Anthocyanin stability and degradation in plants

ABSTRACT

Anthocyanins, a flavonoid group of polyphenolic compounds, have evolved in plants since the land was colonized by plants. These bioactive compounds play critical roles in diverse physiological processes. They are synthesized in the cytosol and transported into the vacuole for storage or to other destinations, where they function as bioactive molecules. The mechanisms of anthocyanin synthesis and transport have been well studied. However, the precise regulation of the mechanisms of anthocyanin degradation remains to be elucidated. In this review, we highlight recent progress in the understanding of the characteristics and functions of anthocyanins and class III peroxidases, as well as of the existing evidence of the effects of class III peroxidases on the degradation of anthocyanins and the possible regulatory mechanisms involved.

1. Characteristics of anthocyanin and factors affecting anthocyanin degradation

Anthocyanins are the largest group of plant flavonoid pigments, with a color ranging widely from pale yellow to blue, but are mainly responsible for the red, purple, and blue colors of flowers, fruits, seeds, and vegetative tissues.^1,2 The content of anthocyanins is fairly high in mature fruits, such as grapes, strawberries, apples, red pears, and blood oranges. More than 600 types of anthocyanins have been identified in nature so far, but six forms of aglycones, including pelargonidin, cyanidin, delphinidin, peonidin, petunidin and malvidin, are the most common in horticultural plants.^3,4

Anthocyanins are soluble phenolic compounds that are most abundant in the cell vacuoles of the epidermis and subcutaneous tissues. The location of anthocyanins in cells is different, which affects the color of various tissues. For example, the anthocyanins accumulated in the cell vacuoles give the tissue a purple-red appearance, whereas the anthocyanins in the cytoplasm are oxidized, resulting in the brown color of the oxidized product.⁵ Anthocyanins are modified by glycosyl and aromatic or aliphatic acyl moieties to produce hundreds of anthocyanin molecules with different colors and stability. Notably, their color is closely related to the structure and mainly to the number of hydroxyl groups on the B-ring. The stability and color of these pigments depend on the specific external conditions of the vacuole, such as pH, the coexisting synthesis of colorless flavonoids, and the formation of complexes with metal ions.^6–8 In vitro, at low pH (generally at pH < 3), anthocyanins are redder and more stable, whereas they are colorless in a weakly acidic (pH 3–6) environment. The increase in alkalinity (pH > 6) causes instability and blue appearance of anthocyanins. The rise of pH in the vacuoles of aging tissue reduces the stability of anthocyanins and leads to their degradation.⁹ Anthocyanins interact with colorless flavonoids coexisting in the vacuoles, an interaction known also as co-pigmentation, which stabilizes anthocyanins and changes their colors, deepening the color to the blue range.^7,10,11 In addition, certain metal ions are also involved in such interactions, such as magnesium, copper, tin, manganese, and iron. Once accumulated in the vacuole to a certain extent, they quickly form a stable complex with anthocyanins, thereby affecting plant colors. This impact on petal coloring has been confirmed in many flowers that form anthocyanin-metal complexes.^12–14 Studies have shown that metal ions such as Fe³⁺ are critically involved in the formation of the blue flowers of tulips.¹⁵ The biological significance of anthocyanins has been widely recognized. Anthocyanin synthesis has been extensively studied, and anthocyanin degradation has also been gradually attracting increasing research interest. Existing evidence has shown that a relationship exists between anthocyanin formation and the temperature of the environment. At low-temperature stress, such as in winter, anthocyanins exert protective functions on the leaves of evergreen plant species, reducing the damage of plants caused by such stress.¹⁶ On the other hand, high-temperature stress also triggers plant protection mechanisms. For example, anthocyanins reduce high temperature injuries to pear peel, leading to the degradation of the respective anthocyanin in the pear peel.¹⁷ Anthocyanin degradation under high-temperature and low-light conditions has been also established in Arabidopsis plants with red color overexpressing the PAP1 transcription factor gene; hence, degradation of anthocyanins occurs in plants encountering environmental changes such as those in the temperature.¹⁸ In addition, the development of various plant organs also affects the degradation of anthocyanins. Studies have shown that such degradation is likely caused by the turnover of pigments in the vacuoles and the activity of related enzymes, which has laid the foundation for a further study of anthocyanin degradation in plants.^19–21 Besides, evidence exists that the inhibition of phenylalanine (PAL) synthesis causes degradation of anthocyanins in plant. For example, PAL pulse-chase treatment led to alterations in anthocyanin metabolism in mustard seedlings. Aminooxy-phenylpropionic acid (AOPP) is a PAL-specific inhibitor that was found to reduce the anthocyanin content in vitro buds of petunia flowers, which most likely affected the degradation of anthocyanins in the specific development stage of petunia flowers.²²

