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Does anthocyanin degradation play a significant role in determining pigment concentration in plants?
Plant Science 177 (2009) 310–316
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Plant Science journal homepage: www.elsevier.com/locate/plantsci
Review
Does anthocyanin degradation play a significant role in determining pigment concentration in plants? Michal Oren-Shamir * Department of Ornamental Horticulture, Agriculture Research Organization, P.O. Box 6, Bet-Dagan 50250, Israel
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 April 2009 Received in revised form 25 June 2009 Accepted 29 June 2009 Available online 7 July 2009
In contrast to the detailed knowledge available on anthocyanin synthesis, very little is known about its stability and catabolism in plants. Here we review evidence supporting in planta turnover and degradation of anthocyanins. Transient anthocyanin accumulation and disappearance during plant development or changes in environmental conditions suggest that anthocyanin degradation is controlled and induced when beneficial to the plant. Several enzymes have been isolated that degrade anthocyanins in postharvest fruit that may be candidates for in vivo degradation. Three enzyme groups that control degradation rates of anthocyanins in fruit extracts and juices are polyphenol oxidases, peroxidases and b-glucosidases. Evidence supporting the involvement of peroxidases and bglucosidases in in vivo anthocyanin degradation in Brunfelsia flowers is presented. Understanding the in vivo anthocyanin degradation process has potential for enabling increased pigmentation and prevention of color degradation in crops. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Anthocyanin degradation Brunfelsia Peroxidase b-Glucosidase Polyphenol oxidase
Contents 1. 2.
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Anthocyanins in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence supporting in planta anthocyanin degradation . . . . . . . . . . . . . . . . . . . . . . . 2.1. Degradation during development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Loss of red pigmentation in young foliage as it matures. . . . . . . . . . 2.1.2. Anthocyanin degradation in developing fruit. . . . . . . . . . . . . . . . . . . 2.1.3. Anthocyanin degradation in developing flowers . . . . . . . . . . . . . . . . 2.2. Degradation due to changes in environmental conditions . . . . . . . . . . . . . . . . 2.3. Is anthocyanin turnover in plants dependent on environmental conditions? . Enzymatic degradation of anthocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Anthocyanin degradation in fruit after harvest . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Enzymatic degradation of anthocyanins in fruit juices . . . . . . . . . . . . . . . . . . . 3.3. Active in planta degradation of anthocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Anthocyanins in plants Anthocyanins are the largest and most diverse group of plant pigments derived from the phenylpropanoid pathway, ranging in color from red to violet and blue [1]. They are water-soluble
* Tel.: +972 3 9683840. E-mail address: [email protected]. 0168-9452/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2009.06.015
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phenolic compounds and part of a large and widespread group of plant flavonoids. There are less than 20 anthocyanidins (aglycones or chromophores of anthocyanins), differing in the number and position of their hydroxyl groups and methyl groups. Anthocyanidins are modified by glycosyl and aromatic or aliphatic acyl moieties, resulting in hundreds of anthocyanin molecules that differ in hue and stability. These pigments accumulate in the vacuoles and their stability and hue depend on intravacuolar conditions such as pH, copigmentation with coexisting colorless
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flavonoids and formation of complexes with metal ions [2,3]. Anthocyanins are located mainly in the epidermal tissue, but are also present in the palisade and spongy mesophyll in leaves and in the flesh of fruits and underground storage organs such as sweet potato [4,5]. The biosynthesis of anthocyanins has been characterized in great detail [6,7]. The basic anthocyanin molecule is comprised of two aromatic rings and an oxygen containing heterocyclic ring. One of the aromatic rings is derived from phenylalanine and the second ring from the action of chalcone synthase (CHS), condensing one molecule of p-counaroyl-coA with three molecules of malonyl-coA to produce tetrahydroxy chalcone [8]. CHS is the first committed enzyme in the anthocyanin biosynthetic pathway. The regulation of anthocyanin biosynthesis has also been studied thoroughly and comprises of basic-helixloop-helix (bHLH) transcription factors, interacting with R2R3 MYB transcription factors to activate either all or part of the anthocyanin genes [9]. Many studies generated transgenic plants with either increased concentration or altered composition of anthocyanins in flowers, foliage or fruit by manipulating the expression of the anthocyanin regulatory genes [10–15]. Conversely, very little is known about the stability and catabolism of anthocyanins in plants. Is there turnover (simultaneous synthesis and degradation) of the pigments in living plant tissue, or are the pigments stable once they have accumulated in the vacuoles? Can plants actively degrade anthocyanins, thereby controlling pigment concentration via both biosynthesis and degradation? Since the color of fruits, flowers and leaves is of the utmost economic importance in a variety of agricultural products, a better understanding of anthocyanin degradation may reveal ways in which to inhibit the process and consequently increase pigmentation under conditions of low synthesis. Here we review evidence supporting the existence of anthocyanin turnover and degradation in planta. In addition, we review the knowledge on active anthocyanin degradation processes in in vitro systems such as fruit juices, in an attempt to understand the in planta enzymatic process.
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2. Evidence supporting in planta anthocyanin degradation Anthocyanins often accumulate transiently, appearing and disappearing during plant development or with changes in environmental conditions [16]. Several examples in which anthocyanins are degraded in plants are described below. 2.1. Degradation during development 2.1.1. Loss of red pigmentation in young foliage as it matures Anthocyanins often accumulate in young/juvenile leaves, and degrade as the leaves mature [16]. Since anthocyanins absorb light in both the visible and UV region, their accumulation in young developing foliage may serve as a ‘sunscreen’, protecting the leaves, and in particular their photosynthetic apparatus, from damaging UV light as well as from photoinhibitory high intensities of visible light [17]. These pigments may also provide antioxidant activity, protecting the cells from oxidative damage [18]. As the leaves mature and form protective waxes that reflect sunlight, thereby providing a photoprotective function [19], they often change color from red to green. This loss of red pigmentation may be due to a combination of increased chlorophyll accumulation during leaf expansion and growth, termination of anthocyanin biosynthesis and dilution by growth, and/or increased actively induced anthocyanin degradation when it is no longer required as a photoprotectant. Anthocyanin degradation is apparent in greening of Chrysobalanus icaco (cocoplum) (Fig. 1A) and Photinia fraseri cv. Red Robin leaves as they mature. In both plants, anthocyanins degrade as the leaves develop, resulting in green foliage [20,21]. In Photinia plants, anthocyanin concentration is directly related to the age of the leaf, with high concentrations in young leaves, and a gradual decrease in concentration as the leaves develop, due both to dilution and degradation, resulting in undetectable concentrations of anthocyanins in the mature foliage [21]. In cocoplum, the decrease in anthocyanin concentration as the leaves mature is due in part to dilution of the pigments in the
Fig. 1. Anthocyanin degradation during development of flowers, fruit and foliage. (A) Degradation in developing leaves of Chrysobalanus icaco. (B) Degradation in pepper (Capsicum annuum) mutant 5226 from mature unripe fruit (4) to ripe fruit (5). The Capsicum figure was slightly altered from Fig. 3 of Borovsky et al. [19] with the kind permission of Springer Science + Business Media. (C) Degradation in Brunfelsia calycina flowers from the day of flower opening (0) until 3 days after opening (3). Values are means of four replications SE. The relative anthocyanin concentration is described by either a spectrophotometric reading at 530 nm (A) or the absorption-peak area of the pigments, when separated by HPLC on an RP-18 column (B and C).
