Role of Anthocyanins in Plant Defence 23 Indeed,Armbruster (2002)suggested that plants fo ocyanins are deployed in the defence of veetative be the more to use anthocyanins to attract pollinators seed dispersers Combinations of traits that simultaneously enhance both pollination d likelyo disproportionate fitness advantage(Herrera t 20 synergistic gains may act for a quicker and more common evolution of the red plant organ colour trait. 2.2 Hypotheses Hypotheses for an anti-herbivory function of anthocyanic plant organs include (i)aposematism (conspicuous colouration serving to parry predators)in poisonous fruits and seeds(Cook et al.1971;Harborne 1982;Williamson 1982),flowers (Hinton 1973),and thorns (Lev-Yadun 2001,2003a,2003b, 2006) (ii)mimicry of dead or foliage(Stone 1979:Jun per 1994),of thorns (. dun 200 of ants,aphids,and poisonous (ii)camouflage of seeds against the background of the soil substrate (Saracino et al.1997.2004).and of variegated foliage in forest understory herbs (Givnish 1990): (iv)the undermining of herbivorous insect crypsis by leaf variegation (Lev- Yadun et al.2004:Lev-Yadun 2006); (v)attraction of herbivores to young,colourful leaves,diverting them from the more costly older leaves(Luttge 1997)and (vi)signalling to insects by red autumn leaves that the trees are well defended (Archetti 2000;Hamilton and Brown 2001;Schaefer and Rolshausen 2006). Each of these hypotheses is discussed in detail belov 2.3 Reluctance to Accept Hypotheses on Defensive Colouration Prior to the much of the published infomat tion on defens ding anthe yanin-ba. had n largel nented in his semina about the ility of defensive colouration operating in botanists have b een surprisingly relucta nt to accept com onplace for zoologists apers or ion in otany as compared to those in zoology was highlight ography on mimicry and aposematism by Komarek (1998).It should be appreciated,however,that that it has taken oologists more than a century to understand the defensive role and the genetic mechanisms of pigmentation in animals (Hoekstra 2006):the effort needed to achieve the same progress in botany
Role of Anthocyanins in Plant Defence 23 selected for by several agents. Indeed, Armbruster (2002) suggested that plants for which anthocyanins are deployed in the defence of vegetative organs would be the more likely also to use anthocyanins to attract pollinators and seed dispersers. Combinations of traits that simultaneously enhance both pollination and defence would likely confer a disproportionate fitness advantage (Herrera et al. 2002). Such synergistic gains may act for a quicker and more common evolution of the red plant organ colour trait. 2.2 Hypotheses Hypotheses for an anti-herbivory function of anthocyanic plant organs include: (i) aposematism (conspicuous colouration serving to parry predators) in poisonous fruits and seeds (Cook et al. 1971; Harborne 1982; Williamson 1982), flowers (Hinton 1973), and thorns (Lev-Yadun 2001, 2003a, 2003b, 2006); (ii) mimicry of dead or senescing foliage (Stone 1979; Juniper 1994), of thorns and spines (Lev-Yadun 2003a), and of ants, aphids, and poisonous caterpillars (Lev-Yadun and Inbar 2002); (iii) camouflage of seeds against the background of the soil substrate (Saracino et al. 1997, 2004), and of variegated foliage in forest understory herbs (Givnish 1990); (iv) the undermining of herbivorous insect crypsis by leaf variegation (LevYadun et al. 2004; Lev-Yadun 2006); (v) attraction of herbivores to young, colourful leaves, diverting them from the more costly older leaves (Lüttge 1997); and (vi) signalling to insects by red autumn leaves that the trees are well defended (Archetti 2000; Hamilton and Brown 2001; Schaefer and Rolshausen 2006). Each of these hypotheses is discussed in detail below. 2.3 Reluctance to Accept Hypotheses on Defensive Colouration Prior to the year 2000, much of the published information on defensive plant colouration, including anthocyanin-based ones, had been largely anecdotal. As Harper (1977) commented in his seminal book about the possibility of defensive colouration operating in plants, botanists have been surprisingly reluctant to accept ideas that are commonplace for zoologists. The relative scarcity of papers on defensive colouration in botany as compared to those in zoology was highlighted in the annotated bibliography on mimicry and aposematism by Komárek (1998). It should be appreciated, however, that that it has taken zoologists more than a century to understand the defensive role and the genetic mechanisms of pigmentation in animals (Hoekstra 2006); the effort needed to achieve the same progress in botany