According to the above studies, many factors have been identified to affect the degradation of anthocyanin. In recent years, a new factor affecting anthocyanin degradation began to appear in the public view. It is well known that anthocyanin is synthesized in the cytoplasm and transported to vacuoles for storage. Reactive oxygen species (ROS) is produced in the mitochondria. No direct connection seems to exist between the two, but hydrogen peroxide can enter the vacuole through the cell membrane.^23–25 There is no superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase, or ascorbic acid-glutathione cycle enzyme in the vacuole.²⁶ Thus, the removal of ROS depends mainly on the activities of antioxidants, such as glutathione, ascorbic acid, and phenolic compounds. Class III peroxidase is thought to reduce the escape of hydrogen peroxide in other organs by using anthocyanins and other substances as substrates.^27,28

2. Class III peroxidase classification and functions

Peroxidase activity can be induced in the process of plant defense, where it exerts a key role in several metabolic reactions. Basically, four peroxidases exist in higher plants: glutathione peroxidase (EC 1.11.1.9), catalase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11, class I peroxidase), and classical secreted plant peroxidase (EC 1.11.17, class III peroxidase, Prxs).^29,30

Class III peroxidase, known as plant secretion peroxidase, is widely distributed in organisms and considered to be a potential multifunctional protein that can participate in auxin metabolism, cell wall extension and hardening, and plant resistance to pathogens.^31–35 This enzymatic class catalyzes the redox reaction between hydrogen peroxide (H₂O₂), as an electron acceptor, and a variety of electron donors, such as auxin, phenolic compounds, and secondary metabolites.^36,37 Studies have shown that class III peroxidase is a ubiquitous enzyme in the plant cell walls and vacuoles, and is also involved in the accumulation of most secondary metabolites, many of which can be used as class III peroxidase substrates.^38,39

Class III peroxidases are plant-specific oxidoreductases. The gene structure of Class III peroxidases, their key amino-acid residues, and protein sizes are highly conserved among orthologs and paralogs.^40,41 There are many peroxidase isomers, which determine the diversity of their enzyme-catalyzed reactions. In the whole life cycle of plants, peroxidases participate in a number of reactions of essential plant physiological processes, such as auxin metabolism, lignin and suberin formation, cross-linking of cell wall components, defense against pathogens or cell elongation, phytoalexin synthesis, and the ROS and reactive nitrogen species (RNS) metabolism.^36,37 Notably, class III peroxidases are involved in numerous plant metabolism processes due to their N- and C-terminal signals and their locations in the vacuoles or cell walls. Class III peroxidases can also identify various substrates with polymorphism.⁴²

Because of the wide range of peroxidase substrates (lignin subunits, lipid membranes, and certain amino-acid side chains) and the highly conserved amino-acid sequence in the multi-gene family with possible redundant functions, the effect of any single peroxidase in vivo is insignificant. And the exact role of any peroxidase in the body is still limited. There are two difficulties in the research of peroxidase gene function. On the one hand, the modification of the expression of a single gene often results in the absence of a visible mutant phenotype, because it is compensated by redundant peroxidase genes. On the other hand, a peroxidase can react with a variety of plant compounds in vitro, although it is still uncertain which compounds are present in plant substrates. Therefore, the in vitro activity cannot provide accurate information concerning the specific activity of different subtypes.⁴³

3. Advances in the research on the effect of class III peroxidase on the anthocyanin degradation

Peroxidases are enzymes that use anthocyanins as a substrate. Anthocyanins can be oxidized by peroxidases and lose their color. In this process, they are likely to undergo polymerization, forming a precipitate and leaving the solution. In addition, anthocyanins may change their colors and become brown due to oxidation.⁴⁴