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Fig. 2. Anthocyanin degradation due to environmental changes. (A) Cotinus coggygria ‘Royal Purple’ mature leaves from plants grown at 17 8C/9 8C day/night (leaves on left) and transferred to 29 8C/21 8C day/night (leaves on right) temperature conditions. (B) ‘Rosemarie’ pear fruit covered with a two-layered ‘Fuji’ apple wrapping bag (Kobayashi Bag Mfg., Nagano, Japan), for 0 (pear on the left), 1, 2, 3 and 4 (pear on the right) weeks. Photograph was kindly provided by Dr. Wiehann Steyn, Dept. of Horticultural Science, Stellenbosch University, South Africa.
growing tissues and in part to actual degradation [20]. In other plants, such as rose, the change in foliage color from young red to mature green leaves does not involve anthocyanin degradation and is due only to increased chlorophyll synthesis, termination of anthocyanin synthesis and dilution of the anthocyanins in the expanding leaves (unpublished, Liat Shahar). 2.1.2. Anthocyanin degradation in developing fruit Transient accumulation of anthocyanins is seen in some fruits. One example is Capsicum spp. in which several lines accumulate anthocyanins in immature fruit and degrade them as the fruits mature [22,23] (Fig. 1B). Anthocyanins may accumulate and protect the photosynthetic apparatus in the developing fruit. As the Capsicum fruit matures, the anthocyanins in the vacuoles are degraded while chlorophyll degradation in the plastids and the synthesis of carotenoids result in the red, orange and yellow colors of the mature pepper fruit. Anthocyanin degradation is also observed in Sicilian sweet orange varieties, known as blood oranges (Tarocco, Moro e Sanquinello). The pigments accumulate in both the rind and flesh of the fruit resulting in these varieties’ characteristic red color. However, at the late stages of ripening, anthocyanin degradation is observed, resulting in a commercially undesirable partial loss of this color [24]. 2.1.3. Anthocyanin degradation in developing flowers Flowers often change color during development, acting as a signal for pollinators. In most cases, the change in color is due to induction of anthocyanin synthesis, but in others, such as Brunfelsia calycina, anthocyanin is degraded, resulting in a change of flower color from dark purple to white after anthesis [25] (Fig. 1C). Further details on anthocyanin degradation in Brunfelsia are described later. The fading Fa mutants of petunia, lose color during flower development [26,27]. However, the change in color in Fa mutants is not due to anthocyanin degradation, but rather to a change in the vacuolar pH resulting in a change of anthocyanin hue and loss of visual coloration with no change in pigment concentration. 2.2. Degradation due to changes in environmental conditions Anthocyanin concentration in foliage is tightly dependent on environmental conditions such as light quality, light intensity and growth temperature. Foliar anthocyanin accumulation is induced at high light intensities, in particular high UV light, and low growth temperatures [16]. This process is often reversible, with greening of red foliage and stems when growth conditions change to low light intensity and warmer temperatures. The dark red pigmentation of both the young and mature leaves of the garden plant Cotinus coggygria ‘Royal Purple’, occurs only with both low temperatures ( 17 8C/9 8C day/night respectively) and high UV irradiation [28,29]. Anthocyanins of red Cotinus plants, grown at low temperatures, are degraded and their foliage turns green when
covered with a UV screen. Similarly, anthocyanin degradation occurs in red-pigmented plants when the temperature is elevated but UV radiation remains high [28,29] (Fig. 2A). The dependence of anthocyanin concentration on environmental conditions was also demonstrated in Arabidopsis thaliana plants transformed to over-express the transcription factor PAP1 (Production of Anthocyanin Pigments 1) [30]. The mutated arabidopsis plants were dark red under room temperature and high light conditions (22 8C, 440 mmol m 2 s 1), but when transferred to conditions of high temperature and low light conditions (30 8C, 150 mmol m 2 s 1) the plants turned green. The change in color was due to the simultaneous down-regulation of synthesis and degradation of the anthocyanins. These results suggest that there are additional mechanisms involved in the environmental control of anthocyanins, one of which may be controlled degradation [30]. Anthocyanin pigmentation also fluctuates in fruits in response to environmental conditions such as growth temperature and light intensity. An increase in growth temperatures causes preharvest degradation of anthocyanins and a significant decrease in the fruit’s market value in both red pears and apples [31]. Rapid anthocyanin degradation has also been reported in ‘Rosemarie’ and ‘Forelle’ pears, still attached to the tree, when the fruits were covered with light-impermeable bags [32] (Fig. 2B). 2.3. Is anthocyanin turnover in plants dependent on environmental conditions? In addition to specific degradation of anthocyanins due to either developmental or environmental changes, there may be turnover of the pigments in plant tissue, even when no visual change in color is observed. The turnover of anthocyanins in living plant tissues has been followed using various techniques. Pulse-chase treatment of mustard seedlings with radioactive phenylalanine revealed a constant turnover of the pigments after reaching a high and steady level of anthocyanins [33]. The turnover rate of anthocyanins was also followed by treating plant tissues with a specific phenylalanine ammonia-lyase (PAL) inhibitor (aminooxyphenylpropionic acid—AOPP) preventing biosynthesis of the pigments: in petunia, low degradation levels were detected at specific developmental stages, while in carrot cells, no anthocyanin turnover was detected [34,35]. Increased anthocyanin turnover in detached Cabernet Sauvignon grape skin was seen at elevated growth temperatures [36]. The ratio between the anthocyanins comprising Cabernet Sauvignon grape skin color varied, with less degradation of the methylated and acylated pigments [36]. This is consistent with the finding that methoxylation, glycosylation and acylation increase the thermal stability of anthocyanins [37]. This finding suggests that the low levels of anthocyanin observed in many plants when grown at elevated temperatures may be a combination of a slower rate of biosynthesis [38–40] and increased catabolism. The catabolic process may be due either to chemical instability of the pigments or to specific enzymatic activity
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decreasing the pigment concentration in plant tissues, often in parallel to the synthesis process. Metal ions such as magnesium, manganese, tin and copper affect the stability of anthocyanins [2]. These metals accumulate in the vacuoles and form stable complexes with the anthocyanins, thereby affecting their hue and increasing their stability [2]. Stable complexes of magnesium ions with anthocyanins have been isolated from Hydrangea sepals [41]. This phenomenon can be exploited to increase anthocyanin concentration in plants under environmental conditions in which the rates of synthesis are low. For example, treatment of Aster flowers with magnesium salts during the synthesis of anthocyanins caused an increase in pigment concentration in the flowers, without inducing the activity of several of the key enzymes along the biosynthetic pathway [42]. This suggests that there is constant turnover of anthocyanins in the aster flowers, and magnesium treatments slow this process by stabilizing the pigments. Magnesium treatment has a similar effect on other ornamentals, increasing anthocyanin concentration in Anigozanthos, Limonium, Gypsophila and Aconitum flowers grown at elevated temperatures [43]. 3. Enzymatic degradation of anthocyanins Some of the examples presented above suggest that the anthocyanin degradation process is controlled and induced when beneficial to the plant, such as in the loss of foliar pigmentation as the leaves mature [20,21] or when light intensity decreases [32], allowing more light to be available for photosynthesis. In other examples, such as in Cabernet Sauvignon grapes, anthocyanin degradation does not result in a dramatic change in color as the pigments continue to be synthesized in parallel to their catabolism [36]. In this case, the degradation may be due to lower chemical stability of the pigments at elevated temperatures. The question still remains as to whether degradation is due to enzymatic activity and whether degradation can be specifically induced as part of a complex system for controlling plant pigment concentration. Enzymatic studies of anthocyanin degradation in postharvest fruit and fruit juices have revealed several enzymes that degrade anthocyanins in these systems and may be candidates for active in vivo degradation. 