Anthocyanin Function in Vegetative Organs 7 However,UV filtering is unlikely to be the primary role of anthocyanins in leaves.Foliar anthocyanins tend not to be acylated,and are therefore less effective absorbers of UV radiation than are certain other flavonoids(Woodall and Stewart 1998).Moreover,to be an efficient screen,anthocyanins must intercept incident UV radiation before it reaches the chloroplasts (Caldwell et al.1983).In the case of leaves,this means that the pigments should reside in the vacuoles and/or cell walls of epidermal or hypodermal tissues(Day et al.1992:Alenius et al.1995:Gorton and Vogelmann 1996:Olsson et al.1999).In some species,anthocyanins can indeed be found in these superficial foliar tissues More commonly however anthocvanin occur in the vacuoles of the chlorenchyma cells themselves (Wheldale 1916:Gould and Quinn 1999:Gould et al.2000:Lee and Collins 2001).a suboptimal location for UV filtering It was shown recently that the presence of anthocyanins might in the long term be osodtnnigcaeneheDre tal rather than beneficial to nts that face high uv lev els (ada et al 2003).Thea le-leafed rice.anthocyanins absorb a portion of the blue/UV-A radiation that w of DNA repair would offse t any short-t n by eyanins 1.5 Free Radical Scavenging nvironmenta stressutratin light fux or high levels of e produ ra in plant cells (Foye ceadealS 0n0 th quan nthocyanins might serve to radical any cr hemical able c existence that contain one more unpair on (Ha utteridge oxygen radic have b st extensively studi ough there is inc reasing awareness the of nitrogen-centred radical The collective te "reactive oxygen specie (ROS)or active oxygen intermediates"(ROI)is often used to include both the oxygen radicals and non-radical derivatives of oxygen which nave simila chemic properties These include the superoxide radical(O2,hydroxyl radical(OH),peroxy radical (ROO),and alkoxyl radical(RO )as well as the non-radical intermediates such as singlet oxygen (O2),hydrogen peroxide(HO).and ozone(O). In plant cells,chloroplasts and mitochondria are the principal sources of ROS which are generated via the aerobic reactions involved in photosynthesis and respiration (Mittler 2002:Rhoads et al.2006).ROS are also produced in the peroxisomes during photorespiration and fatty acid oxidation(Corpas et al.2001) Enzymatic sources of ROS have been identified,including NADPH oxidase in the plasma membrane(Grant and Loake 2000),oxalate oxidase and amine oxidase in the apoplasm (Allan and Fluhr 1997;Dat et al.2000),and peroxidases in the cell wall (Kawano 2003).Under optimal growth conditions the production of ROS from How ver. ncrease levels of ROS three-fold (Polle 2001)
Anthocyanin Function in Vegetative Organs 7 However, UV filtering is unlikely to be the primary role of anthocyanins in leaves. Foliar anthocyanins tend not to be acylated, and are therefore less effective absorbers of UV radiation than are certain other flavonoids (Woodall and Stewart 1998). Moreover, to be an efficient screen, anthocyanins must intercept incident UV radiation before it reaches the chloroplasts (Caldwell et al. 1983). In the case of leaves, this means that the pigments should reside in the vacuoles and/or cell walls of epidermal or hypodermal tissues (Day et al. 1992; Ålenius et al. 1995; Gorton and Vogelmann 1996; Olsson et al. 1999). In some species, anthocyanins can indeed be found in these superficial foliar tissues. More commonly, however, anthocyanins occur in the vacuoles of the chlorenchyma cells themselves (Wheldale 1916; Gould and Quinn 1999; Gould et al. 2000; Lee and Collins 2001), a suboptimal location for UV filtering. It was shown recently that the presence of anthocyanins might in the long term be detrimental rather than beneficial to plants that face high UV levels (Hada et al. 2003). The authors found that in purple-leafed rice, anthocyanins absorb a portion of the blue/UV-A radiation that would otherwise activate the DNA-repairing enzyme photolyase. Such inhibition of DNA repair would offset any short-term gain from UV absorption by anthocyanins. 