The mechanisms of enzymatic degradation of anthocyanins in plants are not yet clear, but three enzyme families in fruit juice are involved in the degradation of anthocyanins: polyphenol oxidase (PPO), class III peroxidase, and β-glycosidase.^20,45,46 Studies have shown that PPOs and peroxidases are the main enzyme families responsible for anthocyanin degradation. The research on the degradation of anthocyanin by PPOs has been mainly conducted in litchi (Litchi chinensis Sonn.). Litchi has a fruit with high anthocyanin content, the color of whose pericarp changes into brown after harvest, accompanied by a certain extent of anthocyanin degradation.^6,47,48 Subsequent studies showed that anthocyanin degradation during browning was also a mechanism by which litchi protects its pericarp color. The activity of PPOs on exogenous substrates such as catechol and 4-methylcatechol was confirmed in the litchi pericarp.^49,50 Further investigations revealed that PPOs could degrade anthocyanins only in the presence of exogenous catechol, and thus an anthocyanin-PPO-phenol model was established.⁵¹ To further elucidate enzymatic anthocyanin degradation in litchi, anthocyanin degradation enzyme (ADE) was obtained by purification and identified as laccase (LAC). Finally, ADE/LAC was identified as an intracellular enzyme, which was involved in the biological processes before pericarp senescence, and epicatechin was involved as an endogenous substrate in anthocyanin degradation coupled with ADE/LAC.^52,53 However, PPO was also found to be located in the cytoplasm, whereas peroxidase was predominantly located in the vacuole. Thus, peroxidase activities are more likely to cause anthocyanin degradation in most plant species.^27,54 Environmental stresses such as high temperature and low light can induce the activity of anthocyanin peroxidase, promoting anthocyanin degradation.^8,55 In this review, we have focused on recent developments in the fIeld and have presented our current understanding of the molecular mechanisms underlying the degradation of anthocyanins by class III peroxidases.

Earlier research established that gamay grapevine vacuole peroxidase participated in the oxidation of anthocyanins. Gamay anthocyanin aglycones were degraded by class III peroxidases, with oxidation that was strictly dependent on the level of H₂O₂. Gamay grapevine cell culture that anthocyanin aglycones can degrade like peonidin, delphinidin, and cyanidin, but the aglycones do not degrade, suggesting that class III peroxidase may play a role in the degradation of anthocyanin in vivo.⁵⁶

Recent evidence showed that heat stress in grape berries activated Prx31 at the transcriptional and enzymatic activity levels, which was correlated with the observed decrease in anthocyanin levels. The overexpression of VviPrx31 under high-temperature stress reduced the anthocyanin content in Petunia hybrida petals, which further confirmed that high temperature stimulates the activity of class III peroxidases, causing the degradation of anthocyanins in ripening grape berries.^57–59

A class III peroxidase, RsPrx1, was isolated in vitro from the roots of Chinese red radish. It can metabolize pelargonidin in plant organs (except for the green outer epidermis of the roots) and is likely to target the vacuoles. RsPrx1 and the homologous sequences were proposed to target the vacuoles, leading to modifications of stored anthocyanins.⁴⁴

Research on litchi showed that the peroxidase isolated from litchi peel can degrade anthocyanins in the presence of guaiacol and H₂O₂; guaiacol is a simple phenol, although the peroxidase in litchi cannot directly oxidize anthocyanins.⁵¹ The requirement of guaiacol for the degradation of anthocyanins indicates a coupled oxidation mechanism: peroxidase-phenol-H₂O₂-anthocyanins, in which the effect of peroxidase on anthocyanins is indirect, and the specific performance is that peroxidase oxidizes guaiacol through H₂O₂, leading to degradation of anthocyanins.⁶⁰

Research on Brunfelsia calycina supported the hypothesis of the enzymatic degradation of anthocyanin degradation in plants. BcPrx01 is a class III peroxidase, which plays an important role in the degradation of complex anthocyanins in flowers. Vacuolar class III peroxidase (Prx) was isolated from B. calycina flowers, which confirmed that Prx is involved in the degradation of anthocyanins in plants. It is co-localized with these pigments in the vacuoles of the petals and significantly induces the mRNA and protein levels of BcPrx01 in the process of anthocyanin degradation. This study showed that the degradation of anthocyanins in B. calycina flowers may be mediated by a new vacuolar class III peroxidase BcPrx01.²⁷

