Mechanisms by Which High Temperatures Suppress Anthocyanin Coloration in Flowers and Fruits, and Discovery of Floricultural Crops that Exhibit High-temperature-tolerant Flower Pigmentation

Abstract

Anthocyanins are ubiquitously accumulated in diverse plant species and play crucial roles in plant development. In addition, anthocyanin pigmentation is associated with significant characteristics in the flowers and fruits of horticultural products. Notably, anthocyanin biosynthesis and storage are known to be affected by environmental factors. High ambient temperatures often suppress anthocyanin accumulation in flowers and fruits, raising concerns regarding the increase in atmospheric temperatures caused by global warming. Therefore, a comprehensive understanding of the mechanisms underlying the effects of high ambient temperatures on the regulation of anthocyanin biosynthesis and storage is necessary to maintain pigment quality and quantity of the products. In this review, we provide comprehensive information on the role of high-temperature-related signaling pathways in the regulation of anthocyanin biosynthesis. The expression of genes involved in anthocyanin biosynthesis is mainly regulated by R2R3-MYB activators, R2R3-MYB repressors, and R3-MYB negative regulators. The expression levels of R2R3-MYB activators decrease under high-temperature conditions, as observed in many flowers and fruits. The upregulation of R2R3-MYB repressors and R3-MYB negative regulators has also been demonstrated in some plant species under high-temperature conditions. The high-temperature-related signaling pathways have been evaluated mainly in the vegetative organs of Arabidopsis and apple fruits. In these organs, light strongly influences anthocyanin biosynthesis in addition to ambient temperatures. The CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1)-ELONGATED HYPOCOTYL 5 (HY5) module and B-box proteins upregulate the R2R3-MYB activators under light conditions, while they downregulate the R2R3-MYB activators under high-temperature conditions. However, the pathways that transduce high-temperature signals in flowers are poorly understood. Unlike in fruits and vegetative organs, light exerts relatively small effects on anthocyanin pigmentation in flowers, suggesting that the COP1-HY5 module-independent pathway could be responsible for the regulation of R2R3-MYB regulators in many flowers. Further research to clarify the related signaling pathways in flowers is needed to find solutions to overcome the problem of color fading caused by high ambient temperatures. In addition, exceptional cases have been reported in which high temperatures do not inhibit or enhance the anthocyanin pigmentation of flowers. Such species can prove helpful in elucidating the mechanisms underlying temperature-mediated regulation of anthocyanin pigmentation and as parental materials for crossbreeding.

Introduction

Colors of flowers and fruits enhance their attractiveness and quality and strongly influence consumers’ inclination to purchase. Therefore, improvements in color and color patterns in these organs have been a major target of breeding programs for floricultural crops, and growers routinely improve horticultural practices to preserve and enhance color intensity. Anthocyanins are among the major visible pigments that accumulate in flowers, fruits, and vegetative organs. The core pathways that biosynthesize diverse classes of anthocyanins have been explored for both applied and basic purposes, and the enzymes and genes associated with anthocyanin biosynthesis have been identified in a wide range of plant species. The transcriptional and post-transcriptional regulation of these genes has also been elucidated. In addition, biotic and abiotic factors, including light, temperature, water, nutrients, wounding, and pathogen infection, influence anthocyanin accumulation in flowers and fruits (Naik et al., 2022; Naing and Kim, 2021). Among these factors, high ambient temperatures often suppress anthocyanin pigmentation in diverse species, as explained below. As air and land temperatures increase due to global climate change, elucidating the molecular mechanisms whereby high ambient temperatures impact anthocyanin biosynthesis and storage is essential for maintaining and improving product quality under atmospheric warming. In this review, we first provide an overview of the transcriptional and post-transcriptional regulation of anthocyanin biosynthesis and also introduce the major MYB transcription factors involved. Subsequently, we review how high ambient temperatures alter regulatory cascades for anthocyanin biosynthesis. This knowledge will improve understanding the molecular mechanisms underlying high-temperature-induced color fading in flowers and fruits and support development of strategies to solve this problem associated with global warming.