24 S Lev-Yadun KS Gould would surely not be smaller.Thus,our explanations for the role of pigments in plant defences remain imperfect.Notwithstanding the difficulties involved in providing concrete evidence for plant defensive colouration,it has therefore been extremely encouraging to note a recent wave of interest in this area,particularly in relation to foliar anthocyanins. 2.4 Colour Vision in Animals A frequent criticism of the anti-herbivory hypotheses for foliar anthocyanins is that herbivorous insects may lack ocular receptors for red light Insects have up to five kinds of photore eptors sensitive to different regions of the visible and UV spectrum (Kelber 2001:Kelber et al.2003).Butterflies of the genera Papilio and Pieris have arguably the most sophisticated colour vision system of the insects studied so far including a red receptor maximally sensitive ar und 610 nm (Arikawa et al.1987 Shimohigashi and Tomi aga 1991)Ho most of the insects that have bee evamined to date including the phyto three types of photorec to en.blue.and ultraviole ectively (Brisco and Chittka 2001:Kirehner e t al 2005).In the ab visual cue presented Chitka s good evide e that red is recog ed by ins s Do rised the in or b mpared in aph In 2 apl n ob po th nters n ed the nd foun stimulus was th red or gree the refore,that elated)and a chromatic (int ty re ted) sed by frugivorous b (Scha r et al a to state with confidence whether aphids tend to avoid red green than to red light. Only one has add in a "no-choice ottinghar et al.(1991)recorded positive phototaxis towards red targets by the bird cherry aphic Rhopalosiphum padi. Although response rates were very low,that experiment suggested that the insects were not innately repelled by red objects. Detail of the mechanism by which insects lacking a red photoreceptor perceive red colours remains to be resolved.A colour opponency mechanism has beer proposed,which may explain colour discrimination in certain aphid species(Doring and Chittka 2007). This requires negative excitation in the blue and UV.and positive excitation in the green waveband.It is unclear,however,how such a mechanism might facilitate perception of anthocyanic leaves which,compared to green leaves,typically reflect smaller quantities of both green light (Neill and Gould 1999)and UV radiation (Lee and Lowry 1980).It may simply be that red leaves are less attractive to insect herbivores because the excitation of their green receptor is
24 S. Lev-Yadun, K.S. Gould would surely not be smaller. Thus, our explanations for the role of pigments in plant defences remain imperfect. Notwithstanding the difficulties involved in providing concrete evidence for plant defensive colouration, it has therefore been extremely encouraging to note a recent wave of interest in this area, particularly in relation to foliar anthocyanins. 2.4 Colour Vision in Animals A frequent criticism of the anti-herbivory hypotheses for foliar anthocyanins is that herbivorous insects may lack ocular receptors for red light. Insects have up to five kinds of photoreceptors sensitive to different regions of the visible and UV spectrum (Kelber 2001; Kelber et al. 2003). Butterflies of the genera Papilio and Pieris have arguably the most sophisticated colour vision system of the insects studied so far, including a red receptor maximally sensitive around 610 nm (Arikawa et al. 1987; Shimohigashi and Tominaga 1991). However, most of the insects that have been examined to date – including the phytophagous aphid Myzus persicae – possess only three types of photoreceptors, maximally sensitive to green, blue, and ultraviolet light, respectively (Briscoe and Chittka 2001; Kirchner et al. 2005). In the absence of a red light receptor, it could be argued that insects would be unable to perceive the visual cue presented by anthocyanins. There is nevertheless