1.5 Free Radical Scavenging Environmental stressors such as saturating light flux or high levels of UV radiation can augment the production of free radicals in plant cells (Foyer et al. 1994; Gould 2003). It has been suggested that by absorbing a proportion of the incident quanta, and by scavenging the free radicals thus formed, foliar anthocyanins might serve to abate this oxidative insult. A free radical is any chemical species capable of independent existence that contains one or more unpaired electron (Halliwell and Gutteridge 1999). The oxygen radicals have been most extensively studied in plants, although there is increasing awareness of the roles of nitrogen-centred radicals. The collective term “reactive oxygen species” (ROS) or “reactive oxygen intermediates” (ROI) is often used to include both the oxygen radicals and non-radical derivatives of oxygen which have similar chemical properties. These include the superoxide radical (O2 − ), hydroxyl radical (OH), peroxyl radical (ROO), and alkoxyl radical (RO− ), as well as the non-radical intermediates such as singlet oxygen (1 O2), hydrogen peroxide (H2O2), and ozone (O3). In plant cells, chloroplasts and mitochondria are the principal sources of ROS, which are generated via the aerobic reactions involved in photosynthesis and respiration (Mittler 2002; Rhoads et al. 2006). ROS are also produced in the peroxisomes during photorespiration and fatty acid oxidation (Corpas et al. 2001). Enzymatic sources of ROS have been identified, including NADPH oxidase in the plasma membrane (Grant and Loake 2000), oxalate oxidase and amine oxidase in the apoplasm (Allan and Fluhr 1997; Dat et al. 2000), and peroxidases in the cell wall (Kawano 2003). Under optimal growth conditions the production of ROS from routine metabolic processes is low: 240 μM s−1 O2 − , and a steady-state level of 0.5 μM H2O2 in chloroplasts (Polle 2001). However, environmental stressors can increase levels of ROS three-fold (Polle 2001)
8 J.-H.B.Hatier,K.S.Gould membran and nuc and been cons detrimental fun Guarding against oxidativ damage,plants have evolved elaborate antioxidant defence mechanisms m the different intracellular compartments. These serve to control concentrations of ROS. to improve the plant's resistance to stressors,to repair damage to proteins particularly those in photosystem II,and to re-activate key enzymes(Halliwell and Gutteridge 1999).An antioxidant may be defined as any substance whic wher present at low concentrations compared with those of an oxidisable substrate significantly delays or prevents oxidation of that substrate.The major antioxidants are enzymes,and include superoxide dismutase (SOD),catalase (CAT),various peroxida s such as ascorbate peroxidase (APX),and glutathione reductase (GR) (Polle 1997)There are in addition a number of low molecular weight antioxidants (LMWAs)in plant cells:ascorbate (vitamin C),tocopherols(vitamin E),glutathione, B-carotene,and phenolic compounds such as the flavonoids. Certain flavonoids including the more common anthocvanin pigments have ROS-scavenging capacities up to four times greater than those of vitamin E and C analogues(Rice-Evans et al.1997:Wang et al.1997).Their potency stems from a high reactivity as roton and electron donors,from their ability to stabilize and delocalize unpaired electrons,and from their capacity to chelate transition metal ions (Rice-Evans et al.1996:van Acker et al.1996;Brown et al.1998).Flavonoids have heen shown in vitro to neutralise most of the biologically important ROS and nitroge -centred radicals Recently.compelling evidence was nted for the scavenging of ROS by flavonoids in vivo. Agati et al.(2007)infused leaves of Phillyrea latifolia with DanePy.a fluorochro whose fluore nched exclusively byOz.Microscopi c examinations of cross-sections through thos evealed that the of'O.which had beer ated leaves to strong light.was la table to flay onols and flav associated with chloroplasts in the mesophyll cells Could antioxidan act of anth presenc in tiss s?Two mechanis 69 t xida ves have bee First.by edu the nu bers of high- cident on the neht-d pho thetic cel e ROS Thi prev an eldal 1916) "generat chlorophyll, en I 0n. nage (Ka and Shimiz 985 hloroplasts suspended in a buffered produced fewer h radicals, and were bleached less. when irradiated with monochromatic red light than with white light of comparable intensity. However the benefits of anthocyanin as an optical shield have yet to be demonstrated in situ. Second,anthocyanins might directly scavenge ROS. Anthocvanins are usually colourless or light blue at the pH of the cvtoplasm.but they turn red after being transported into the vacuole.Both the colourless and the red tautomers of cyanidin glycosides have been demonstrated to scavenge O,produced by a suspension of