Generally, high temperatures adversely affect anthocyanin pigmentation, but do not inhibit or enhance the anthocyanin pigmentation in the flowers of a few plant species. In the third section of this article, we describe such species as they could be useful as parental materials for crossbreeding. Moreover, genetic evaluations using these materials can advance understanding of the mechanisms underlying high-temperature-mediated regulation of anthocyanin pigmentation.

Carotenoids are other major visible pigments accumulated in plant organs. Several studies have elucidated the impact of high temperatures on carotenoid accumulation in flowers. High temperatures suppress the expression of carotenoid biosynthesis genes, but activate the expression of carotenoid degradation genes, resulting in the paler coloration of sweet osmanthus flowers (Wang et al., 2022). High temperatures cause low levels of carotenoids in the flowers of an epiphytic orchid (Psygmorchis pusilla; Vaz et al., 2004). In contrast, temperature does not affect the total amount of flower carotenoids in spray chrysanthemum (Nozaki et al., 2006). Thus, the effects of high temperatures on carotenoid colors of flowers are relatively less clear than those on anthocyanin colors. In this review, we focus on the effects of high temperatures on anthocyanin pigmentation.

Transcriptional and Post-transcriptional Regulation of Anthocyanin Biosynthesis

Roles and biosynthesis pathway of anthocyanin pigments

Anthocyanins play various roles in tolerance to abiotic and biotic stresses, and their biosynthesis can be activated by cold, ultraviolet (UV)-B, excess light, nutrient deficiency, drought, salinity, metal toxicity, and pest and pathogen attacks. In addition, anthocyanins are key for signaling to pollinators and seed dispersal agents (Davies et al., 2018; Ferreyra et al., 2021; Landi et al., 2015). Anthocyanin biosynthesis pathways and the genes and enzymes involved in these pathways have been well-evaluated (Tanaka et al., 2008; Tohge et al., 2017). Anthocyanins are biosynthesized from malonyl-CoA and p-coumaroyl-CoA as precursors using chalcone synthase (CHS), chalcone isomerase, flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonoid-3',5'-hydroxylase, dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and uridine diphosphate-glucose: flavonoid (or anthocyanidin) 3-o-glucosyltransferase (UFGT). They are further modified by the addition of sugars and acyl side groups and are transported to, and stored in, vacuoles.

R2R3-MYB activators predominantly regulate anthocyanin biosynthesis

Anthocyanin biosynthesis in flowers and fruits is principally regulated at the transcriptional level. Notably, MYB-bHLH-WD repeat (MBW) complexes consisting of R2R3-MYB transcription factors, basic helix-loop-helix (bHLH) transcription factors, and WD40 proteins are involved in regulating the expression of the genes implicated in anthocyanin biosynthesis (Lloyd et al., 2017; Xu et al., 2015). The R2R3-MYBs derived from the subgroup (SG) 6 class of R2R3-MYBs (the definition of R2R3-MYB SGs follows Stracke et al., 2001) activate anthocyanin biosynthesis in many species (Table 1). The SG5 class of R2R3-MYBs in orchids and grasses positively regulates anthocyanin biosynthesis (Chiou and Yeh, 2008), and those in bilberries, kiwifruits, and apples regulate both anthocyanin and proanthocyanidin biosynthesis (Lafferty et al., 2022; Peng et al., 2020; Wang et al., 2018). However, those in other plant species are usually involved in the regulation of proanthocyanidin biosynthesis. These R2R3-MYB activators conserve the amino acid signature of the bHLH-interacting motif ([D/E]Lx₂[R/K]x₃Lx₆Lx₃R) at the R3 repeat and physically interact with bHLH partners. Among the transcription factors comprising the MBW complex, R2R3-MYB activators often exhibit spatially and temporally restricted expression, whereas bHLHs are expressed in a relatively expanded area of plant surfaces, determining the identification of epidermal cells. Thus, the expression profiles of these R2R3-MYB activators predominantly correlate with spatially and temporally restricted pigment deposition in flowers and often create color patterns in single petals, such as spots, venation, and bud-blush (Table 1; Albert et al., 2011; Hsu et al., 2015; Shang et al., 2011; Yamagishi, 2018; Yamagishi et al., 2014; Yuan et al., 2014).

