induced enhancement in ROS concentration - the antioxidant enzymes - have been reported to be ineffective to protect cells from oxidative damage during severe stress conditions (Polle 2001; Apel and Hirt 2004; Hatier and Gould 2008). Hatier and Gould (2008) have recently suggested that the very conditions that lead to the accumulation of flavonoids are those that may inactivate key antioxidant enzymes

(Casano et al. 1997; Streb et al. 1997). In other words, the actual significance of stress-responsive "antioxidant" flavonoids in the context of the well-coordinated antioxidant defenses operating in stressed plants has to be explored on the basis of the stress severity at which plants are faced with.

Flavonoids with the greatest antioxidant potential have the additional capacity to inhibit the polar auxin transport (PAT, Jacobs and Rubery 1988; Brown et al. 2001; Besseau et al. 2007; Peer and Murphy 2007), and, hence, capable of regulating the development of individual organs and the whole plant (Taylor and Grotewold 2005; Lazar and Goodman 2006; Agati and Tattini 2010). Flavonoids have long been shown to inhibit the activity of PIN and MDR P-glycoproteins, that regulate the cellular auxin homeostasis and the cell-to-cell auxin transport (Geissler et al. 2005; Peer and Murphy 2007; Friml and Jones 2010). In this regard, flavonoids do not perform any reducing activity (i.e., antioxidant activity sensu stricto), but the chemical features that confer the antioxidant potential are required to effectively interact with the auxin transport proteins (Peer and Murphy 2006; Agati and Tattini 2010) ; These "internal regulatory" or "physiological" functions of flavonoids, early hypothesized by Stafford (1991) as the most prominent i n planta, have to be regarded with special attention. In fact, an increasing body of evidence suggests that the health-promoting effect of flavonoids in mammals depends not only upon their ability to scavenge a wide array of reactive oxygen species - free radicals and H2O2 - but on their affinity with several proteins (including the mitogen activated protein kinases, MAPK) that supersede key steps of cell growth and differentiation (Williams et al. 2004; Taylor and Grotewold 2005; Peer and Murphy 2006; Lamoral-Theys et al. 2010). Flavonoids might behave, therefore, as "signaling molecules" (Peer and Murphy 2006) or "developmental regulators" (Taylor and Grotewold 2005) in plants, as well as in animals, and their functional roles going well beyond their ability to merely scavenge reactive oxygen.

In this article, we (1) review the pertinent literature on the effect of most common abiotic stresses on the biosynthesis of "UV-absorbing" flavonoids (2) discuss on the potential contribution of flavonoids in the antioxidant machinery of plants under severe stress conditions and (3) their functional roles in the control of plant growth.

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