good evidence that red is recognised by insects. Döring and Chittka (2007) recently summarised the results from 38 studies in which the behavioural responses to red or green stimuli were compared in aphid species. In 28 of those studies, the aphids had been observed to move preferentially towards the green stimulus, and in only one of the reports had aphids not demonstrated a colour preference. In the remaining studies, the experimenters had varied the shade of the green stimulus, and found that insects moved preferentially towards whichever stimulus was the brighter, red or green. It is likely, therefore, that both chromatic (wavelength related) and achromatic (intensity related) information is involved, as is known also to be used by frugivorous birds (Schaefer et al. 2006). There are insufficient data to state with confidence whether aphids tend to avoid red light or, instead, are simply more attracted to green than to red light. Only one publication has addressed this issue, albeit indirectly: in a “no-choice” experiment, Nottingham et al. (1991) recorded positive phototaxis towards red targets by the bird cherry aphid Rhopalosiphum padi. Although response rates were very low, that experiment suggested that the insects were not innately repelled by red objects. Detail of the mechanism by which insects lacking a red photoreceptor perceive red colours remains to be resolved. A colour opponency mechanism has been proposed, which may explain colour discrimination in certain aphid species (Döring and Chittka 2007). This requires negative excitation in the blue and UV, and positive excitation in the green waveband. It is unclear, however, how such a mechanism might facilitate perception of anthocyanic leaves which, compared to green leaves, typically reflect smaller quantities of both green light (Neill and Gould 1999) and UV radiation (Lee and Lowry 1980). It may simply be that red leaves are less attractive to insect herbivores because the excitation of their green receptor is
Role of Anthocyanins in Plant Defence 25 ower than when bygreen eaves (Thomas Doring.personal arelikely ascbe important aini et ves and the visual background Con 2.5 Anthocyanins and Other Red Pigments Anthocyanins usually appear red in leaf cells,but depending on their chemical nature and concentration,the vacuolar pH,and interactions with other pigments,they can result in pink k,purple,blue,orange,brown,and even black leaf colours(Schwinn and Davies 2004;Andersen and Jordheim 2006;Hatier and Gould 2007). Many of the published articles on plant defensive colouration have assumed red foliage to be the outcome of the production of anthocyanins,this despite the fact that other pigments- carotenoids,apocarotenoids,betalains,condensed tannins, quinones and phvtomelanins-can also contribute to plant vermilion (Davies 2004).There is moreover,a dearth of systematic information on the full complement of pigments in all plant organs at all developmental stages.This lack of data precludes detailed taxon-wide comparisons of the involvement of anthocyanin,or indeed any pigment in plant defence.Clearly,if only visible cues(hue,lightness,and colour saturation) are involved in defence,the chemical nature of a pigment would be unimportant to a herbivore:red warnings would be similarly effective irrespective of whether they were generated by anthocyanins,carotenoids,or betalains.If,on the other hand,the efficacy of the warning relied on a combination of attributes,for example the reflection of red light plus the presence of a toxic or olfactory phenolic derived from an offshoot in the anthocyanin biosynthetic pathway,then the pigment type could be critical 2.6 Olfactory Signals nation whe tein insects are lured to flowers but recei w that signalling to ombination of both visual and olfactor ents (Dafni 1984 are ood t to think that the sar ativ and Gersh n2002 007 ent(e.g,Jurgens 2004:Jurg s et al..2002,2003,P0 hersky and Dudar in the ield It is in addi tha among the many nly if plants omn and s the po evera that no animals respond similarly to any chemical signal or cue,shou considered