Table 1

R2R3-MYB and R3-MYB transcription factors positively or negatively regulating anthocyanin biosynthesis.

R2R3-MYB repressors of anthocyanin biosynthesis

The R2R3-MYB repressors of anthocyanin biosynthesis precisely modulate pigment accumulation (LaFountain and Yuan, 2021; Ma and Constabel, 2019). Notably, the R2R3-MYB transcription factors derived from a C2 repressor motif clade (Table 1), including an SG4 subclade and additional subclades, conserve the bHLH-interacting motif at the R3 repeat and share a C2 repressor motif, also called the ethylene response factor-associated amphiphilic repression (EAR) motif (DLNxxP or LxLxL), in the C-terminal half (Cavallini et al., 2015). In addition to the C2/EAR repressor motif, approximately half of the R2R3-MYBs in this clade possess a TLLLFR repressor motif (Cavallini et al., 2015; Ma et al., 2018; Zhao et al., 2023). These R2R3-MYB repressors are assumed to compete with R2R3-MYB activators to form MBW complexes with bHLH and WD40 partners or to directly repress the expression of anthocyanin biosynthesis genes using repressor motifs or both. However, the expression of these repressors is often activated by R2R3-MYB activators. Thus, these repressors are involved in a negative regulatory feedback loop, attenuating the activity of R2R3-MYB activators to avoid excess accumulation of anthocyanins (Huang et al., 2020; Li et al., 2020b; Zhou et al., 2019).

Repressors of other classes of R2R3-MYBs have also been identified. For example, StMYB44-1 and StMYB44-2 repress the DFR promoter activity in potato tubers, resulting in suppression of anthocyanin biosynthesis. StMYB44-1 and StMYB44-2 belong to SG22 type R2R3-MYBs and possess the C2/EAR repressor motif, but do not have the bHLH-interacting motif. Notably, StMYB44-1 shows a stronger repressive ability than StMYB44-2, and the C2/EAR motif in the latter is relatively less conserved (Liu et al., 2019). Moreover, CmMYB012 in chrysanthemum is an atypical SG7 class R2R3-MYB that lacks the SG7-1 and SG7-2 motifs conserved in this class of R2R3-MYBs. Although SG7 class R2R3-MYBs in other species often upregulate the expression of the flavonol synthase or flavone synthase (FNS) gene and induce the accumulation of colorless pigments, CmMYB012 inhibits flavone and anthocyanin biosynthesis by suppressing the expression of FNS, CHS, DFR, ANS, and UFGT. However, typical repressor motifs are not found in its sequence (Zhou et al., 2021).

Roles of R3-MYBs in anthocyanin pigmentation

R3-MYBs have a single R3 repeat motif at the N-terminus and include R3-MYB proteins that negatively regulate anthocyanin biosynthesis (Table 1). These R3-MYBs maintain the amino acid sequence motif required for interaction with bHLH proteins and often disrupt the activity of the MBW protein complex by competing with R2R3-MYB activators for binding to bHLH, resulting in the passive regulation of anthocyanin biosynthesis (Albert et al., 2014; Colanero et al., 2018; Wang and Chen, 2014). In addition, AtMYBL2 (R3-MYB) in Arabidopsis contains the repressor motifs TLLLFR and C2/EAR (Matsui et al., 2008). Similarly, LhR3MYB1 and LhR3MYB2 in lilies (Sakai et al., 2019) and IlMYBL1 in Iochroma (Gates et al., 2018) have a C2/EAR motif at the C-terminus. Therefore, the R3-MYBs in Arabidopsis, lilies, and Iochroma are likely to actively repress the expression of the genes implicated in anthocyanin biosynthesis.