3.1. Anthocyanin degradation in fruit after harvest Anthocyanin degradation due to changing environmental conditions has been observed in fruit after harvest. One example of this is anthocyanin degradation in red-skinned pears and apples when the fruit are stored in warm (>10 8C) temperatures [32]. This is similar to the effect of increased growth temperature on the pigmentation of red-skinned pears while still attached to the tree [32]. The search for anthocyanin-degrading enzymes was performed on postharvest fruit, because of the market value of high pigmentation. Postharvest fruit is intermediate between the fruit still attached to the tree and the in vitro systems of fruit juices: postharvest fruit still carry out many of the enzymatic processes occurring before harvest. Intracellular decompartmentation and cell layer reparation begins during storage, and the pigments may be exposed to microenvironmental conditions that differ from those in planta, including enzymes that are not located in the vacuoles when the plant cells are intact. Anthocyanins in litchi fruit are degraded after harvest, accompanied by fruit browning [44–48]. This change in color lowers the commercial value. Polyphenol oxidase (PPO) and peroxidase are thought to be responsible for the anthocyanin degradation in litchi. Peroxidase activity initially increases in the exocarp and during longer-term storage in the endocarp, while
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PPO activity increases during long-term storage in the exocarp [44–48]. Treatment of the fruit after harvest with oxalic acid, known to reduce PPO activity by binding with the active sites of copper to form an inactive complex [49], reduces oxidation levels in litchi fruit and helps maintain low peroxidase activity and delay anthocyanin degradation [48]. Postharvest loss of red color has also been detected in eggplant [50]. In eggplant fruit subjected to chilling injury, browning with concomitant vacuolar disruption, electrolyte leakage, increased pH and decreased anthocyanin concentration were observed in the skin tissue [50]. A b-glucosidase was extracted from eggplant peels that were capable of degrading anthocyanins [51]. 3.2. Enzymatic degradation of anthocyanins in fruit juices The most detailed studies on anthocyanin stability and degradation have been carried out with fruit extracts. These studies form the basis for understanding the processes leading to pigment loss in both postharvest fruit and in juices and wine. One of the main enzyme groups that oxidize anthocyanins in fruit extracts is the PPOs. These ubiquitous enzymes in higher plants are located in the plastids of both photosynthetic and nonphotosynthetic tissues [52]. Both PPO in conjunction with phenolic extracts [53] and anthocyanin b-glucosidase [54] are able to degrade the pigments in ethanolic extracts from the lichti fruit periderm in vitro. It was proposed that anthocyanins are first hydrolyzed by an anthocyanase (b-glucosidase), forming anthocyanidin [53]. These compounds can then be oxidized by PPO and/ or peroxidase. Oxidative products of phenolics, such as 4methylcatechol, resulting from PPO activity may then accelerate anthocyanin degradation via a coupled oxidative reaction [55]. Furthermore, PPO can oxidize anthocyanin degradation products resulting in tissue browning. PPO activity is the main factor responsible for anthocyanin degradation in juice prepared from red muscadine grapes (Vitis rotundifolia cv. Noble). Treating the juice with increased CO2 pressure (dense-phase CO2 processing) partially inhibited PPO and decreased the rate of degradation of anthocyanin, as well as that of other polyphenolic compounds, during refrigerated storage [56]. A specific b-glucosidase was isolated from the fruit juice of Tarocco Sicilian blood oranges and anthocyanin degradation kinetics was followed physiochemically [24]. This enzyme is responsible for the degradation of anthocyanins in both the juice and the ripening fruit [24]. In the presence of chlorogenic acid or caffeoyltartaric acid, PPO increased the rate of anthocyanin degradation in both grape and blueberry extracts and juices [57–60]. PPO also degrades anthocyanins during the drying of plums in the presence of chlorogenic acid [61]. Plastid located PPOs are not likely to degrade vacuolar anthocyanins in living tissue unless decompartmentation occurs. The peroxidases also degrade anthocyanins in fruit extracts, under mildly oxidizing conditions provoked by exogenous application of H2O2. A peroxidase isolated from a Gamay grapevine cell culture degraded anthocyanins in solution and was suggested to do so in the grape fruit as well [62]. Peroxidases are also involved in the degradation of anthocyanins, resulting in loss of color in processed strawberries [63]. Exogenous application of H2O2 on strawberry slices caused a more rapid decrease in anthocyanin concentration during aging relative to non-treated slices, suggesting the involvement of peroxidase in this process. A proposed scheme summarizing the enzymatic processes involved in anthocyanin degradation is presented in Fig. 3. Since peroxidases are present in cell vacuoles [64], unlike the oxidizing PPO enzymes, they are more likely candidates for in planta anthocyanin degradation.
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Fig. 3. Proposed scheme describing anthocyanin degradation and tissue browning in fruit extracts. POD, peroxidase; PPO, polyphenol oxidase. POD is dependant on H2O2 for its activity while PPO is dependant on O2. Three alternative pathways for enzymatic anthocyanin degradation are presented. (1) Coupled oxidation, i.e. reduction of quinones of phenolic compounds to the original phenolic compounds in parallel to oxidation of anthocyanin to anthocyanin quinone. (2) Degradation in two steps: de-glycosylation with anthocyanase (b-glucosidase) and then oxidation with polyphenol oxidase or peroxidase. (3) Direct oxidation of anthocyanin with peroxidase.
3.3. Active in planta degradation of anthocyanins Evidence has been presented of in planta degradation of anthocyanins in ornamentals and fruits. We have also outlined extensive studies on enzymatic anthocyanin degradation in fruit extracts, juices and wine. However, until recently, it was not known if anthocyanin degradation in intact plants involves active enzymatic or merely chemical reactions. Active anthocyanin degradation was first reported in living plant tissue of Brunfelsia calycina flowers. Brunfelsia, a shrub in the Solanaceae family is native to Brazil, and is an ideal model plant for studying anthocyanin degradation because of the dramatic and rapid color changes occurring within 2–3 days after flower opening (Fig. 1C). This loss of color from dark purple to white is dependent on anthocyanin degradation and de novo synthesis of mRNAs and proteins during the different stages of development, well before the start of flower senescence [25]. An additional advantage of this model system is that the same dramatic decrease in pigment concentration occurs in flowers detached on the day of flower opening and grown in a sucrose solution [25]. Similar to what has been shown in fruit juices and wine, oxidative reactions are crucial for anthocyanin degradation in Brunfelsia, since treatment of detached flowers with the reducing reagents dithiothreitol (DTT) or glutathione inhibit degradation [25]. Assuming that in planta anthocyanin degradation occurs in the cell vacuoles, oxidizing enzymes found in the vacuole, such as peroxidases are more likely candidates than those accumulating elsewhere in the cells, such as PPO located in the plastids. A dramatic increase of total peroxidase activity was detected in correlation with the onset of anthocyanin degradation in Brunfelsia flower petals, strengthening the notion that these enzymes are involved in the process [25]. Nevertheless, the possibility that anthocyanins are transferred out of the vacuoles before degradation exists and further studies are needed to determine the site of degradation.