Role of Anthocyanins in Plant Defence 25 lower than when excited by green leaves (Thomas Döring, personal communication). Contrasts in colour and/or brightness between red leaves and the visual background are likely also to be important (Dafni et al. 1997). 2.5 Anthocyanins and Other Red Pigments Anthocyanins usually appear red in leaf cells, but depending on their chemical nature and concentration, the vacuolar pH, and interactions with other pigments, they can result in pink, purple, blue, orange, brown, and even black leaf colours (Schwinn and Davies 2004; Andersen and Jordheim 2006; Hatier and Gould 2007). Many of the published articles on plant defensive colouration have assumed red foliage to be the outcome of the production of anthocyanins, this despite the fact that other pigments – carotenoids, apocarotenoids, betalains, condensed tannins, quinones and phytomelanins – can also contribute to plant vermilion (Davies 2004). There is, moreover, a dearth of systematic information on the full complement of pigments in all plant organs at all developmental stages. This lack of data precludes detailed taxon-wide comparisons of the involvement of anthocyanin, or indeed any pigment, in plant defence. Clearly, if only visible cues (hue, lightness, and colour saturation) are involved in defence, the chemical nature of a pigment would be unimportant to a herbivore; red warnings would be similarly effective irrespective of whether they were generated by anthocyanins, carotenoids, or betalains. If, on the other hand, the efficacy of the warning relied on a combination of attributes, for example the reflection of red light plus the presence of a toxic or olfactory phenolic derived from an offshoot in the anthocyanin biosynthetic pathway, then the pigment type could be critical. 2.6 Olfactory Signals An important, if not critical issue is whether or not olfactory signals are involved along with the visual ones. From studies of deceptive pollination, wherein insects are lured to flowers but receive no sugar reward, we know that signalling to animals can involve a combination of both visual and olfactory components (Dafni 1984; Ayasse et al. 2000; Schiestl et al. 2000); there are good reasons to think that the same may be true in the defence of vegetative organs (Pichersky and Gershenzon 2002). The identification of olfactory volatiles is achievable using modern laboratory equipment (e.g., Jürgens 2004; Jürgens et al. 2002, 2003; Pichersky and Dudareva 2007), but such procedures are difficult to accomplish in the field. It is, in addition, very difficult to identify the specific molecules that deter specific herbivores from among the many volatile molecules that plants omit, and there is the possibility that deterrence operates only if several molecules are sensed simultaneously. The fact that not all animals respond similarly to any chemical signal or cue, should also be considered
26 S Lev-Yadun KS Gould 2.7 Aposematic Colouration Aposematic colouration,a well-kn mbas wn phe until recently i ar with black ma is da ngerous o unpalatabletopreda that c rs a selective adva with unplea 940 Gittleman rvey 980;Harvey and 198 Wi und and Jarv 1982:Ruxton et al.2004 Although seve author d a simila between conspicuous colouration and toxicity 971:Hintor Harper 1977;Wiens 1980 1982: 1982;Knight and Siegfried 1983;Smith 1986;Lee et al.1987;C ey an IAide 198 Givnish 1990;Tuomi and Augner 1993),only in the past de cade h ave the scope and significance of this phenomenon been appreciated. Indeed.the possibility o aposematic colouration was discounted in some of these earlier studies(Knight and Siegfried 1983;Smith 1986;Lee et al.1987;Coley and Aide 1989). A related phenomenon,olfactory aposematism in poisonous plants,has also been proposed (e.g.,Eisner and Grant 1981;Harborne 1982;Launchbaugh and Provenza 1993: Provenza et al.2000)although this has received scant attention. 