R3-MYB negative regulators occasionally create unique color features. Atroviolacea (R3-MYB) in tomato fruits (Colanero et al., 2018) and IlMYBL1 (R3-MYB) in Iochroma flowers (Gates et al., 2018) strongly prevent anthocyanin biosynthesis, creating anthocyanin-less phenotypes, and ROSE INTENSITY1 (R3-MYB) determines the color intensity of monkeyflower (Mimulus) petals (Yuan et al., 2013). The RED TONGUE (RTO) inhibitors (R3-MYB) and NECTAR GUIDE ANTHOCYANIN (NEGAN) activators (SG6 R2R3-MYB) are involved in spot formation on monkeyflower petals (Ding et al., 2020). Notably, the expression of the RTO gene is activated by NEGAN, and the RTO protein diffuses from the source cells to neighboring cells. As NEGAN enhances its own expression in source cells and RTO inhibits the function of NEGAN in neighboring cells, RTO generates distinct NEGAN activity, inducing distinct pigment contents between source cells and neighboring cells, resulting in anthocyanin spot patterning on the petals. Other R3-MYB negative regulators are often expressed when R2R3-MYB activators are highly expressed; therefore, these are likely involved in fine-tuning anthocyanin biosynthesis to prevent the excess accumulation of pigments.

Post-transcriptional regulation of anthocyanin biosynthesis

Post-transcriptional regulation of anthocyanin biosynthesis has recently been recognized. MicroRNAs (miRNAs) prevent the function of target genes by cleaving mRNA, suppressing translation, or both. MiR828 and miR858 negatively regulate the VvMYB114 repressor, promoting anthocyanin biosynthesis in grapevine berries (Tirumalai et al., 2019). Similarly, an abundance of miR828 is correlated with high anthocyanin content in potato tubers, in which miR828 downregulates MYB repressors (Bonar et al., 2018). In lily flowers, miR828 directly inhibits the expression of the LhMYB12 activator. As a lot of miR828 accumulates in the lower half of single tepals, but only slightly in the upper half, the spatially distinct accumulation levels of miR828 create bicolor tepals in Asiatic hybrid lilies and their parental wild species, Lilium dauricum (Yamagishi, 2022a; Yamagishi and Sakai, 2020). Notably, the same microRNA is involved in the regulation of grapevine berries, potato tubers, and lily flowers, but its target genes and the abilities of its target genes to enhance or prevent pigmentation differ. MiR828 in lilies inhibits the activator to prevent pigment accumulation, whereas miR828 in grapevines and potatoes downregulates the repressors to enhance pigmentation. The color characteristics caused by miR828, via the post-transcriptional regulation of anthocyanin biosynthesis, vary among species, although miR828 is among the most highly conserved microRNAs in plant species.

MiR828 in Arabidopsis and apples negatively regulates anthocyanin accumulation via different routes. In Arabidopsis, miR828 cleaves the transcripts of trans-acting small interfering RNA gene 4 (TAS4) to produce a small interfering RNA TAS4-siR81(−), which post-transcriptionally represses the PAP1, PAP2, and MYB113 activator genes (Rajagopalan et al., 2006). In apple, miR828 cleaves TAS4 transcripts to produce TAS4-siR81(−). Although the anthocyanin-related MYB activator genes, MdMYBA/MdMYB1/MdMYB10, have no siR81(−)-recognition sites, the siR81(−) targets the MdbHLH3 activator gene (Zhang et al., 2020b), which mainly acts under low-temperature conditions in fruits (Xie et al., 2012). Accumulation of miR828 in apples increases in response to high temperatures, thereby suppressing MdbHLH3 expression and, in turn, anthocyanin accumulation (Zhang et al., 2020b).

