Model for light signaling regulation of anthocyanin biosynthesis in light-dependent horticultural plants. GST = glutathione S-transferase; UVR8 = ultraviolet resistance locus 8; CRYs = cryptochromes; PHOTs = phototropins; PHYs = phytochromes; PIF = phytochrome interacting factor; COP1 = constitutively photomorphogenic 1; DET1 = de-etiolated; FUS = fusca; HY5 = elongated hypocotyl 5.
The five mechanisms of light-independent anthocyanin biosynthesis. × denotes regulatory failure, ? denotes regulatory relationship unknown, dashed red lines denote inhibition, and green arrows denote activation. ABG = anthocyanin biosynthetic gene; TE = transposable elements.
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Anthocyanins constitute an important class of plant flavonoids responsible for color formation and antioxidant processes in flowers, fruits, and other plant organs. Anthocyanin biosynthesis during the maturation of horticultural plant products is influenced by environmental factors and plant hormones. In most horticultural plants, anthocyanin biosynthesis is light-dependent. When subjected to artificial darkness using bags, the fruits and flowers of light-dependent horticultural plants cease anthocyanin production, thereby turning white or yellow. In contrast, fruits and flowers of light-independent horticultural plants do not entirely depend on light for coloring, and anthocyanins accumulation occurs even in complete darkness, suggesting distinct pathways of anthocyanin biosynthesis. This review explores the molecular mechanisms of light-independent anthocyanin biosynthesis in horticultural plants, such as fruits, vegetables, and ornamental plants. In addition, horticultural plants have been categorized into five broad classes based on their underlying mechanisms of action of light-independent anthocyanin.
Anthocyanins comprise an important class of flavonoid compounds in plants and play a vital role in color formation of plant reproductive organs, such as flowers and fruits. Anthocyanins constitute a class of secondary metabolites in plants that are well known for attracting pollinating insects, rendering color to the vegetative tissue of plants, and protecting plants from ultraviolet irradiation and pest attacks. In addition, anthocyanins display a strong antioxidant capacity, which can reduce the damage to plant tissues from reactive oxygen species and other stresses (Kong et al. 2003). Anthocyanins provide health benefits to humans in terms of anti-ageing effects and in the prevention and treatment of cardiovascular diseases (Kong et al. 2003; Lin et al. 2017; Martin et al. 2011; Salehi et al. 2020). The direct precursor of anthocyanin is phenylalanine, and its conversion to anthocyanin involves the catalytic reactions of phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H), 4-coumarate CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT). Structural genes encoding anthocyanin enzymes are transcriptionally regulated by well-characterized transcription factors belonging to the MYB, basic helix–loop–helix (bHLH), and WD40 transcription families. This MYB–bHLH–WD40 (MBW) regulatory complex plays a crucial role in coordinating the activity of the abovementioned structural enzyme genes, ensuring anthocyanin production in all horticultural plants that have been characterized and addressed in several reviews to date (Allan et al. 2008; Cappellini et al. 2021; Chaves-Silva et al. 2018; Liu et al. 2018).
Anthocyanin biosynthesis is not only affected by the MBW complex but also by various environmental factors, such as light and temperature. These factors alter the coloration of plant reproductive organs by regulating the MBW complex at both the transcription and post-translational levels. Light is one of the most important environmental factors affecting anthocyanin biosynthesis (Jiao et al. 2007). Plants receive light signals through photoreceptors, which form a cascade of intracellular secondary messenger systems via signal transduction to regulate anthocyanin biosynthesis (Jaakola 2013). Currently, the identified photoreceptors include phytochromes (PHYs), cryptochromes (CRYs), ultraviolet-B photoreceptor UVR8, and phototropins (PHOTs), which receive light signals from ultraviolet-A and ultraviolet-B to red/far-red light (Christie et al. 2012; Li et al. 2012; Rizzini et al. 2011). Upon receiving a light signal, the photoreceptor undergoes signal transduction and transmission to activate the downstream light signal transduction elements. In light signal transduction, constitutive photomorphogenic 1 (COP1) (Osterlund et al. 2000), suppressor of PhyA (SPA1) (Zuo et al. 2011), de-etiolated 1 (DET1) (Yanagawa et al. 2004), and phytochrome kinase substrate 1 (PKS1) (Gyula et al. 2003) are involved in the post-transcriptional and -translational regulation of various growth and developmental responses, including photomorphogenesis, circadian rhythm, and secondary metabolite synthesis (Casal 2013). For instance, in Arabidopsis, COP1 interacts with upstream photoreceptor proteins while participating in downstream transcription factor degradation and target gene expression. Therefore, they are considered to be central regulators of light signal transduction (Ma et al. 2002). Under dark conditions, COP1 is located in the nucleus and forms a complex with SPA, targeting bZIP-like transcription factor HY5, and degrades transcription factors via proteasome-mediated ubiquitination (Stracke et al. 2010). Under light conditions, COP1 is inhibited by activated photoreceptor proteins, such as under blue light. CRY1 physically interacts with SPA, isolating it from COP1, thereby preventing the formation of a COP1–SPA protein complex. Subsequently, visible light promotes COP1 transport from the nucleus to the cytoplasm and inhibits COP1 expression for a long period of time, leading to a decrease in COP1 activity and HY5 and its binding to target proteins, thus promoting photomorphogenesis in plants (Lau and Deng 2012). In pears, PpyCOP1 may be involved in regulating anthocyanin accumulation through direct interactions with PpyHY5 (Tao et al. 2018). In addition, MdCOP1 in apples is involved in its degradation by the ubiquitinated MdMYB1 protein, which in turn inhibits apple peel coloration (Li et al. 2012). These light-signaling factors directly bind to the MBW transcription factors that regulate anthocyanin biosynthesis and activate or inhibit their transcription (An et al. 2017; Nguyen et al. 2015; Shin et al. 2013).