4. Environmental stress and peroxidases correlating to anthocyanin degradation

Plants in nature inevitably suffer from various environmental threats, they would produce a series of reactions. Secondary metabolites play an indispensable role in this process. To ensure plant homeostasis and stabilize peroxidase activity, plants have evolved a variety of ‘ self-help ‘ mechanisms to defend, such as accelerated degradation of anthocyanin.

Prxs have a wide range of physiological functions, mainly in assisting plants to resist external injuries.⁶¹ Many studies have shown that the activity of most Prxs in plant systems is vacuolar and is composed of a vacuolar basic Prx isoenzyme.^62–64 The vacuole is not only an important signal transduction and defense base of plants, but also a significant part of secondary metabolites accumulation in plants. And secondary metabolites (e.g., anthocyanins) are reducing substrates of class III peroxidases.^37,65 In plants, H₂O₂ is a potentially harmful molecule, which is produced under several types of stress, but it also has a signal transmission function in important biological processes.⁶⁶ In addition, H₂O₂ is a key molecular substance that can react timely in response to changing environmental conditions.^40,67

Two important characteristics of Prxs valuable in the response to changing environments are to be noted. On the one hand, a large number of plant secondary metabolites are reductant substrates that supply cell walls and vacuoles. On the other hand, the level of H₂O₂ present/produced in plant cells can diffuse and be used by the cell wall or vacuole Prxs to complete its peroxidation catalytic cycle.⁶⁴

Under high light, reactive oxygen species such as H₂O₂ are mainly produced in chloroplasts.⁶⁸ When plants encounter too much light stress, chloroplasts could not timely defense H₂O₂, H₂O₂ is easy to spread out from biological membranes, which requires vacuole coupling Prx/secondary metabolites to stabilize the level of H₂O₂.⁶⁹ In other words, Prxs in green plant leaves oxidize H₂O₂ through secondary metabolite substrates such as anthocyanins under excessive light, and Prxs coupled with secondary metabolite substrates act as H₂O₂ sink/buffer. Evidence exists that phenols may form a synergistic regeneration cycle with ascorbic acid, enabling Prx/phenols to remove high doses of H₂O₂. Prxs oxidize phenol compound to phenolic radical, and Prxs can also oxidize ascorbic acid to monodehydroascorbate. MDHA will spontaneously interact to produce dehydroascorbic acid, which can be reduced to ASA through cytosolic dehydroascorbic acid reductase of cytosolic, thus forming a synergistic cycle of Prxs/Ph/AsA and H₂O₂. In addition, the coupling oxidation of H₂O₂ and Prxs promoted the degradation of anthocyanin in the vacuole, which also maintained the stability of H₂O₂ to a certain extent.⁶⁴ Class III peroxidase CrPrx1 in the leaves of the medicinal plant Catharanthus roseus is a multifunctional enzyme that can oxidize a series of secondary metabolites (such as phenols) presenting an important sink/buffer for excessive H₂O₂.⁷⁰

Another kind of environmental factor temperature also has a noticeable relationship with plant anthocyanin. When plants are exposed to low or high temperature stress, plants can respond to these environmental stresses by regulating anthocyanin accumulation and degradation. For example, the leaves of Galax urceolata rapidly accumulate anthocyanin and exert corresponding antioxidant effects, so that the color of the leaves change from green to red to resist winter low temperature.¹⁶ Studies have shown that under high temperature conditions, the pericarp temperature is increased by 15°C by radiation heating, resulting in the degradation of anthocyanin.⁷¹ It was further proved that the degradation of anthocyanin in ‘Forelle pears’ and ‘Royal Gala apples’ was accelerated at high temperature.⁷²