2.7.1 Poisonous Plants The first detailed hypothesis for a possible defence from herbivory attributable to red colouration(and other colours)was published by Hinton(1973),who proposed that colourful poisonous flowers should be considered aposematic,and that they probably have mimics.His review about deception in nature was published in a book about illusion;this was not a biological book,but rather dealt with art.His hypothesis was briefly referred to by Rothschild(1980)in her discussion on the roles of carotenoids. but otherwise did not stir botanists or ecologists to pursue this issue.Indeed.Harper (1977).who had written the comment about botanists being reluctant to accept things that were commonplace for zoologists,omitted to explain why zoologists who dealt with animal apo matism,and who were also involved in research on plant-animal interactions.had not recognized how common are these phenomena in plants Harborne (1982)p proposed that the brightly coloured,purple-black berries of the deadly Atropa belladonna warn grazing mammals of the danger to consume then Williamson (1982)also proposed that brightly coloured (red.or red and black)seeds lacking an arillate or fleshy eward (e.g osia and Abrus)might be aposematically coloured to titten only as short parag aphs within long reviews,ho further effort to study the functior colouration 2.7.2 Thorny Plants In English there are three terms for pointed plant organs:spines (modified leaves) eg in P we refer r to plants as
26 S. Lev-Yadun, K.S. Gould 2.7 Aposematic Colouration Aposematic colouration, a well-known phenomenon in animals, has until recently been given little attention in plants. Often, a brightly-coloured animal (red, orange, yellow, white with black markings, or combinations of these colours) is dangerous or unpalatable to predators – a trait that confers a selective advantage because predators learn to associate the colouration with unpleasant qualities (Cott 1940; Edmunds 1974; Gittleman and Harvey 1980; Harvey and Paxton 1981; Wiklund and Järvi 1982; Ruxton et al. 2004). Although several authors had noted a similar association between conspicuous colouration and toxicity in plants (Cook et al. 1971; Hinton 1973; Harper 1977; Wiens 1978; Rothschild 1980; Harborne 1982; Williamson 1982; Knight and Siegfried 1983; Smith 1986; Lee et al. 1987; Coley and Aide 1989; Givnish 1990; Tuomi and Augner 1993), only in the past decade have the scope and significance of this phenomenon been appreciated. Indeed, the possibility of aposematic colouration was discounted in some of these earlier studies (Knight and Siegfried 1983; Smith 1986; Lee et al. 1987; Coley and Aide 1989). A related phenomenon, olfactory aposematism in poisonous plants, has also been proposed (e.g., Eisner and Grant 1981; Harborne 1982; Launchbaugh and Provenza 1993; Provenza et al. 2000) although this has received scant attention. 2.7.1 Poisonous Plants The first detailed hypothesis for a possible defence from herbivory attributable to red colouration (and other colours) was published by Hinton (1973), who proposed that colourful poisonous flowers should be considered aposematic, and that they probably have mimics. His review about deception in nature was published in a book about illusion; this was not a biological book, but rather dealt with art. His hypothesis was briefly referred to by Rothschild (1980) in her discussion on the roles of carotenoids, but otherwise did not stir botanists or ecologists to pursue this issue. Indeed, Harper (1977), who had written the comment about botanists being reluctant to accept things that were commonplace for zoologists, omitted to explain why zoologists who dealt with animal aposematism, and who were also involved in research on plant-animal interactions, had not recognized how common are these phenomena in plants. Harborne (1982) proposed that the brightly coloured, purple-black berries of the deadly Atropa belladonna warn grazing mammals of the danger to consume them. Williamson (1982) also proposed that brightly coloured (red, or red and black) seeds lacking an arillate or fleshy reward (e.g. Erythrina, Ormosia, and Abrus) might be aposematically coloured to warn seed eaters of their toxicity. These hypotheses were written only as short paragraphs within long reviews, however, and there has been no further effort to study the function of their colouration. 2.7.2 Thorny Plants In English there are three terms for pointed plant organs: spines (modified leaves), thorns (modified branches), and prickles (comprising cortical tissues, e.g. in roses). For the purposes of this discussion, we refer to plants as “thorny” if they produce any