Squamosa promoter-binding protein-like (SPL) 9 in Arabidopsis (AtSPL9) directly prevents the expression of anthocyanin biosynthesis genes and destabilizes MBW complexes, thereby repressing anthocyanin biosynthesis. Expression of the AtSPL9 gene is post-transcriptionally suppressed by miR156, which exhibits a high accumulation during the juvenile phase (Gou et al., 2011). Thus, the miR156-SPL9 module controls the pigmentation of vegetative organs during this juvenile phase. In addition, the miR156-SPL9 module is implicated in the pigment accumulation necessary for stress resistance, as the expression of primary miR156 transcripts is triggered by abiotic stresses (Cui et al., 2014). In blueberries, miR156 accumulates at relatively later stages of fruit development and inhibits the expression of the VcSPL12 gene, which has an miR156 recognition site. As VcSPL12 directly prevents the expression of the DFR gene and represses the function of VcMYBPA1, positively regulating anthocyanin biosynthesis in fruits, the miR156-SPL12 module in blueberries accelerates anthocyanin biosynthesis during fruit maturation (Li et al., 2020a).

Post-transcriptional regulation is likely involved in the precise modulation of pigment accumulation in response to environmental changes or plant development. We believe these novel findings regarding the post-transcriptional regulation of pigment biosynthesis will deepen our understanding of pigment fine-tuning in flowers and fruits.

Mechanisms Underlying High-temperature-mediated Suppression of Anthocyanin Accumulation

Inhibition of the R2R3-MYB activator is the main cause of color fading in flowers and fruits

High ambient temperatures suppress anthocyanin biosynthesis and accumulation in diverse plant species (Man et al., 2015; Shi et al., 2022; Tan et al., 2023; Zhang et al., 2019). Reduced expression of R2R3-MYB activators is often found in such species under high ambient temperatures (Table 2), such as that of MdMYBA/MdMYB1/MdMYB10 in apples (Lin-Wang et al., 2011), FaMYB10 in strawberries (Matsushita et al., 2016), LhMYB12 in Oriental hybrid lilies (Lilium spp.; Lai et al., 2011), and CyMYB1 in Cymbidium flowers (Nakatsuka et al., 2019). In the Oriental hybrid lily cultivar ‘Marrero’, high-temperature treatment in stages 2 and 3 during flower development causes poor flower coloration, whereas such treatment in other stages does not affect anthocyanin coloration. In stages 2 and 3, the LhMYB12 activator exhibits an intrinsic high expression in control flowers, but high temperatures suppress this expression (Lai et al., 2011). In the tepals (petal-like sepals and lateral petals) of Cymbidium flowers, the expression levels of CyMYB1 and CybHLH2 activators, as well as of major anthocyanin biosynthesis genes, are strongly suppressed by high temperatures, resulting in color fading (Nakatsuka et al., 2019). Therefore, the suppression of R2R3-MYB activator expression is likely among the major reasons for the suppression of anthocyanin biosynthesis at high temperatures.

Table 2

Effects of high temperatures on anthocyanin pigmentation.

In addition, the R2R3-MYB repressors and R3-MYB negative regulators are involved in downregulating anthocyanin biosynthesis (Table 2). In the vegetative organs of Arabidopsis, the expression of at least three repressors, AtMYB3 (SG4 R2R3-MYB), AtMYB6 (SG4 R2R3-MYB), and AtMYBL2 (R3-MYB), is activated by high temperatures, resulting in accelerated color fading (Rowan et al., 2009). Similarly, the expression levels of CsMYBL2 (SG4 R2R3-MYB) in tea leaves are increased under high-temperature conditions, inhibiting anthocyanin biosynthesis (Zhao et al., 2023). In purple potato tubers, heat stress reduces the expression of the StAN1 activator gene together with that of StbHLH1 and simultaneously induces the expression of StMYB44-1 and StMYB44-2 repressors (SG22 R2R3-MYB), resulting in the creation of white areas in tuber flesh (Liu et al., 2019). Anthocyanin contents in chrysanthemum tepals dramatically decreased at 35°C. Notably, CmMYB012, atypical SG7 R2R3-MYB, acts as a negative regulator of anthocyanin biosynthesis, and its expression is markedly induced by prolonged high-temperature treatment. As the suppression of the CmMYB012 gene in RNAi-CmMYB012 transgenic plants partially restores the anthocyanin-based phenotype under high-temperature conditions, the activation of CmMYB012 expression is a major mechanism underlying heat-induced suppression of anthocyanin biosynthesis (Zhou et al., 2021).