In nature, anthocyanin biosynthesis of fruit crops is influenced by environmental factors, including light (duration, intensity, quality), temperature, water, nutrient, and plant hormones. Each environmental factor has a specific underlying mechanism for regulating anthocyanin accumulation, and they have cross-purposes. This has been extensively reviewed in previous studies (Espley and Jaakola 2023; Jaakola 2013; Shi et al. 2023) and will not be covered here. In this review, we focus on stress factors associated with dark-grown conditions that influence anthocyanin accumulation in horticultural plants. The light-dependent synthesis of anthocyanins occurs in most horticultural plants (Fig. 1). However, in the last few decades, light-independent anthocyanin biosynthesis has been observed in several naturally grown horticultural plants subjected to bagging. This raises the question of how these plants synthesize anthocyanins. In this review, we discuss the progress of studies on the mechanisms of light-independent anthocyanin biosynthesis in horticultural plants, such as fruits, vegetables, and, ornamental plants, and categorize horticultural plants into five broad classes based on the underlying mechanisms of anthocyanin biosynthesis (Fig. 2).
Citation: HortScience 60, 5; 10.21273/HORTSCI18449-25
Citation: HortScience 60, 5; 10.21273/HORTSCI18449-25
Bagging technology has been widely used in fruit production globally. This method protects fruit from insects and birds and, to a certain extent, inhibits the occurrence and spread of pathogens. This, in turn, reduces the amount of pesticides used, thereby solving the problem of pesticide residues while improving the color and fine-tuning the fruit skin (Jia et al. 2005). Because light constitutes a key environmental factor for anthocyanin accumulation in fruit skin (light-dependent), it is necessary to remove the bag before harvesting to re-expose the fruit to sunlight and induce anthocyanin accumulation and the consequent red pigmentation (Maier and Hoecker 2015). Unlike the light-dependent fruit cultivar, some varieties (light-independent) have stable anthocyanin biosynthesis in the dark (bagging), displaying a lighter or equal red pigmentation than that under light conditions.
For example, the expression levels of VvUFGT, VvMYBA1, and some candidate genes [e.g., anthocyanin-O-methyltransferase (AOMT), glutathione S-transferase (GST), and anthocyanin permease (ANP)] remained almost unchanged in the grape ‘Jingyan’ with or without sunlight, suggesting that anthocyanin biosynthesis in ‘Jingyan’ is independent of light (Wu et al. 2014). Notably, the photomorphogenic positive regulator HY5, which regulates anthocyanin-related gene expression, showed no significant differences in transcript levels regardless of light conditions. Immunohistochemical localization revealed that the VvCOP1 protein, which degrades HY5, remains dispersed in the cytoplasm regardless of the light conditions. This suggests that the nuclear and cytoplasmic transport of VvCOP1 may be involved in the regulation of light-independent grape anthocyanin biosynthesis (Guo 2016). This contrasts with findings in other species, wherein COP1 is localized in the nucleus in darkness to ubiquitinate and degrade HY5 (Li et al. 2012; Shin et al. 2013; Zhao et al. 2022). This phenomenon represents mechanism ‘a’ (Fig. 2a), in which the misregulation of COP1 subcellular localization, a central repressor of photomorphogenic signaling, leads to constitutive photomorphogenesis and light-independent activation of the regulatory pathway of anthocyanin biosynthesis.