Other studies found that Vitis vinifera L. cv was used as the material, and it was found that high temperature could lead to the degradation of anthocyanin in grape skin. By detecting the changes of anthocyanin components in grape skin under high temperature, the mRNA and UFGT enzyme activities of anthocyanin synthesis-related genes, and the ¹³C-labeled anthocyanin experiment, it was further explained that high temperature caused the degradation of anthocyanin rather than inhibiting the accumulation of anthocyanin.⁷³ There are various reasons for anthocyanin degradation, and enzymatic degradation has gradually entered people ‘ s vision. Grape cells studies have shown that peroxidase in vacuoles is involved in anthocyanin degradation in the presence of H₂O₂,⁵⁶ and under high temperature stress, reactive oxygen species such as H₂O₂ are mainly induced in mitochondria through respiration,^74,75 so we summarize a simple pathway. High temperature stress stimulated mitochondria to produce more H₂O₂ and induced the expression of class III peroxidase gene, thereby degrading anthocyanin.

Peroxidase is a multifunctional enzyme. When it is transferred into apple (Malus domestica), the increase of peroxidase activity can provide protection for H₂O₂ and improve plant tolerance to high temperature.⁷⁶ The peroxidase activity of strawberry (Fragaria x ananassa cv. Camarosa) also increased significantly under high temperature stress. Peroxidase is likely to repair the cell membrane damage caused by high temperature.⁷⁷ In addition, high temperatures also caused fading of the red fleshed fruit of Malus profusion, stimulated plant mitochondria to produce more H₂O₂, and induced an increase in peroxidase activity. In crabapple fruit, the coupling oxidation of peroxidase and H₂O₂ promoted the degradation of anthocyanin in the peel. Excessive H₂O₂ can cause lipid peroxidation injuries to cells, whereas the production of peroxidase effectively protects apple tissues from H₂O₂ damage.⁷⁸

5. Conclusions and perspectives

At present, there are detailed molecular mechanisms for anthocyanin synthesis, but little is known about anthocyanin degradation and stability in plants. Anthocyanin degradation exist in different plant organs and is affected by plant development conditions and environmental conditions. For example, due to the increase of temperature and the decrease of light intensity, red pigments in flowers, leaves and fruits are lost.^79,80 The stability of anthocyanin depends largely on the microenvironment of vacuoles. Vacuole is a key participant in plant response to various stress conditions and plays a key role in maintaining cell homeostasis, such as regulating cell PH, maintaining metal ion balance, osmotic regulation, and storing primary and secondary metabolites.⁸¹ Therefore, changes in the vacuole microenvironment, such as the increase of PH, changes in the concentration of co-pigments or metal ions may reduce the stability of anthocyanin and further cause anthocyanin degradation.^8,9 The factors causing anthocyanin degradation are chemical degradation and enzymatic degradation. Chemical degradation was affected by pH, temperature, light and anthocyanin structure. As for enzymatic degradation, among the candidate enzymes for anthocyanin degradation, such as polyphenol oxidase (PPO) and peroxidase are involved in the degradation of anthocyanin activity. PPO is mainly located in the plastids of photosynthetic and non-photosynthetic tissues, so it is unlikely to degrade anthocyanin in living tissues, in which no cell decompartmentation occurs.¹⁹ Therefore, the enzymatic degradation of anthocyanin in plant tissues is likely to play a key role in maintaining plant pigmentation regulation. It was also confirmed in grape cells that peroxidase in vacuole was involved in anthocyanin degradation in the presence of H₂O₂.⁵⁶ Moreover, when plants encounter stress such as high temperature or excessive light, they will induce mitochondria and chloroplasts to produce more H₂O₂.^69,74,75 Evidence exists that phenols may form a synergistic regeneration cycle with ascorbic acid, enabling Prx/phenols to consume high doses of H₂O₂.⁶⁴