The b-glucosidases may also be involved in the in planta degradation process, since they increased the degradation rate of anthocyanins in fruit extracts of both litchi and eggplant [51,54]. DGluconic acid, a specific b-glucosidase inhibitor, significantly decreased the rate of degradation in Brunfelsia flowers (unpublished, Hila Vaknin and Raya Liberman). One possible explanation is that b-glucosidase activity precedes the oxidation reaction with peroxidase as a candidate oxidizing enzyme. Stripping the anthocyanin molecules of their glucose residues may enable better access for the peroxidase enzyme and faster degradation. Hence, it is likely that anthocyanin degradation is a complex process involving more than one enzyme. 4. Concluding remarks Anthocyanin degradation occurs in different plant organs due to a variety of environmental and developmental conditions. In some cases, where there is no apparent benefit to the plant, anthocyanin degradation occurs due to changes in the vacuoles that decrease the stability of the pigments and cause either chemical degradation or increased vulnerability to degrading enzymes (e.g. b-glucosidases, peroxidases) present in the vacuoles. For example, the degradation of anthocyanins in Cabernet Sauvignon grape skin exposed to high temperatures may be due to chemical degradation, but may also be a product of the activity of peroxidase enzymes induced due to the thermal stress [36]. A second example is the effect of vacuolar pH on the stability of the pigments. Changes in the vacuolar pH, such as increased pH in senescing tissue, may decrease the stability of the anthocyanins and cause chemical degradation [2]. In other cases, such as in Brunfelsia flowers or in photosynthetic tissues, there are clear reasons why the degradation of anthocyanins benefits the plant. In these cases the process may be regulated by and dependant on specific genes and proteins. In Brunfelsia
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calycina flowers, the dramatic change in color due to anthocyanin degradation is most probably a signal for the pollinators and is regulated at the gene and protein levels [25]. In photosynthetic leaves, since anthocyanin accumulation decreases their photosynthesis rate but protects the tissue from free radical scavenging [65], degradation under low light and high temperatures will enable more efficient photosynthesis under conditions that are not prone to photo-oxidation. Revealing the different catabolic processes occurring in plants may allow for improved treatments that control plant tissue color. Increased stability of the pigment molecules may be achieved either by treatments such as increasing the concentration of the stable metal–anthocyanin complexes [42], or molecular manipulation of the pigments to form more stable anthocyanins. Recently, genes encoding anthocyanin acyl transferases in Arabidopsis thaliana were identified, and their activities were shown to increase the stability of the pigments. These may be candidate genes for engineering more stable pigment molecules [66]. Understanding the regulation and enzymatic processes involved in degradation may facilitate genetic manipulation in plants in order to enable increased anthocyanin concentrations under broader environmental conditions. Acknowledgement The author would like to thank Rinat Ovadia for helpful comments and suggestions. References [1] A.J. van Tunen, J.N.M. Mol, Control of flavonoids synthesis and manipulation of flower color, Plant Biotechnol. 2 (1991) 94–125. [2] G. Mazza, E. Miniati, Color stabilization and intensification, in: G. Mazza, E. Miniati (Eds.), Anthocyanins in Fruits, Vegetables, and Grains, CRC Press, Boca Raton, FL, 1993, pp. 1–20. [3] Y. Tanaka, N. Sasaki, A. Ohmiya, Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids, Plant J. 54 (2008) 733–749. [4] T. Sugawara, K. Igarashi, Cultivar variation and anthocyanins and rutin content in sweet cherries (Prunus avium L.), J. Jpn. Soc. Food Sci. Technol.—Nippon Shokuhin Kagaku Kogaku Kaishi 55 (2008) 239–244. [5] N.M. Hughes, C.B. Morley, W.K. Smith, Coordination of anthocyanin decline and photosynthetic maturation in juvenile leaves of three deciduous tree species, New Phytol. 175 (2007) 675–685. [6] B. Winkel-Shirley, Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology, Plant Physiol. 126 (2001) 485–493. [7] E. Grotewold, The genetics and biochemistry of floral pigments, Annu. Rev. Plant Biol. 57 (2006) 761–780. [8] O. Yu, M. Matsuno, S. Subramanian, Flavonoid compounds in flowers: genetics and biochemistry, in: J.A. Teixeira da Silva (Ed.), Floriculture, Ornamental and Plant Biotechnology, vol. I, Global Science Books, Ltd., UK, 2006. [9] A.C. Allan, R.P. Hellens, W.A. Laing, MYB transcription factors that colour our fruit, Trends Plant Sci. 13 (2008) 99–102. [10] M.M. Ben Zvi, N.Z. Florence, T. Masci, M. Ovadis, E. Shklarman, H. Ben-Meir, T. Tzfira, N. Dudareva, A. Vainstein, Interlinking showy traits: co-engineering of scent and colour biosynthesis in flowers, Plant Biotechnol. J. 6 (2008) 403–415. [11] J.M. Bradley, S.R. Rains, J.L. Manson, K.M. Davies, Flower pattern stability in genetically modified lisianthus (Eustoma grandiflorum) under commercial growing conditions, N. Z. J. Crop Hortic. Sci. 28 (2000) 175–184. [12] K.M. Davies, K.E. Schwinn, S.C. Deroles, D.G. Manson, D.H. Lewis, S.J. Bloor, J.M. Bradley, Enhancing anthocyanin production by altering competition for substrate between flavonol synthase and dihydroflavonol 4-reductase, Euphytica 131 (2003) 259–268. [13] F. Quattrocchio, J.F. Wing, K. van der Woude, J.N.M. Mol, R. Koes, Analysis of bHLH and MYB domain proteins: species-specific regulatory differences are caused by divergent evolution of target anthocyanin genes, Plant J. 13 (1998) 475–488. [14] E. Butelli, L. Titta, M. Giorgio, H.P. Mock, A. Matros, S. Peterek, E. Schijlen, R.D. Hall, A.G. Bovy, J. Luo, C. Martin, Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors, Nat. Biotechnol. 26 (2008) 1301–1308. [15] Y. Tanaka, A. Ohmiya, Seeing is believing: engineering anthocyanin and carotenoid biosynthetic pathways, Curr. Opin. Biotechnol. 19 (2008) 190–197. [16] L. Chalker-Scott, Environmental significance of anthocyanins in plant stress responses, Photochem. Photobiol. 70 (1999) 1–9. [17] W.J. Steyn, S.J.E. Wand, D.M. Holcroft, G. Jacobs, Anthocyanins in vegetative tissues: a proposed unified function in photoprotection, New Phytol. 155 (2002) 349–361.