8 J.-H. B. Hatier, K.S. Gould A superabundance of ROS potentially causes cellular damage to phospholipid membranes, proteins, and nucleic acids, and this has traditionally been considered detrimental to plant functioning (Alscher et al. 1997). Guarding against oxidative damage, plants have evolved elaborate antioxidant defence mechanisms in the different intracellular compartments. These serve to control concentrations of ROS, to improve the plant’s resistance to stressors, to repair damage to proteins, particularly those in photosystem II, and to re-activate key enzymes (Halliwell and Gutteridge 1999). An antioxidant may be defined as any substance which, when present at low concentrations compared with those of an oxidisable substrate, significantly delays or prevents oxidation of that substrate. The major antioxidants are enzymes, and include superoxide dismutase (SOD), catalase (CAT), various peroxidases such as ascorbate peroxidase (APX), and glutathione reductase (GR) (Polle 1997). There are in addition a number of low molecular weight antioxidants (LMWAs) in plant cells: ascorbate (vitamin C), tocopherols (vitamin E), glutathione, β-carotene, and phenolic compounds such as the flavonoids. Certain flavonoids, including the more common anthocyanin pigments, have ROS-scavenging capacities up to four times greater than those of vitamin E and C analogues (Rice-Evans et al. 1997; Wang et al. 1997). Their potency stems from a high reactivity as proton and electron donors, from their ability to stabilize and delocalize unpaired electrons, and from their capacity to chelate transition metal ions (Rice-Evans et al. 1996; van Acker et al. 1996; Brown et al. 1998). Flavonoids have been shown in vitro to neutralise most of the biologically important ROS and nitrogen-centred radicals. Recently, compelling evidence was presented for the scavenging of ROS by flavonoids in vivo. Agati et al. (2007) infused leaves of Phillyrea latifolia with DanePy, a fluorochrome whose fluorescence is quenched exclusively by 1 O2. Microscopic examinations of cross-sections through those leaves revealed that the scavenging of 1 O2, which had been generated by subjecting the leaves to strong light, was largely attributable to flavonols and flavones specifically associated with chloroplasts in the mesophyll cells. Could antioxidant activity explain the presence of anthocyanins in vegetative tissues? Two mechanisms by which anthocyanins might reduce the oxidative load in leaves have been proposed. First, by reducing the numbers of high-energy quanta incident on the photosynthetic cells, anthocyanins might prevent or moderate the light-driven reactions that generate ROS. This is an old concept. Indeed, Wheldale (1916) herself described an experiment in which a solution of chlorophyll, when illuminated behind a glass vessel containing a red solution, remained green for longer than when illuminated behind a colourless solution. Chlorophyll bleaching is a classic symptom of oxidative damage (Kato and Shimizu 1985). More recently, Neill and Gould (2003) showed that chloroplasts suspended in a buffered solution produced fewer O2 − radicals, and were bleached less, when irradiated with monochromatic red light than with white light of comparable intensity. However, the benefits of anthocyanin as an optical shield have yet to be demonstrated in situ. Second, anthocyanins might directly scavenge ROS. Anthocyanins are usually colourless or light blue at the pH of the cytoplasm, but they turn red after being transported into the vacuole. Both the colourless and the red tautomers of cyanidin glycosides have been demonstrated to scavenge O2 − produced by a suspension of
