Stafford (1991) raised serious concerns about the primary UV-B screening function served by flavonoids during the evolution of early terrestrial plants. She speculated that the concentration of flavonoids would have been very low in liverworts and mosses, because ‘early’ (in the sense proposed by
Rausher, 2006) flavonoid enzymes were not as efficient as current enzymes at constituting an effective filter against UV-B irradiance.
Agati and Tattini (2010) have recently noted that a leaf flavonoid concentration as low as a few micromoles, on a dry mass basis, may result in a much greater concentration, on a molar basis, in the epidermal cells, as actually required for constituting an effective shield against the UV-B wavelengths (
Edwards et al., 2008). Nevertheless, a primary UV-B screening function for flavonols in the photoprotection of early land plants is actually questionable for several reasons (
Winkel-Shirley, 2002).
Early terrestrial plants lost the mycosporin-like amino acid (MAA) in favour of flavonol metabolism, although MAAs are more effective than flavonols in absorbing the short solar wavelengths reaching the leaf surface.
Cockell and Knowland (1999) argued that UV-screening flavonoids evolved from other physiological roles to later fulfil a UV screening function, probably following the evolution of different branches of both the general phenylpropanoid (which may lead, for example, to the synthesis of effective UV-absorbers, such as acylated flavonoids;
Strack et al., 1988;
Harborne and Williams, 2000;
Tattini et al., 2007) and the flavonoid biosynthetic branch pathways. Their suggestion is consistent with the ancient class of flavonols, particularly the dihydroxy B-ring quercetin derivatives (the almost ubiquitous flavonoid in higher plants) having molar extinction coefficients in the 290–390 nm spectral region, 35 % smaller than that of monohydroxy B-ring flavones, such as derivatives of apigenin (
Tattini et al., 2004).
It may not be a mere coincidence that UV-B-responsive flavonols display the greatest antioxidant potential, but not the greatest UV-B-attenuating capacity (
Harborne and Williams, 2000;
Ryan et al., 2002;
Tattini et al., 2004;
Gerhardt et al., 2008).
Stafford (1991) argued that the epidermal cells, the vacuole of which has long been reported (erroneously) to be the exclusive site of flavonoid accumulation, themselves have to be protected, not only aimed at preserving the underlying (sensitive) tissues from photo-oxidative damage. Her suggestion is strongly corroborated by the steep increase in the ratio of dihydroxy B-ring-substituted flavonoids (which display
min in the 290–320 nm spectral region) to hydroxycinnamates (
max between 290 and 320 nm) in tissues and organs exposed to the greatest flux of UV-B radiation (
Olsson et al., 1999;
Tattini et al., 2000;
Agati et al., 2002).
Tattini et al. (2000) and
Agati et al. (2002) suggested that in highly specialized glandular trichomes of
Phillyrea latifolia, which are autonomous in phenylpropanoid biosynthesis, the exclusive UV-induced accumulation of flavonoids (namely, dihydroxy B-ring-substituted quercetin 3-
O-glycosides and luteolin 7-
O-glycosides), apparently at the expense of caffeic acid derivatives, was primarily for protecting glandular trichomes from oxidative damage, while losing the greatest effectiveness in screening out the highly energetic solar short wavelengths from reaching the underlying tissues.
The capacity of flavonoids to inhibit the generation of ROS (through the complexation of Cu and Fe ions, which may lead to the catalytic production of both the hydroxyl radical and the hydroxyl anion, in the well-known Fenton/Haber–Weiss reactions; see
Hernández et al., 2009) and to reduce ROS, once formed, was considered of key value during the colonization of land by plants (
Swain, 1986). Swain's idea conforms to (1) radiation and desiccation, common themes in early land plant evolution, imposing a very severe oxidative stress (
Rothschild and Mancinelli, 2001); and (2) the ancient class of flavonols displaying an effective antioxidant capacity (
Winkel-Shirley, 2002). The presence of the OH group in the 3-position of the flavonoid skeleton (Fig. ) is the key structural feature responsible for the peculiar ability of flavonols to chelate transition metal ions, and, hence, to inhibit the generation of free radicals, as well as to reduce ROS once formed (
Rice-Evans et al., 1996;
Brown et al., 1998;
Melidou et al., 2005;
Agati et al., 2007). Nevertheless, the flavonols usually found in leaf tissues are the glycosylated forms, so that the most reactive/antioxidant group (the OH group in the 3-position in the A-ring of the flavonoid skeleton) is actually ‘silenced’ (Fig. ). Noticeably, in response to various environmental stimuli (
Gerhardt et al., 2008;
Lillo et al., 2008;
Jaakola and Hohtola, 2010), plants almost exclusively synthesize quercetin 3-
O-glycosides, in which the presence of a catechol group in the B-ring of the flavonoid skeleton is responsible for the superior capacity to chelate transition metal ions and to reduce various forms of ROS, as compared with monohydroxy B-ring-substituted flavone or flavonol glycosides (Fig. 1;
Tattini et al., 2004;
Melidou et al., 2005;
Agati et al., 2009).