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Review
Does anthocyanin degradation play a significant role in determining pigment concentration in plants? Michal Oren-Shamir * Department of Ornamental Horticulture, Agriculture Research Organization, P.O. Box 6, Bet-Dagan 50250, Israel
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 April 2009 Received in revised form 25 June 2009 Accepted 29 June 2009 Available online 7 July 2009
In contrast to the detailed knowledge available on anthocyanin synthesis, very little is known about its stability and catabolism in plants. Here we review evidence supporting in planta turnover and degradation of anthocyanins. Transient anthocyanin accumulation and disappearance during plant development or changes in environmental conditions suggest that anthocyanin degradation is controlled and induced when beneficial to the plant. Several enzymes have been isolated that degrade anthocyanins in postharvest fruit that may be candidates for in vivo degradation. Three enzyme groups that control degradation rates of anthocyanins in fruit extracts and juices are polyphenol oxidases, peroxidases and b-glucosidases. Evidence supporting the involvement of peroxidases and bglucosidases in in vivo anthocyanin degradation in Brunfelsia flowers is presented. Understanding the in vivo anthocyanin degradation process has potential for enabling increased pigmentation and prevention of color degradation in crops. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Anthocyanin degradation Brunfelsia Peroxidase b-Glucosidase Polyphenol oxidase
Contents 1. 2.
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Anthocyanins in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence supporting in planta anthocyanin degradation . . . . . . . . . . . . . . . . . . . . . . . 2.1. Degradation during development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Loss of red pigmentation in young foliage as it matures. . . . . . . . . . 2.1.2. Anthocyanin degradation in developing fruit. . . . . . . . . . . . . . . . . . . 2.1.3. Anthocyanin degradation in developing flowers . . . . . . . . . . . . . . . . 2.2. Degradation due to changes in environmental conditions . . . . . . . . . . . . . . . . 2.3. Is anthocyanin turnover in plants dependent on environmental conditions? . Enzymatic degradation of anthocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Anthocyanin degradation in fruit after harvest . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Enzymatic degradation of anthocyanins in fruit juices . . . . . . . . . . . . . . . . . . . 3.3. Active in planta degradation of anthocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Anthocyanins in plants Anthocyanins are the largest and most diverse group of plant pigments derived from the phenylpropanoid pathway, ranging in color from red to violet and blue [1]. They are water-soluble
* Tel.: +972 3 9683840. E-mail address: [email protected]. 0168-9452/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2009.06.015
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phenolic compounds and part of a large and widespread group of plant flavonoids. There are less than 20 anthocyanidins (aglycones or chromophores of anthocyanins), differing in the number and position of their hydroxyl groups and methyl groups. Anthocyanidins are modified by glycosyl and aromatic or aliphatic acyl moieties, resulting in hundreds of anthocyanin molecules that differ in hue and stability. These pigments accumulate in the vacuoles and their stability and hue depend on intravacuolar conditions such as pH, copigmentation with coexisting colorless
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flavonoids and formation of complexes with metal ions [2,3]. Anthocyanins are located mainly in the epidermal tissue, but are also present in the palisade and spongy mesophyll in leaves and in the flesh of fruits and underground storage organs such as sweet potato [4,5]. The biosynthesis of anthocyanins has been characterized in great detail [6,7]. The basic anthocyanin molecule is comprised of two aromatic rings and an oxygen containing heterocyclic ring. One of the aromatic rings is derived from phenylalanine and the second ring from the action of chalcone synthase (CHS), condensing one molecule of p-counaroyl-coA with three molecules of malonyl-coA to produce tetrahydroxy chalcone [8]. CHS is the first committed enzyme in the anthocyanin biosynthetic pathway. The regulation of anthocyanin biosynthesis has also been studied thoroughly and comprises of basic-helixloop-helix (bHLH) transcription factors, interacting with R2R3 MYB transcription factors to activate either all or part of the anthocyanin genes [9]. Many studies generated transgenic plants with either increased concentration or altered composition of anthocyanins in flowers, foliage or fruit by manipulating the expression of the anthocyanin regulatory genes [10–15]. Conversely, very little is known about the stability and catabolism of anthocyanins in plants. Is there turnover (simultaneous synthesis and degradation) of the pigments in living plant tissue, or are the pigments stable once they have accumulated in the vacuoles? Can plants actively degrade anthocyanins, thereby controlling pigment concentration via both biosynthesis and degradation? Since the color of fruits, flowers and leaves is of the utmost economic importance in a variety of agricultural products, a better understanding of anthocyanin degradation may reveal ways in which to inhibit the process and consequently increase pigmentation under conditions of low synthesis. Here we review evidence supporting the existence of anthocyanin turnover and degradation in planta. In addition, we review the knowledge on active anthocyanin degradation processes in in vitro systems such as fruit juices, in an attempt to understand the in planta enzymatic process.
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2. Evidence supporting in planta anthocyanin degradation Anthocyanins often accumulate transiently, appearing and disappearing during plant development or with changes in environmental conditions [16]. Several examples in which anthocyanins are degraded in plants are described below. 2.1. Degradation during development 2.1.1. Loss of red pigmentation in young foliage as it matures Anthocyanins often accumulate in young/juvenile leaves, and degrade as the leaves mature [16]. Since anthocyanins absorb light in both the visible and UV region, their accumulation in young developing foliage may serve as a ‘sunscreen’, protecting the leaves, and in particular their photosynthetic apparatus, from damaging UV light as well as from photoinhibitory high intensities of visible light [17]. These pigments may also provide antioxidant activity, protecting the cells from oxidative damage [18]. As the leaves mature and form protective waxes that reflect sunlight, thereby providing a photoprotective function [19], they often change color from red to green. This loss of red pigmentation may be due to a combination of increased chlorophyll accumulation during leaf expansion and growth, termination of anthocyanin biosynthesis and dilution by growth, and/or increased actively induced anthocyanin degradation when it is no longer required as a photoprotectant. Anthocyanin degradation is apparent in greening of Chrysobalanus icaco (cocoplum) (Fig. 1A) and Photinia fraseri cv. Red Robin leaves as they mature. In both plants, anthocyanins degrade as the leaves develop, resulting in green foliage [20,21]. In Photinia plants, anthocyanin concentration is directly related to the age of the leaf, with high concentrations in young leaves, and a gradual decrease in concentration as the leaves develop, due both to dilution and degradation, resulting in undetectable concentrations of anthocyanins in the mature foliage [21]. In cocoplum, the decrease in anthocyanin concentration as the leaves mature is due in part to dilution of the pigments in the
Fig. 1. Anthocyanin degradation during development of flowers, fruit and foliage. (A) Degradation in developing leaves of Chrysobalanus icaco. (B) Degradation in pepper (Capsicum annuum) mutant 5226 from mature unripe fruit (4) to ripe fruit (5). The Capsicum figure was slightly altered from Fig. 3 of Borovsky et al. [19] with the kind permission of Springer Science + Business Media. (C) Degradation in Brunfelsia calycina flowers from the day of flower opening (0) until 3 days after opening (3). Values are means of four replications SE. The relative anthocyanin concentration is described by either a spectrophotometric reading at 530 nm (A) or the absorption-peak area of the pigments, when separated by HPLC on an RP-18 column (B and C).