Anthocyanin Function in Vegetative Organs 9 chloroplasts under light stress (Neill and Gould 2003).Clearly,cytosolic anthocyanins would be better located than would vacuolar anthocyanins for scavenging ROS produced by organelles such as chloroplasts,mitochondria,and peroxisomes.However,the question of the common occurrence of anthocyanins in both cytosol and vacuole is one that requires further attention.It remains unclear how anthocyanins are transported to the vacuole from their site of synthesis at the endoplasmic reticulum.If they move to the vacuole by diffusion,then they would transiently pass through the cytosol.Alternatively,there is growing evidence of a route from the endoplasmic reticulum directly into vesicles which then migrate to sing the cytosol (Poustka et al.2007).Irrespective of theirmpletel eatelbe observed under the microscone to remove ho.more swiftly than acvanic cells (Gould et al.2002a).It is possible,therefore,that antioxidant activity may be one of the ns in y ative tis available data contribute to the total antioxidan pool more in some species than For in Flat nd s fro red le tha n did those ich could arily to e presenc of al.2002b nil e canopy plan ant pote Ne 02 map in extra from s cing lea es (v erg and ins 2007 hese differ are n nown,though een suggeste he leaf tissu may be s and Ma hich folar n the effects of methyl vi de,on va 10 in different bits pho synthetic ele genera 1g ad to th of chloroplas membranes The authors claimed tha leaves which anthocyanins were located in the me sistant to methyl viologer than were those that held anthoanin teeermis antioxidant activities were not measured in that study,the data are consistent with the hypothesis that when anthocvanins are located in the mesophyll.they can contribute to the LMWA pool. In addition to the anthocyanins,concentrations of other LMWAs,as well as certain enzymatic antioxidants,can also be higher in red than in green leaves.For example,the red-leafed morphs of Elatostema rugosum had higher levels of caffeic acid derivatives and greater sod and CAT activities than had the green-leafed morphs (Neill et al.2002b).Similarly,leaves of maize cultivars that had been exposed to toxic copper concentrations upregulated the production of anthocyanin as well as the activities of superoxide dismutase,ascorbate peroxidase,and glutathione reductase (Tanyolac et al.2007). Anthocvanins might well supplement the antioxidant potential in such plants,but they clearly do not substitute the major LMWA and enzymatic antioxidants
Anthocyanin Function in Vegetative Organs 9 chloroplasts under light stress (Neill and Gould 2003). Clearly, cytosolic anthocyanins would be better located than would vacuolar anthocyanins for scavenging ROS produced by organelles such as chloroplasts, mitochondria, and peroxisomes. However, the question of the common occurrence of anthocyanins in both cytosol and vacuole is one that requires further attention. It remains unclear how anthocyanins are transported to the vacuole from their site of synthesis at the endoplasmic reticulum. If they move to the vacuole by diffusion, then they would transiently pass through the cytosol. Alternatively, there is growing evidence of a route from the endoplasmic reticulum directly into vesicles, which then migrate to the vacuole, completely bypassing the cytosol (Poustka et al. 2007). Irrespective of their intracellular location, however, anthocyanin-containing leaf cells have been observed under the microscope to remove H2O2 more swiftly than acyanic cells (Gould et al. 2002a). It is possible, therefore, that antioxidant activity may be one of the major functions of anthocyanins in vegetative tissues. The available data indicate that anthocyanins contribute to the total antioxidant pool more in some species than in others. For example, in Elatostema rugosum, a sprawling understorey herb from New Zealand, extracts from red leaves had a significantly greater LMWA activity than did those from green leaves, which could be attributed primarily to the presence of anthocyanins (Neill et al. 2002b). In contrast, in the canopy plant Quintinia serrata, extracts from red and green leaves showed similar ranges in antioxidant potential (Neill et al. 2002c). Similarly, in the sugar maple (Acer saccharum), antioxidant activity correlated strongly with anthocyanin content in extracts from juvenile leaves, but the correlation was only weak in extracts from senescing leaves (van den Berg and Perkins 2007). Reasons for these differences are not known, though it has been suggested that the location of anthocyanic cells within the leaf tissues may be important. Kytridis and Manetas (2006) compared the effects of methyl viologen, a herbicide, on various species for which foliar anthocyanins were located in different cell types. Methyl viologen inhibits photosynthetic electron transport, generating ROS that lead to the destruction of chloroplast membranes. The authors claimed that red leaves for which anthocyanins were located in the mesophyll were more resistant to methyl viologen treatment