Based on the findings of previous studies, we summarized a model diagram of anthocyanin degradation by class III peroxidases (Figure 1). Anthocyanins are synthesized in the cytoplasm by its synthetic precursor phenylalanine through flavonoid metabolic pathways, and then transported to vacuoles through further processing of endoplasmic reticulum.⁸² This transport process is mainly completed by glutathione S-transferase (GST). GST combine with anthocyanin to protect anthocyanin from being transported to the vacuole membrane along the way, and then transported into vacuoles through the anthocyanin transporters of the multidrug resistance-associated proteins (MRP), a specific family of ABC proteins, and multidrug and toxic compound extrusion transporter (MATE) transporters.⁸³ Anthocyanins are not stable after entering vacuoles, and some of them degrade to some extent due to abiotic stresses. When plants encounter high temperature and excessive light stress, they stimulate mitochondria and chloroplasts to produce more H₂O₂.^69,74,75 Excessive H₂O₂ will lead to membrane oxidation, such as lipid peroxidation.⁸⁴ Another part of H₂O₂ enters the vacuole, which carries out a series of regulation of H₂O₂. On the one hand, Prxs combine with secondary metabolite substrates (SM) to buffer H₂O₂, Prxs oxidize secondary metabolite substrates (SM) to secondary metabolite radicals (SM·), and reduce H₂O₂ to water. On the other hand, the regeneration cycle of Prxs and ascorbic acid/phenols also has the effect of maintaining the steady state of H₂O₂.⁶⁴ oxidize phenol compound (Ph) to phenolic radical (Ph·), and Prxs can also oxidize ascorbic acid (ASA) to monodehydroascorbate (MDHA). MDHA will spontaneously interact to produce dehydroascorbic acid (DHA), which can be reduced to ASA through cytosolic dehydroascorbic acid reductase (cDHAR) of cytosolic, thus forming a synergistic cycle of Prxs/Ph/AsA and H₂O₂. In addition, the coupling oxidation of H₂O₂ and Prxs promoted the degradation of anthocyanin in the vacuole, which also maintained the stability of H₂O₂ to a certain extent.

Figure 1.

Class III peroxidases (Prxs) in the vacuoles coupled with H₂O₂ oxidation to promote the degradation of anthocyanin. Anthocyanins are synthesized in the cytoplasm by phenylalanine through flavonoid metabolic pathways and are then transported to the vacuoles through further processing in the endoplasmic reticulum. Anthocyanins are transported into the vacuoles by anthocyanin transporter including glutathione S-transferase (GST), multidrug resistance-associated proteins (MRPs) and MATEs (multidrug and toxic compound extrusion transporters). However, anthocyanin is easily degraded due to high temperature or excessive light stress after entering the vacuole. The main manifestation was the increase of H₂O₂ content. Excessive H₂O₂ entered the vacuole leads to a coupled oxidative reaction Prxs and anthocyanin resulting in the degradation of anthocyanin. Besides, multiple cycles centered on Prxs were formed under these stress conditions to maintain the balance of H₂O₂ in plants.For one thing, Prxs oxidize secondary metabolite substrates (SM) to secondary metabolite radicals (SM·). For another, Prxs oxidize phenol compound (Ph) to phenolic radical (Ph·), and oxidize ascorbic acid (ASA) to monodehydroascorbate (MDHA), MDHA will spontaneously interact to produce dehydroascorbic acid (DHA), which can be reduced to ASA through cytosolic dehydroascorbic acid reductase (cDHAR) of cytosolic, thus forming a synergistic cycle of Prxs/Ph/AsA and H₂O_2.

The appearance quality of horticultural crops has important economic value. The color change of related horticultural crops caused by anthocyanin degradation will affect their sales. Therefore, improving the stability of related crops and reducing the degradation of anthocyanin are of great significance to the development of horticultural crops industry. However, at present, the mechanisms of anthocyanin degradation are still poorly understood. Future research will focus on which anthocyanin aglycone degrades more easily, which enzymes are involved in the degradation of these aglycones, and which signal network transduction pathways these enzymes are involved in. These are of great significance for us to understand the cascade of physiological and molecular signals in the process of anthocyanin degradation and explore the methods to improve the quality of horticultural crops.

Funding Statement

This project was supported by grants from National Key Research and Development Program of China [2018YFD1000200]; the National Natural Science Foundation of China [32122080, 31972375]; and Shandong Province [ZR2020YQ25]

Disclosure statement

The authors declare that they have no coflict of interest.

Author contributions

Y.W.Z., C.K.W. and X.Y.H. Investigation, Writing - original draft. D.G.H. Visualization, Writing - review & Editing, Supervision, Funding acquisition.

Highlights

• We summarize the factors affecting anthocyanin degradation and elaborated the research advances on class III peroxidase effects on anthocyanins degradation.