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Fig. 2. Anthocyanin degradation due to environmental changes. (A) Cotinus coggygria ‘Royal Purple’ mature leaves from plants grown at 17 8C/9 8C day/night (leaves on left) and transferred to 29 8C/21 8C day/night (leaves on right) temperature conditions. (B) ‘Rosemarie’ pear fruit covered with a two-layered ‘Fuji’ apple wrapping bag (Kobayashi Bag Mfg., Nagano, Japan), for 0 (pear on the left), 1, 2, 3 and 4 (pear on the right) weeks. Photograph was kindly provided by Dr. Wiehann Steyn, Dept. of Horticultural Science, Stellenbosch University, South Africa.
growing tissues and in part to actual degradation [20]. In other plants, such as rose, the change in foliage color from young red to mature green leaves does not involve anthocyanin degradation and is due only to increased chlorophyll synthesis, termination of anthocyanin synthesis and dilution of the anthocyanins in the expanding leaves (unpublished, Liat Shahar). 2.1.2. Anthocyanin degradation in developing fruit Transient accumulation of anthocyanins is seen in some fruits. One example is Capsicum spp. in which several lines accumulate anthocyanins in immature fruit and degrade them as the fruits mature [22,23] (Fig. 1B). Anthocyanins may accumulate and protect the photosynthetic apparatus in the developing fruit. As the Capsicum fruit matures, the anthocyanins in the vacuoles are degraded while chlorophyll degradation in the plastids and the synthesis of carotenoids result in the red, orange and yellow colors of the mature pepper fruit. Anthocyanin degradation is also observed in Sicilian sweet orange varieties, known as blood oranges (Tarocco, Moro e Sanquinello). The pigments accumulate in both the rind and flesh of the fruit resulting in these varieties’ characteristic red color. However, at the late stages of ripening, anthocyanin degradation is observed, resulting in a commercially undesirable partial loss of this color [24]. 2.1.3. Anthocyanin degradation in developing flowers Flowers often change color during development, acting as a signal for pollinators. In most cases, the change in color is due to induction of anthocyanin synthesis, but in others, such as Brunfelsia calycina, anthocyanin is degraded, resulting in a change of flower color from dark purple to white after anthesis [25] (Fig. 1C). Further details on anthocyanin degradation in Brunfelsia are described later. The fading Fa mutants of petunia, lose color during flower development [26,27]. However, the change in color in Fa mutants is not due to anthocyanin degradation, but rather to a change in the vacuolar pH resulting in a change of anthocyanin hue and loss of visual coloration with no change in pigment concentration. 2.2. Degradation due to changes in environmental conditions Anthocyanin concentration in foliage is tightly dependent on environmental conditions such as light quality, light intensity and growth temperature. Foliar anthocyanin accumulation is induced at high light intensities, in particular high UV light, and low growth temperatures [16]. This process is often reversible, with greening of red foliage and stems when growth conditions change to low light intensity and warmer temperatures. The dark red pigmentation of both the young and mature leaves of the garden plant Cotinus coggygria ‘Royal Purple’, occurs only with both low temperatures ( 17 8C/9 8C day/night respectively) and high UV irradiation [28,29]. Anthocyanins of red Cotinus plants, grown at low temperatures, are degraded and their foliage turns green when
covered with a UV screen. Similarly, anthocyanin degradation occurs in red-pigmented plants when the temperature is elevated but UV radiation remains high [28,29] (Fig. 2A). The dependence of anthocyanin concentration on environmental conditions was also demonstrated in Arabidopsis thaliana plants transformed to over-express the transcription factor PAP1 (Production of Anthocyanin Pigments 1) [30]. The mutated arabidopsis plants were dark red under room temperature and high light conditions (22 8C, 440 mmol m 2 s 1), but when transferred to conditions of high temperature and low light conditions (30 8C, 150 mmol m 2 s 1) the plants turned green. The change in color was due to the simultaneous down-regulation of synthesis and degradation of the anthocyanins. These results suggest that there are additional mechanisms involved in the environmental control of anthocyanins, one of which may be controlled degradation [30]. Anthocyanin pigmentation also fluctuates in fruits in response to environmental conditions such as growth temperature and light intensity. An increase in growth temperatures causes preharvest degradation of anthocyanins and a significant decrease in the fruit’s market value in both red pears and apples [31]. Rapid anthocyanin degradation has also been reported in ‘Rosemarie’ and ‘Forelle’ pears, still attached to the tree, when the fruits were covered with light-impermeable bags [32] (Fig. 2B). 2.3. Is anthocyanin turnover in plants dependent on environmental conditions? In addition to specific degradation of anthocyanins due to either developmental or environmental changes, there may be turnover of the pigments in plant tissue, even when no visual change in color is observed. The turnover of anthocyanins in living plant tissues has been followed using various techniques. Pulse-chase treatment of mustard seedlings with radioactive phenylalanine revealed a constant turnover of the pigments after reaching a high and steady level of anthocyanins [33]. The turnover rate of anthocyanins was also followed by treating plant tissues with a specific phenylalanine ammonia-lyase (PAL) inhibitor (aminooxyphenylpropionic acid—AOPP) preventing biosynthesis of the pigments: in petunia, low degradation levels were detected at specific developmental stages, while in carrot cells, no anthocyanin turnover was detected [34,35]. Increased anthocyanin turnover in detached Cabernet Sauvignon grape skin was seen at elevated growth temperatures [36]. The ratio between the anthocyanins comprising Cabernet Sauvignon grape skin color varied, with less degradation of the methylated and acylated pigments [36]. This is consistent with the finding that methoxylation, glycosylation and acylation increase the thermal stability of anthocyanins [37]. This finding suggests that the low levels of anthocyanin observed in many plants when grown at elevated temperatures may be a combination of a slower rate of biosynthesis [38–40] and increased catabolism. The catabolic process may be due either to chemical instability of the pigments or to specific enzymatic activity
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decreasing the pigment concentration in plant tissues, often in parallel to the synthesis process. Metal ions such as magnesium, manganese, tin and copper affect the stability of anthocyanins [2]. These metals accumulate in the vacuoles and form stable complexes with the anthocyanins, thereby affecting their hue and increasing their stability [2]. Stable complexes of magnesium ions with anthocyanins have been isolated from Hydrangea sepals [41]. This phenomenon can be exploited to increase anthocyanin concentration in plants under environmental conditions in which the rates of synthesis are low. For example, treatment of Aster flowers with magnesium salts during the synthesis of anthocyanins caused an increase in pigment concentration in the flowers, without inducing the activity of several of the key enzymes along the biosynthetic pathway [42]. This suggests that there is constant turnover of anthocyanins in the aster flowers, and magnesium treatments slow this process by stabilizing the pigments. Magnesium treatment has a similar effect on other ornamentals, increasing anthocyanin concentration in Anigozanthos, Limonium, Gypsophila and Aconitum flowers grown at elevated temperatures [43]. 3. Enzymatic degradation of anthocyanins Some of the examples presented above suggest that the anthocyanin degradation process is controlled and induced when beneficial to the plant, such as in the loss of foliar pigmentation as the leaves mature [20,21] or when light intensity decreases [32], allowing more light to be available for photosynthesis. In other examples, such as in Cabernet Sauvignon grapes, anthocyanin degradation does not result in a dramatic change in color as the pigments continue to be synthesized in parallel to their catabolism [36]. In this case, the degradation may be due to lower chemical stability of the pigments at elevated temperatures. The question still remains as to whether degradation is due to enzymatic activity and whether degradation can be specifically induced as part of a complex system for controlling plant pigment concentration. Enzymatic studies of anthocyanin degradation in postharvest fruit and fruit juices have revealed several enzymes that degrade anthocyanins in these systems and may be candidates for active in vivo degradation. 