In pears, ‘Starkrimson’ developed a good red coloration when subjected to bagging treatment. Real-time polymerase chain reaction indicated that PsDFR and PsUFGT are important structural genes for anthocyanin biosynthesis (Zhu et al. 2018). In the mango cultivar ‘Ruby’, the fruit skin could accumulate anthocyanins even at the later stages of development under bagging treatment, suggesting a light-independent anthocyanin accumulation pattern. This is attributed to the expression of the key anthocyanin biosynthetic structural genes MiUFGT1 and MiUFGT3, and the regulatory gene MiMYB1 in the skin of bagged ‘Ruby’ fruit (Shi et al. 2021). In the sweet cherry cultivar ‘Hongdeng’, the transcript levels of anthocyanin biosynthetic (CHS, F3H, ANS, and UFGT), regulatory (MYB10), and signaling pathway-activated (HY5) genes were consistently high under light and dark conditions, which may explain why ‘Hongdeng’ fruit color is independent of light conditions (Guo et al. 2018). This phenomenon belongs to mechanism ‘b’ (Fig. 2b-1), suggesting the possible existence of novel interacting partners that protect HY5 from COP1-triggered degradation in darkness, thereby relaying positive signals for anthocyanin biosynthesis.
Some varieties of fruit trees, such as red-fleshed apples, blood oranges, red-fleshed strawberries, and blood-fleshed peaches, exhibit unique red flesh pigmentation. For example, red-fleshed apples have six 23-bp R6 repeats (broadly considered as transposons) upstream of the MdMYB10 promoter, which is self-activating and constitutively expressed, conferring red coloration to the entire plant, including leaves, fruits, flowers, and branches (Espley et al. 2009). In blood oranges, a Copia LTR retrotransposon, Tcs1, inserted upstream of the MYB transcription factor Ruby gene, provides a new promoter for Ruby, which induces its expression at low temperatures, resulting in a remarkable red pulp coloration (Butelli et al. 2012). The red-fleshed strawberry berry mutant FaEnSpm-2 is due to the insertion of a CACAT-like DNA transposon into the promoter region of MYB10-2, which enhances MYB10 and MYB10-2 expression in strawberry pulp and anthocyanin biosynthesis, leading to a red-fleshed strawberry (Castillejo et al. 2020). The red flesh trait in fruits is essentially based on transposon-mediated regulation of anthocyanin biosynthesis by the MYBs, wherein light signaling is not involved, which is quite different from the mechanism of the red color trait in the pericarp (Fig. 2a and b-1). The abovementioned phenomenon corresponds to mechanism ‘c’ (Fig. 2c), in which variations in the MYB promoter or the action of novel cofactors lead to the upregulation or constitutive expression of MBW transcription factors, resulting in elevated levels of anthocyanin accumulation in fruit flesh.
The mechanism of blood flesh in the peach is significantly different from the above-mentioned fruits. The blood-flesh trait in the peach is a dominant trait controlled by a single gene, BLOOD (BL), which promotes PpMYB10.1 expression by forming a heterodimer with PpNAC1, thereby inducing anthocyanin accumulation in ‘Dahongpao’ (Zhou et al. 2015). A transposon insertion mutation in the promoter region of BL is the only mutation cosegregating with the red-fleshed trait, indicating that anthocyanin accumulation in ‘Tenshin-suimitsuto’ is caused by this mutation and that the transposon could be a simple and accurate marker of the red coloration (Hara-Kitagawa et al. 2020). The red flesh around the stone in peach fruit is formed by the interaction of PpHY5 with PpBBX10 to activate the transcription of PpMYB10.1, which enhances anthocyanin accumulation (Zhao et al. 2023) (Fig. 2b-2), probably due to the presence of unknown upstream regulators involved in both the regulation of PpCOP1 function and the transcriptional regulation of PpHY5. This also belongs to mechanism ‘b’.
The light-independent anthocyanin accumulation pattern has also been reported in vegetables. The existing light-independent vegetables are classified into two major types: genetically modified and naturally grown plants that are uniformly colored under shade conditions. Anthocyanin biosynthesis in both is barely affected by light.