than were those that held anthocyanin in the epidermis. Although antioxidant activities were not measured in that study, the data are consistent with the hypothesis that when anthocyanins are located in the mesophyll, they can contribute to the LMWA pool. In addition to the anthocyanins, concentrations of other LMWAs, as well as certain enzymatic antioxidants, can also be higher in red than in green leaves. For example, the red-leafed morphs of Elatostema rugosum had higher levels of caffeic acid derivatives and greater SOD and CAT activities than had the green-leafed morphs (Neill et al. 2002b). Similarly, leaves of maize cultivars that had been exposed to toxic copper concentrations upregulated the production of anthocyanin as well as the activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase (Tanyolaç et al. 2007). Anthocyanins might well supplement the antioxidant potential in such plants, but they clearly do not substitute the major LMWA and enzymatic antioxidants
10 J.-H.B.Hatier,K.S.Gould 1.6 Paradigm Shift Recent genetic studies have indicated that the potential for ros to cause unrestricted damage to plant cell components is realised far less commonly than had been eviousy Ros may serve many useful functonpded Ros contrary there is a growing body of empirical evidence to be actively produced by plant cells for their use as signalling molecul in processes as di e as or th and dovelor ent stomatal clo 005) grammed cell death.and abiotic stres This evide ce led Foyer Noctor "The nt ha to th enconc of Thev which RoS are ad an xidativ allin and c sociated mecha :by adjustment exp The (Dat t0 role for ROS have been re2003 2 2004 Ape 2004 Mitler et al e for Among comp Wagner et a on the f en transferred from ng【och rophyl or to th arrest of grov more matur However,when a ingle gene. EXECUTERI,is inactivated in this species (as in the flu/executerl double mutant),both seedlings and mature plants grov despite the continued production ofO2. The authors concluded that in wild type plants. 'O2 does not damage cellular components directly,but rather, genetic switch that initiates a signalling cascade leading to programmed cell suicide ROS-induced programmed cell death may be useful for plants facing pathogenic attack since it potentially limits the spread of disease from the point of infection However,cell death would probably not be beneficial under conditions of abiotic stress such as those imposed by strong light and elevated UV-B. Mittler (2002 argued that the steady state levels of ROS may be used by plants as a gauge of intracellular stress.When levels of ROS rise in response to abiotic stress,plants face the challenge of removing excess ROS to avoid programmed cell death,yet retaining sufficient low levels of the different types of ROS for signalling purposes.This would require the finely-tuned modulation of ROS production and scavenging mechanisms. Specificity in response may be achievable by the coordinated production of LMWAs such as ascorbate and glutathione(Foyer and Noctor 2005). The flavonoids,too,seem likely to play a role in this. 1.7 Modulation of Signalling Cascades:A New Hypothesis That ROS can be at once the products of plant stress as well as mediators in plant stress responses presents the possibility for a new functional hypothesis for the
10 J.-H. B. Hatier, K.S. Gould 1.6 Paradigm Shift Recent genetic studies have indicated that the potential for ROS to cause unrestricted damage to plant cell components is realised far less commonly than had been previously thought. On the contrary, there is a growing body of empirical evidence to suggest that ROS may serve many useful functions in plants. Indeed, ROS appear to be actively produced by plant cells for their use as signalling molecules in processes as diverse as growth and development, stomatal closure, pathogen defence, programmed cell death, and abiotic stress responses. This evidence led Foyer and Noctor (2005) to state, “The moment has come to re-evaluate the concept of oxidative stress.” They proposed that the processes by which ROS are generated and scavenged might better be described as “oxidative signalling”, and should be regarded as “an important and critical function associated with the mechanisms by which plant cells sense the environment and make appropriate adjustments to gene expression, metabolism and physiology.” The arguments in favour of a signalling