3.1. Anthocyanin degradation in fruit after harvest Anthocyanin degradation due to changing environmental conditions has been observed in fruit after harvest. One example of this is anthocyanin degradation in red-skinned pears and apples when the fruit are stored in warm (>10 8C) temperatures [32]. This is similar to the effect of increased growth temperature on the pigmentation of red-skinned pears while still attached to the tree [32]. The search for anthocyanin-degrading enzymes was performed on postharvest fruit, because of the market value of high pigmentation. Postharvest fruit is intermediate between the fruit still attached to the tree and the in vitro systems of fruit juices: postharvest fruit still carry out many of the enzymatic processes occurring before harvest. Intracellular decompartmentation and cell layer reparation begins during storage, and the pigments may be exposed to microenvironmental conditions that differ from those in planta, including enzymes that are not located in the vacuoles when the plant cells are intact. Anthocyanins in litchi fruit are degraded after harvest, accompanied by fruit browning [44–48]. This change in color lowers the commercial value. Polyphenol oxidase (PPO) and peroxidase are thought to be responsible for the anthocyanin degradation in litchi. Peroxidase activity initially increases in the exocarp and during longer-term storage in the endocarp, while
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PPO activity increases during long-term storage in the exocarp [44–48]. Treatment of the fruit after harvest with oxalic acid, known to reduce PPO activity by binding with the active sites of copper to form an inactive complex [49], reduces oxidation levels in litchi fruit and helps maintain low peroxidase activity and delay anthocyanin degradation [48]. Postharvest loss of red color has also been detected in eggplant [50]. In eggplant fruit subjected to chilling injury, browning with concomitant vacuolar disruption, electrolyte leakage, increased pH and decreased anthocyanin concentration were observed in the skin tissue [50]. A b-glucosidase was extracted from eggplant peels that were capable of degrading anthocyanins [51]. 3.2. Enzymatic degradation of anthocyanins in fruit juices The most detailed studies on anthocyanin stability and degradation have been carried out with fruit extracts. These studies form the basis for understanding the processes leading to pigment loss in both postharvest fruit and in juices and wine. One of the main enzyme groups that oxidize anthocyanins in fruit extracts is the PPOs. These ubiquitous enzymes in higher plants are located in the plastids of both photosynthetic and nonphotosynthetic tissues [52]. Both PPO in conjunction with phenolic extracts [53] and anthocyanin b-glucosidase [54] are able to degrade the pigments in ethanolic extracts from the lichti fruit periderm in vitro. It was proposed that anthocyanins are first hydrolyzed by an anthocyanase (b-glucosidase), forming anthocyanidin [53]. These compounds can then be oxidized by PPO and/ or peroxidase. Oxidative products of phenolics, such as 4methylcatechol, resulting from PPO activity may then accelerate anthocyanin degradation via a coupled oxidative reaction [55]. Furthermore, PPO can oxidize anthocyanin degradation products resulting in tissue browning. PPO activity is the main factor responsible for anthocyanin degradation in juice prepared from red muscadine grapes (Vitis rotundifolia cv. Noble). Treating the juice with increased CO2 pressure (dense-phase CO2 processing) partially inhibited PPO and decreased the rate of degradation of anthocyanin, as well as that of other polyphenolic compounds, during refrigerated storage [56]. A specific b-glucosidase was isolated from the fruit juice of Tarocco Sicilian blood oranges and anthocyanin degradation kinetics was followed physiochemically [24]. This enzyme is responsible for the degradation of anthocyanins in both the juice and the ripening fruit [24]. In the presence of chlorogenic acid or caffeoyltartaric acid, PPO increased the rate of anthocyanin degradation in both grape and blueberry extracts and juices [57–60]. PPO also degrades anthocyanins during the drying of plums in the presence of chlorogenic acid [61]. Plastid located PPOs are not likely to degrade vacuolar anthocyanins in living tissue unless decompartmentation occurs. The peroxidases also degrade anthocyanins in fruit extracts, under mildly oxidizing conditions provoked by exogenous application of H2O2. A peroxidase isolated from a Gamay grapevine cell culture degraded anthocyanins in solution and was suggested to do so in the grape fruit as well [62]. Peroxidases are also involved in the degradation of anthocyanins, resulting in loss of color in processed strawberries [63]. Exogenous application of H2O2 on strawberry slices caused a more rapid decrease in anthocyanin concentration during aging relative to non-treated slices, suggesting the involvement of peroxidase in this process. A proposed scheme summarizing the enzymatic processes involved in anthocyanin degradation is presented in Fig. 3. Since peroxidases are present in cell vacuoles [64], unlike the oxidizing PPO enzymes, they are more likely candidates for in planta anthocyanin degradation.
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Fig. 3. Proposed scheme describing anthocyanin degradation and tissue browning in fruit extracts. POD, peroxidase; PPO, polyphenol oxidase. POD is dependant on H2O2 for its activity while PPO is dependant on O2. Three alternative pathways for enzymatic anthocyanin degradation are presented. (1) Coupled oxidation, i.e. reduction of quinones of phenolic compounds to the original phenolic compounds in parallel to oxidation of anthocyanin to anthocyanin quinone. (2) Degradation in two steps: de-glycosylation with anthocyanase (b-glucosidase) and then oxidation with polyphenol oxidase or peroxidase. (3) Direct oxidation of anthocyanin with peroxidase.
3.3. Active in planta degradation of anthocyanins Evidence has been presented of in planta degradation of anthocyanins in ornamentals and fruits. We have also outlined extensive studies on enzymatic anthocyanin degradation in fruit extracts, juices and wine. However, until recently, it was not known if anthocyanin degradation in intact plants involves active enzymatic or merely chemical reactions. Active anthocyanin degradation was first reported in living plant tissue of Brunfelsia calycina flowers. Brunfelsia, a shrub in the Solanaceae family is native to Brazil, and is an ideal model plant for studying anthocyanin degradation because of the dramatic and rapid color changes occurring within 2–3 days after flower opening (Fig. 1C). This loss of color from dark purple to white is dependent on anthocyanin degradation and de novo synthesis of mRNAs and proteins during the different stages of development, well before the start of flower senescence [25]. An additional advantage of this model system is that the same dramatic decrease in pigment concentration occurs in flowers detached on the day of flower opening and grown in a sucrose solution [25]. Similar to what has been shown in fruit juices and wine, oxidative reactions are crucial for anthocyanin degradation in Brunfelsia, since treatment of detached flowers with the reducing reagents dithiothreitol (DTT) or glutathione inhibit degradation [25]. Assuming that in planta anthocyanin degradation occurs in the cell vacuoles, oxidizing enzymes found in the vacuole, such as peroxidases are more likely candidates than those accumulating elsewhere in the cells, such as PPO located in the plastids. A dramatic increase of total peroxidase activity was detected in correlation with the onset of anthocyanin degradation in Brunfelsia flower petals, strengthening the notion that these enzymes are involved in the process [25]. Nevertheless, the possibility that anthocyanins are transferred out of the vacuoles before degradation exists and further studies are needed to determine the site of degradation.