It is worth noting that the whole set of genes responsible for the biosynthesis of quercetin derivatives –
CHS,
CHI,
F3H,
FLS and
F3'H (encoding chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonol synthase and flavonoid 3'-hydroxylase, respectively) – was already present in liverworts and mosses (Fig. 1;
Markham, 1988;
Rausher, 2006). Interestingly, these early/old genes (
sensu Rausher, 2006) are induced early by high light, at least in
Arabidopsis (
van Tunen et al., 1988;
Vanderauwera et al., 2005), and are the most responsive genes in current-day plants suffering from a wide range of environmentally induced oxidative damage (Fig. 1;
Walia et al., 2005;
Hannah et al., 2006;
Lillo et al., 2008;
Olsen et al., 2009;
Akhtar et al., 2010;
Agati et al., 2011). R2R3 MYB transcription factors, which control the biosynthesis of flavonols, were already present in mosses, are strongly induced by UV-B radiation and are themselves controlled by changes in cellular redox homeostasis (
Rabinowicz et al., 1999;
Heine et al., 2006;
Falcone Ferreyra et al., 2010).
R2R3 Myb genes have been proposed as having been involved in the protection of early land plants from pathogens (Rabinowicz, 1999), but new evidence leads to hypothesizing for them other regulatory functions, through the flavonol-mediated control of plant form and, possibly, of ROS homeostasis (
Close and McArthur, 2002;
Taylor and Grotewold, 2005;
Fujita et al., 2006;
Dubos et al., 2010). The observation that the flavonol metabolic pathway has remained intact for millions of years is consistent with natural selection having favoured secondary metabolites with multiple functional roles to protect plants from unpredictable injuries of different origin (
Izhaki, 2002). We therefore conclude that the flavonol biosynthetic branch pathway represents a robust character in land plants, as having conferred adaptability to species in an ever-changing environment, over an extraordinarily extended time scale (
Lesne, 2008).
Stafford (1991) also hypothesized flavonoids as having served an ‘internal’ function during the evolution of early land plants, based upon their ability to inhibit polar auxin transport (PAT;
Jacobs and Rubery, 1988), a role fully accomplished by flavonols in the manomolar range. This issue has been explored in depth during the last decade (for reviews, see
Peer and Murphy, 2007;
Buer et al., 2010), and flavonols have been conclusively shown to behave as endogenous regulators of auxin movement, at the inter- and intracellular level.
Arabidopsis mutants defective in the first enzyme of flavonoid biosynthesis, CHS, display phenotypes with altered growth (
Brown et al., 2001;
Buer and Muday, 2004;
Besseau et al., 2007). It is noted that quercetin is a much more potent inhibitor of PAT as compared with kaempferol (
Jacobs and Rubery, 1988), as a consequence of a greater ability to inhibit the activity of protein kinases (
DeLong et al., 2002) – which is conferred by the catechol group in the B-ring of the flavonoid skeleton – and, hence, of both PIN and MDR-glycoproteins (multidrug-resistant proteins), the auxin efflux facilitator proteins (
Peer et al., 2004;
Geisler et al., 2005;
Bandyopadhyay et al., 2007).