Light independence is observed in constitutive photomorphogenic/de-etiolated/fusca (cop/det/fus) recessive mutants in Arabidopsis, showing a photomorphogenic phenotype in the dark, short hypocotyls, expanded cotyledons, and expression of photoinduced and chloroplast-related genes (Chory 1992; Hardtke and Deng 2000; Miséra et al. 1994; Wei and Deng 1996). Deleting a key component of the ubiquitinated multimeric protein complex, such as CUL4–DDB1COP1–SPA E3 ligase, CUL4–C3D, and CSN, in the mutant population affects COP1 ubiquitination, which invalidates COP1 stability, and HY5 overaccumulation exhibits accumulation of high anthocyanin levels with occasional fusca phenotype (Cañibano et al. 2021; Lau and Deng 2012) (Fig. 2a). A study on the partial phenotype of the tomato high pigment (hp-2) (homologue of Arabidopsis DET1) found that seedlings accumulated high anthocyanin levels when grown in complete darkness, similar to observations in Arabidopsis det1 mutants (Mustilli et al. 1999). The abovementioned phenomenon also belongs to mechanism ‘a.’
Additionally, a study on mutated turnips observed anthocyanin accumulation in the epidermis of the swollen underground storage roots. The transcript levels of all the structural and regulatory genes and the light signaling pathway factors (BrHY5 and BrCOP1) were much higher in r15 (a red mutant grown with no light) when compared with the wild type (Yang et al. 2017) (Fig. 2b-1). This phenomenon also belongs to mechanism ‘b.’
In addition to the mutated light-independent vegetables, several naturally occurring vegetables exist that are light independent, including crops such as pod, pepper, eggplant, tomato, and root vegetables. For example, similar to pods grown under natural environmental conditions, ‘Zi Bawang’ pods grown in the dark (covered with bags) accumulated anthocyanins. This was attributed to the expression of PvCHS, PvF3'5′H, PvDFR, PvANS, PvMYB1, and PvTTG1 involved in anthocyanin biosynthesis in the purple pods grown in the dark (Hu et al. 2015). A study on peppers found that under shade stress, anthocyanin pigment accumulation was visible to the naked eye, but the anthocyanin content was substantially lower than that in the light (Tang et al. 2020). In eggplants, a large amount of anthocyanin was still synthesized in the skin of light-independent eggplant ‘145’ after bagging. This may be related to the expression of anthocyanin biosynthesis structural genes and the fact that the corresponding gene products were not ubiquitinated by SmCOP1 (He et al. 2019) (Fig. 2b-1). This phenomenon also belongs to mechanism ‘b.’
In some cases, constitutively active photoreceptor mutants, such as CRY1, UVR8, and PHYB, have been shown to exhibit light-independent anthocyanin biosynthesis. For example, the CRY1-W400A variant in Arabidopsis seedlings resulted in constitutive accumulation of high anthocyanin levels after 5 d of darkness (Gao et al. 2015). In addition, UVR8W285A and XVE:UVR88G101S, W285A double mutant Arabidopsis seedlings exhibited extreme photomorphogenic phenotypes, including elevated anthocyanin concentration, following 4 d of estradiol treatment (Heijde et al. 2013; Podolec et al. 2021). In tomato, the substitution of tyrosine, the 276th amino acid of Arabidopsis AtPHYB, to histidine (also called “YHB”), produced a light-insensitive “signaling-active” phytochrome B protein. Heterologous overexpression of the dominant missense allele AtYHB in tomato seedlings resulted in a cop mutant phenotype, in which the dark-growing hypocotyl accumulated large amounts of anthocyanins (Hu et al. 2020). The above phenomenon belongs to mechanism ‘d’ (Fig. 2d), in which mutations in CRY1, UVR8, and PHYB result in constitutively active photoreceptors, leading to elevated anthocyanin levels in darkness, thus eliminating the light requirement for the entire pathway.
Flower color is the most important ornamental quality. However, weak-light stress in greenhouses often results in color fading, or even no color, which adversely affects its ornamental quality and landscaping effects. Therefore, investigating the cultivars that retain their color under weak-light conditions is important for application value.
Light-independent ornamental plant species are relatively rare. Among them, the chrysanthemum cultivar 2001402 stands out, with its anthocyanin content increasing almost twice as much under shade treatment than under control conditions. The transcriptional levels of anthocyanin biosynthesis genes, including CmCHS2, CmDFR, CmANS, and CmMYB6, did not change under shade treatment when compared with the control. Yeast one-hybrid showed that a key light signal transduction factor, CmBIC1 [blue light inhibitors of cryptochromes (BIC)] can directly promote the expression of the anthocyanin structural gene, CmCHS2 (Huang et al. 2017; Liu et al. 2024). The above phenomenon belongs to mechanism ‘e’ (Fig. 2e), in which mutations in promoters or the action of novel cofactors in biosynthetic structural genes enable protein expression independent of light conditions.