role for ROS have been expertly summarised in several reviews (Dat et al. 2000; Mittler 2002; Vranová et al. 2002; Mahalingam and Fedoroff 2003; Apel and Hirt 2004; Laloi et al. 2004; Mittler et al. 2004; Foyer and Noctor 2005; Pitzschke et al. 2006). Among the more compelling lines of evidence for ROS signalling is the work by Wagner et al. (2004) on the flu mutant of Arabidopsis thaliana. The flu mutant, when transferred from darkness to the light, generates singlet oxygen in the plastids, ultimately leading to chlorophyll bleaching and death of seedlings, or to the arrest of growth in more mature plants. However, when a single gene, EXECUTER1, is inactivated in this species (as in the flu/executer1 double mutant), both seedlings and mature plants grow normally despite the continued production of 1 O2. The authors concluded that in wild type plants, 1 O2 does not damage cellular components directly, but rather, activates a genetic switch that initiates a signalling cascade leading to programmed cell suicide. ROS-induced programmed cell death may be useful for plants facing pathogenic attack since it potentially limits the spread of disease from the point of infection. However, cell death would probably not be beneficial under conditions of abiotic stress such as those imposed by strong light and elevated UV-B. Mittler (2002) argued that the steady state levels of ROS may be used by plants as a gauge of intracellular stress. When levels of ROS rise in response to abiotic stress, plants face the challenge of removing excess ROS to avoid programmed cell death, yet retaining sufficient low levels of the different types of ROS for signalling purposes. This would require the finely-tuned modulation of ROS production and scavenging mechanisms. Specificity in response may be achievable by the coordinated production of LMWAs such as ascorbate and glutathione (Foyer and Noctor 2005). The flavonoids, too, seem likely to play a role in this. 1.7 Modulation of Signalling Cascades: A New Hypothesis That ROS can be at once the products of plant stress as well as mediators in plant stress responses presents the possibility for a new functional hypothesis for the
Anthocyanin Function in Vegetative Organs 11 presence of anthocyanins in vegetative tissues.We propose that the anthocyanins along with some other flavonoids,provide multifarious mechanisms for the modulation of signalling cascades that mitigate the effects of abiotic and biotic stressors.As explained below,this role is achievable in three interrelated ways:(i) by protecting antioxidant enzymes;(ii)by scavenging ROS directly;and (iii)by interactions with other molecules in the signal transduction pathways. Many of the putative roles of anthocvanins in plant physiology could equally be achieved by antioxidant enzymes.For example,like the anthocyanins,the ROS- ging enzymes of the so-called "water-water cycle"(SOD and APX) chlo sts result in a reduced pr nsity for photoinhibition and photo-oxidation (Asada 1999.2000:Rizhsky et al.2003).These enzymes scavenge O,and H.O with extreme efficiency.and are undoubtedly key plavers in the modulation of ROS signalling cascades.Under certain conditions how er thes enzymes may be activated.Stron light combined with chilling for ex reduces the efficie of APx leading to the inactiv APX SOD.and CAT (ahnke et al.1991:Wise 1995:Casan et al.1997:Streb e t al 1997 Asada 1999)For the to function properly,its enzym ti antioxidants need to be tected fro dical attack It is coincidenc that lead to the at ant is nth n enzyme may also timula e the on of f ROS) of ligh ereb edu erary RO、apa efor me from ivat fo product s,plants migh hi ac ong-tem prote the compon Indeed,the capa city of plants to mai ntain o the enzym as a fplant tissue stre (Bo al.199 Pinheroet al.1997 Scebba et al.19 Kuk et al.2003). It is possible that anthocyanins interact with stress signal transduction cascades more directly. This has been demonstrated already in human tumour cells;two anthocyanin aglycones,cyanidin and delphinidin,were found to inhibit tumour cell growth by shutting off downstream signalling cascades that would otherwise lead to the production of growth factors(Meiers et al.2001).Interactions between phenolic compounds and ROS signalling have also been documented for plants.For example the softening of plant cell walls.which is necessary for cell expansion.results partly from OH radical