The b-glucosidases may also be involved in the in planta degradation process, since they increased the degradation rate of anthocyanins in fruit extracts of both litchi and eggplant [51,54]. DGluconic acid, a specific b-glucosidase inhibitor, significantly decreased the rate of degradation in Brunfelsia flowers (unpublished, Hila Vaknin and Raya Liberman). One possible explanation is that b-glucosidase activity precedes the oxidation reaction with peroxidase as a candidate oxidizing enzyme. Stripping the anthocyanin molecules of their glucose residues may enable better access for the peroxidase enzyme and faster degradation. Hence, it is likely that anthocyanin degradation is a complex process involving more than one enzyme. 4. Concluding remarks Anthocyanin degradation occurs in different plant organs due to a variety of environmental and developmental conditions. In some cases, where there is no apparent benefit to the plant, anthocyanin degradation occurs due to changes in the vacuoles that decrease the stability of the pigments and cause either chemical degradation or increased vulnerability to degrading enzymes (e.g. b-glucosidases, peroxidases) present in the vacuoles. For example, the degradation of anthocyanins in Cabernet Sauvignon grape skin exposed to high temperatures may be due to chemical degradation, but may also be a product of the activity of peroxidase enzymes induced due to the thermal stress [36]. A second example is the effect of vacuolar pH on the stability of the pigments. Changes in the vacuolar pH, such as increased pH in senescing tissue, may decrease the stability of the anthocyanins and cause chemical degradation [2]. In other cases, such as in Brunfelsia flowers or in photosynthetic tissues, there are clear reasons why the degradation of anthocyanins benefits the plant. In these cases the process may be regulated by and dependant on specific genes and proteins. In Brunfelsia
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calycina flowers, the dramatic change in color due to anthocyanin degradation is most probably a signal for the pollinators and is regulated at the gene and protein levels [25]. In photosynthetic leaves, since anthocyanin accumulation decreases their photosynthesis rate but protects the tissue from free radical scavenging [65], degradation under low light and high temperatures will enable more efficient photosynthesis under conditions that are not prone to photo-oxidation. Revealing the different catabolic processes occurring in plants may allow for improved treatments that control plant tissue color. Increased stability of the pigment molecules may be achieved either by treatments such as increasing the concentration of the stable metal–anthocyanin complexes [42], or molecular manipulation of the pigments to form more stable anthocyanins. Recently, genes encoding anthocyanin acyl transferases in Arabidopsis thaliana were identified, and their activities were shown to increase the stability of the pigments. These may be candidate genes for engineering more stable pigment molecules [66]. Understanding the regulation and enzymatic processes involved in degradation may facilitate genetic manipulation in plants in order to enable increased anthocyanin concentrations under broader environmental conditions. Acknowledgement The author would like to thank Rinat Ovadia for helpful comments and suggestions. References [1] A.J. van Tunen, J.N.M. Mol, Control of flavonoids synthesis and manipulation of flower color, Plant Biotechnol. 2 (1991) 94–125. [2] G. Mazza, E. Miniati, Color stabilization and intensification, in: G. Mazza, E. Miniati (Eds.), Anthocyanins in Fruits, Vegetables, and Grains, CRC Press, Boca Raton, FL, 1993, pp. 1–20. [3] Y. Tanaka, N. Sasaki, A. Ohmiya, Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids, Plant J. 54 (2008) 733–749. [4] T. Sugawara, K. Igarashi, Cultivar variation and anthocyanins and rutin content in sweet cherries (Prunus avium L.), J. Jpn. Soc. Food Sci. Technol.—Nippon Shokuhin Kagaku Kogaku Kaishi 55 (2008) 239–244. [5] N.M. Hughes, C.B. Morley, W.K. Smith, Coordination of anthocyanin decline and photosynthetic maturation in juvenile leaves of three deciduous tree species, New Phytol. 175 (2007) 675–685. [6] B. Winkel-Shirley, Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology, Plant Physiol. 126 (2001) 485–493. [7] E. Grotewold, The genetics and biochemistry of floral pigments, Annu. Rev. Plant Biol. 57 (2006) 761–780. [8] O. Yu, M. Matsuno, S. Subramanian, Flavonoid compounds in flowers: genetics and biochemistry, in: J.A. Teixeira da Silva (Ed.), Floriculture, Ornamental and Plant Biotechnology, vol. I, Global Science Books, Ltd., UK, 2006. [9] A.C. Allan, R.P. Hellens, W.A. Laing, MYB transcription factors that colour our fruit, Trends Plant Sci. 13 (2008) 99–102. [10] M.M. Ben Zvi, N.Z. Florence, T. Masci, M. Ovadis, E. Shklarman, H. Ben-Meir, T. Tzfira, N. Dudareva, A. Vainstein, Interlinking showy traits: co-engineering of scent and colour biosynthesis in flowers, Plant Biotechnol. J. 6 (2008) 403–415. [11] J.M. Bradley, S.R. Rains, J.L. Manson, K.M. Davies, Flower pattern stability in genetically modified lisianthus (Eustoma grandiflorum) under commercial growing conditions, N. Z. J. Crop Hortic. Sci. 28 (2000) 175–184. [12] K.M. Davies, K.E. Schwinn, S.C. Deroles, D.G. Manson, D.H. Lewis, S.J. Bloor, J.M. Bradley, Enhancing anthocyanin production by altering competition for substrate between flavonol synthase and dihydroflavonol 4-reductase, Euphytica 131 (2003) 259–268. [13] F. Quattrocchio, J.F. Wing, K. van der Woude, J.N.M. Mol, R. Koes, Analysis of bHLH and MYB domain proteins: species-specific regulatory differences are caused by divergent evolution of target anthocyanin genes, Plant J. 13 (1998) 475–488. [14] E. Butelli, L. Titta, M. Giorgio, H.P. Mock, A. Matros, S. Peterek, E. Schijlen, R.D. Hall, A.G. Bovy, J. Luo, C. Martin, Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors, Nat. Biotechnol. 26 (2008) 1301–1308. [15] Y. Tanaka, A. Ohmiya, Seeing is believing: engineering anthocyanin and carotenoid biosynthetic pathways, Curr. Opin. Biotechnol. 19 (2008) 190–197. [16] L. Chalker-Scott, Environmental significance of anthocyanins in plant stress responses, Photochem. Photobiol. 70 (1999) 1–9. [17] W.J. Steyn, S.J.E. Wand, D.M. Holcroft, G. Jacobs, Anthocyanins in vegetative tissues: a proposed unified function in photoprotection, New Phytol. 155 (2002) 349–361.
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