Role of Anthocyanins in Plant Defence 27 of the three types of sharp appendages ndages pro vide mechanical an Janzen 1986;Myers anc 02)becau e they ca n wound mouths digestive systems (Janzen and Martin 1982:Janzen 1986),and other body parts of They might also inject pathogenic bacteria into herbivores(Halpern et al.2007).Thus,once herbivores learn to identify thorns-and their bright colours and associated markings should help in their recognition-they can avoid harmful plants displaying them. The flora of countries such as Israel,which has a millennia-long history of large. scale grazing,clearly and "sharply"indicates the ecological benefit of being thorny when grazing pressure is high.A continuous blanket of spiny shrubs such as Sarcopoterium spinosum,as well as many types of thistles,covers large tracts of the land.The thorns effectively impede the rate at which an herbivore feeds within the canopy of the individual plant,and this presents an overall considerable advantage to such plants over non-detended ones Spiny plants.such as Echinops sp (Asteraceae),which normally grow as individuals or in small groups,sometimes become the most common perennial plant over many acres in heavily grazed lands. The same is true for many other taxa. Thousands of thorny species have colourful or otherwise conspicuous markings (e.g.Fig.2.1A).many of which can be considered to be aposematic (Lev-Yadun 2001).We will not discuss this common phenomenon in the thorniest taxon-the Cactaceae-since they lack anthocvanins and use betalains instead (Stafford 1994) 。n2o,at(h (e n colourful stripes,in leaves and stems associated with the thoms.Both types have imately 2.000 species originating from several continents ir been ord o award2200 2006 Bua McCarthy 2004:Halpern et al.2007;Lev-Yadun unpublished).It has been proposed A ⊙ Fig.2.1 Thorns and their mimics.(A)Anthocy for colour version of these photographs
Role of Anthocyanins in Plant Defence 27 of the three types of sharp appendages. Thorny appendages provide mechanical protection against herbivory (Janzen and Martin 1982; Janzen 1986; Myers and Bazely 1991; Grubb 1992; Rebollo et al. 2002) because they can wound mouths, digestive systems (Janzen and Martin 1982; Janzen 1986), and other body parts of herbivores. They might also inject pathogenic bacteria into herbivores (Halpern et al. 2007). Thus, once herbivores learn to identify thorns – and their bright colours and associated markings should help in their recognition – they can avoid harmful plants displaying them. The flora of countries such as Israel, which has a millennia-long history of largescale grazing, clearly and “sharply” indicates the ecological benefit of being thorny when grazing pressure is high. A continuous blanket of spiny shrubs such as Sarcopoterium spinosum, as well as many types of thistles, covers large tracts of the land. The thorns effectively impede the rate at which an herbivore feeds within the canopy of the individual plant, and this presents an overall considerable advantage to such plants over non-defended ones. Spiny plants, such as Echinops sp. (Asteraceae), which normally grow as individuals or in small groups, sometimes become the most common perennial plant over many acres in heavily grazed lands. The same is true for many other taxa. Fig. 2.1 Thorns and their mimics. (A) Anthocyanic thorns on a rose stem. (B) Red mucron at the apex of a leaf of Limonium angustifolium. (C) Red fruit of Erodium laciniatum. See Plate 1 for colour version of these photographs Thousands of thorny species have colourful or otherwise conspicuous markings (e.g. Fig. 2.1A), many of which can be considered to be aposematic (Lev-Yadun 2001). We will not discuss this common phenomenon in the thorniest taxon – the Cactaceae – since they lack anthocyanins, and use betalains instead (Stafford 1994). Lev-Yadun (2001) categorised two types of thorn ornamentation, which are typical of many thorny plant species: (i) colourful thorns, and (ii) white spots, or white and colourful stripes, in leaves and stems associated with the thorns. Both types have been recorded for approximately 2,000 species originating from several continents in both the Old and New World (Lev-Yadun 2001, 2003a, 2003b, 2006; Rubino and McCarthy 2004; Halpern et al. 2007; Lev-Yadun unpublished). It has been proposed