Jansen et al. (2001) have suggested that the widely reported UV-B-induced increase in the quercetin to kaempferol ratio may offer protection against UV-B stress, as a consequence of the contrasting effects of the two flavonols on the peroxidase-mediated oxidation of indole acetic acid (IAA). Quercetin is an inhibitor and kaempferol is a cofactor of IAA oxidase (
Furuya et al., 1962), and flavonols might have served these ancestral functions to regulate the levels of free IAA in early land plants, such as in the liverworts (
Cooke et al., 2002).
Recently,
Friml and Jones (2010) have reported that PIN5, an atypical member of the PIN protein family, is associated with the endoplasmic reticulum (ER), the putative site of flavonoid biosynthesis (Fig. ). The finding that these ‘short’ PIN proteins (which also include PIN6 and PIN8), which have been suggested to mediate intracellular auxin homeostasis (
Mravec et al., 2009), were the only PINs present in mosses is consistent with Stafford's idea of flavonoids as physiological regulators during the evolution of early terrestrial plants (Fig. ), although a direct effect of flavonols on the activity of short PINs has not been proven yet. The new evidence of ER-located PINs also addresses the important question of ‘how much free flavonoids remains in the cytoplasm to modulate the trafficking or the activity of auxin transporters’ posed by
Taylor and Grotewold (2005). The ‘long’ PIN and MDR-P glycoproteins that act in concert at the plasma membrane (PM) to regulate the cell–cell movement of auxin (Fig. 1;
Geisler et al., 2005,
Titapiwatanakum et al., 2009) and, hence, basipetal auxin transport, occurred at a later stage during the evolution of land plants (
Friml and Jones, 2010).
Actually, flavonols are good candidates to affect greatly the stress-induced redistribution of growth, the so-called ‘flight’ strategy of sessile organisms (
Potters et al., 2009). Stress-induced morphogenic responses (
Potters et al., 2007) have been reported to reflect molecular processes, such as increased ROS production and altered phytohormone transport and metabolism, which can be tightly controlled by the stress-responsive antioxidant flavonols (
Peer and Murphy, 2006;
Pritzsche and Hirt, 2006;
Beveridge et al., 2007).
Thibaud-Nissen et al. (2003) have suggested that flavonoids play a role in the regulation of the redox activity associated with the induction of cell division and somatic embryogenesis. We note that antioxidant flavonols in the high nanomolar to low micromolar concentration range may perform these regulatory functions – which depend upon their ‘antioxidant structure’, but go beyond their mere ability to scavenge ROS (Fig. ) – as earlier speculated by
Stafford (1991).
Nevertheless, how the control exerted by flavonols on auxin movement directly translates to developmental events at the level of the whole plant is still to be explored in depth and little, merely correlative, evidence has been shown for
Arabidopsis only (
Besseau et al., 2007;
Buer and Djordjevic, 2009). This complex issue will be unlikely to be addressed simply by analysing the growth responses of
Arabidopsis mutants lacking or not the ability to synthesize flavonols, particularly when grown under unnatural sunlight irradiance (
Jansen, 2002). Indeed, high sunlight induces the synthesis of both auxin and quercetin derivatives, and increases the activity of phenol-oxidizing peroxidases (
Jansen et al., 2001;
Friml, 2003;
Buer and Muday, 2004;
Besseau et al., 2007). Quercetin displays a great capacity for fine regulating auxin gradients as well as the local auxin concentrations – by inhibiting PAT and peroxidase-mediated IAA oxidation – that represent the actual determinants for different morphological responses (
Jansen, 2002), such as the outgrowth of axillary buds (
Bennet et al., 2006;
Dun et al., 2006;
Lazar and Goodman, 2006). Actually, low doses of UV-B irradiance have been reported to alter the whole-plant architecture profoundly, with more axillary branching being associated with an increase in UV-B-absorbing compounds (
Hectors et al., 2007). Above-ground biomass production and leaf size have been shown to correlate negatively with both the quercetin glycoside concentration and the ratio of quercetin to kaempferol in
Trifolium repens, and ecotypes with a constitutively superior quercetin concentration were more resistant to both UV-B and drought stresses than the fast-growing ecotypes (
Hofmann et al., 2001;
Hofmann and Jahufer, 2011).
This article has been 
DAY TERRESTRIAL PLANTS: OLD HYPOTHESES AND NEW EVIDENCE FOR THEIR FUNCTIONAL ROLES