attack on cell wall polysaccharides(Fry 1998),and is terminated by the cross-linking of phenolic compounds (Rodriguez et al. 2002). anthocyanins can scavenge a variety of free radicals and oxidants such as H2Oz (Takahama 2004),they have the potential directly to influence the balance between ROS production and ROS scavenging in stress responses.H2O2 is considered a particularly important molecule in plant signalling because of its relative stability,as well as its ability to diffuse rapidly across membranes and between different cell compartments (Droge 2002:Neill et al.2002a).H2O2 is a known activator of MAP kinase cascades,and has been shown to regulate the expression of certain genes
Anthocyanin Function in Vegetative Organs 11 presence of anthocyanins in vegetative tissues. We propose that the anthocyanins, along with some other flavonoids, provide multifarious mechanisms for the modulation of signalling cascades that mitigate the effects of abiotic and biotic stressors. As explained below, this role is achievable in three interrelated ways: (i) by protecting antioxidant enzymes; (ii) by scavenging ROS directly; and (iii) by interactions with other molecules in the signal transduction pathways. Many of the putative roles of anthocyanins in plant physiology could equally be achieved by antioxidant enzymes. For example, like the anthocyanins, the ROSscavenging enzymes of the so-called “water-water cycle” (SOD and APX) in the chloroplasts result in a reduced propensity for photoinhibition and photo-oxidation (Asada 1999, 2000; Rizhsky et al. 2003). These enzymes scavenge O2 − and H2O2 with extreme efficiency, and are undoubtedly key players in the modulation of ROS signalling cascades. Under certain conditions, however, these enzymes may be inactivated. Strong light combined with chilling stress, for example, reduces the efficiency of APX, leading to the accumulation of H2O2 to levels can inactivate APX, SOD, and CAT (Jahnke et al. 1991; Wise 1995; Casano et al. 1997; Streb et al. 1997; Asada 1999). For the water-water cycle to function properly, its enzymatic antioxidants need to be protected from free-radical attack. It is perhaps no coincidence that the very conditions that can lead to the inactivation of such enzymes may also stimulate the formation of anthocyanins in plant tissues. Anthocyanins may prevent the inactivation of antioxidant enzymes by restricting the amount of light within a photosynthetic cell (thereby reducing the production of ROS). The strong antioxidant capacities of anthocyanins mean that they could also scavenge supernumerary ROS and therefore spare the antioxidant enzymes from inactivation. Thus, for a one-time investment in the production of anthocyanin pigments, plants might achieve the long-term protection of these pivotal components of plant stress responses. Indeed, the capacity of plants to maintain or enhance their antioxidant enzyme activities is regarded as a key feature in the acclimation of plant tissues to environmental stress (Bowler et al. 1992; Anderson et al. 1995; Pinhero et al. 1997; Scebba et al. 1999; Kuk et al. 2003). It is possible that anthocyanins interact with stress signal transduction cascades more directly. This has been demonstrated already in human tumour cells; two anthocyanin aglycones, cyanidin and delphinidin, were found to inhibit tumour cell growth by shutting off downstream signalling cascades that would otherwise lead to the production of growth factors (Meiers et al. 2001). Interactions between phenolic compounds and ROS signalling have also been documented for plants. For example, the softening of plant cell walls, which is necessary for cell expansion, results partly from OH radical attack on cell wall polysaccharides (Fry 1998), and is terminated by the cross-linking of phenolic compounds (Rodríguez et al. 2002). Because anthocyanins can scavenge a variety of free radicals and oxidants such as H2O2 (Takahama 2004), they have the potential directly to influence the balance between ROS production and ROS scavenging in stress responses. H2O2 is considered a particularly important molecule in plant signalling because of its relative stability, as well as its ability to diffuse rapidly across membranes and between different cell compartments (Dröge 2002; Neill et al. 2002a). H2O2 is a known activator of MAP kinase cascades, and has been shown to regulate the expression of certain genes