This review systematically summarized the five mechanisms along the signal transduction pathway involved in light-independent anthocyanin biosynthesis. The key findings were as follows:
In Arabidopsis and tomato, mutations in CRY1, UVR8, and PHYB result in constitutively active photoreceptors, resulting in elevated anthocyanin accumulation in darkness, thus eliminating the light requirement for the entire biosynthetic pathway (Fig. 2d).
Functional disruption (either by misregulation of COP1 protein subcellular location, as in ‘Jingyan’ grape, or genetic mutation, as in Arabidopsis) of central repressors of photomorphogenic signaling, such as COP1/DET1/CSN/SPA, leads to constitutive photomorphogenesis and light-independent activation of the regulatory pathway of anthocyanin biosynthesis (Fig. 2a).
Novel interacting partners that protect HY5 from COP1-triggered degradation in darkness relay positive signals for anthocyanin biosynthesis (Fig. 2b).
Variations in the MYB promoter or the action of novel cofactors lead to the upregulation or constitutive expression of MBW transcription factors, increasing anthocyanin accumulation in fruit fleshes (Fig. 2c).
Mutations in the promoters or novel cofactor action in biosynthesis structural genes allow protein expression independent of light conditions (Fig. 2e).
Anthocyanin biosynthesis under bagging conditions is affected by MBW complexes and anthocyanin biosynthetic genes. In addition, the photoreceptors CRY1, UVR8, and PHYB and the light signaling regulators COP1 and HY5 play decisive roles in light-independent anthocyanin accumulation in plants. Therefore, their corresponding genes can be used to breed plants adapted to weak-light or greenhouse environments in the future. Incorporating these improved light-independent plants in production can save labor and provide economic benefits.
Although researchers have widely explored the regulatory mechanisms of anthocyanin biosynthesis in light-independent plants, studies on the regulatory mechanisms of light signaling on phycocyanin biosynthesis remain in their infancy. Furthermore, many questions warrant further analysis as described below:
Although it is clear that the MBW complex regulates the biosynthesis of anthocyanins in light-dependent and -independent plants, the reason for the different anthocyanin biosynthesis mechanisms in such plants under dark conditions remains largely unknown. We hypothesize that either the content of some enzymes decreases during light avoidance or the light signaling transduction factors undergo mutations. While further research on the translational regulation of COP1 concerning anthocyanins accumulation in the dark or light is required, analysis of other light signaling factors and their interaction with the MBW complex is important for establishing the light-independence axis for regulating anthocyanin biosynthesis.
The accumulation of anthocyanins in the organs of horticultural plants is usually affected by the combined action of environmental factors, such as light and temperature. To explore the interaction between light and other environmental factors in regulating anthocyanin biosynthesis and to rationally use the greenhouse conditions, precise regulation of anthocyanin formation in horticultural plants is crucial. Such studies can subsequently improve the color of ornamental plants and the nutritional value of horticultural products.
Model for light signaling regulation of anthocyanin biosynthesis in light-dependent horticultural plants. GST = glutathione S-transferase; UVR8 = ultraviolet resistance locus 8; CRYs = cryptochromes; PHOTs = phototropins; PHYs = phytochromes; PIF = phytochrome interacting factor; COP1 = constitutively photomorphogenic 1; DET1 = de-etiolated; FUS = fusca; HY5 = elongated hypocotyl 5.
The five mechanisms of light-independent anthocyanin biosynthesis. × denotes regulatory failure, ? denotes regulatory relationship unknown, dashed red lines denote inhibition, and green arrows denote activation. ABG = anthocyanin biosynthetic gene; TE = transposable elements.
Contributor Notes
We are deeply grateful to Yong Li for his help with the idea of the article. This work was supported by Grant 32402523 from the National Natural Science Foundation of China and Grants 222102110440 and 232102111083 from the Science and Technology Program of Henan Province.
Model for light signaling regulation of anthocyanin biosynthesis in light-dependent horticultural plants. GST = glutathione S-transferase; UVR8 = ultraviolet resistance locus 8; CRYs = cryptochromes; PHOTs = phototropins; PHYs = phytochromes; PIF = phytochrome interacting factor; COP1 = constitutively photomorphogenic 1; DET1 = de-etiolated; FUS = fusca; HY5 = elongated hypocotyl 5.
The five mechanisms of light-independent anthocyanin biosynthesis. × denotes regulatory failure, ? denotes regulatory relationship unknown, dashed red lines denote inhibition, and green arrows denote activation. ABG = anthocyanin biosynthetic gene; TE = transposable elements.