Upstream factors that affect MYB expression in vegetative organs and fruits

Upstream factors that affect the expression of R2R3-MYB activators, R2R3-MYB repressors, and R3-MYB negative regulators, which in turn interact to regulate anthocyanin biosynthesis, have been evaluated in the vegetative organs of Arabidopsis and the fruits of several species, including apples (Fig. 1). In addition to ambient temperatures, light (visible light and UV-B) strongly influences anthocyanin biosynthesis in these organs. High temperatures suppress anthocyanin accumulation, whereas light enhances it. The CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1)-ELONGATED HYPOCOTYL 5 (HY5) module is involved in the transduction of light and temperature signals and plays a central role in the regulation of photomorphogenesis, including anthocyanin accumulation. Notably, AtHY5 in Arabidopsis upregulates anthocyanin biosynthesis by directly binding to the promoters of biosynthesis genes, such as CHS and F3H (Shin et al., 2007) and the PAP1 activator (Shin et al., 2013). The COP1 protein forms complexes with SUPPRESSOR OF PHYTOCHROME A-105 proteins and degrades AtHY5 proteins (Gangappa and Botto, 2016; Podolec and Ulm, 2018). Light induces the translocation of COP1 proteins from the nucleus to the cytoplasm using photoreceptors such as phytochromes, cryptochromes, and the UV receptor protein UV RESISTANCE LOCUS 8, thus reducing the levels of the nuclear COP1 protein. In contrast, high temperatures enhance the levels of the nuclear COP1 protein. Consequently, the amount of HY5 proteins in the nucleus and the rate of HY5-regulated anthocyanin biosynthesis are increased by exposure to light, but reduced by high temperatures (Kim et al., 2017; Park et al., 2017; Xiao et al., 2022). Simultaneously, AtHY5 downregulates the expression of AtMYBL2 (R3-MYB) using miR858. Therefore, high temperatures stimulate AtMYBL2 activity, enhancing the suppression of anthocyanin biosynthesis (Kim et al., 2017; Wang et al., 2016a).

Fig. 1

Putative molecular mechanisms for high-temperature-induced suppression of anthocyanin biosynthesis in fruits and vegetative organs. High temperatures inhibit the activity of HY5 and BBX (COL) positive regulators (including MdCOL11 and AtBBX20/21/22) and activate that of BBX (COL) negative regulators (including MdCOL4) using COP1 or HSF. HY5 interacts with the BBX positive regulators to stimulate the expression of the R2R3-MYB positive regulators and anthocyanin biosynthesis genes, while dimers of HY5 and the BBX negative regulators suppress that of the R2R3-MYB positive regulators and anthocyanin biosynthesis genes. Abbreviations: BBX, B-box transcription factor; COL, CONSTANS-like transcription factor; COP1, CONSTITUTIVE PHOTOMORPHOGENIC 1; HSF, heat shock transcription factors; HY5, ELONGATED HYPOCOTYL 5.

In the fruits of apples (An et al., 2017; Peng et al., 2013), eggplants (Jiang et al., 2016), and pears (Tao et al., 2018) and the leaves and fruits of tomatoes (Liu et al., 2018; Qiu et al., 2019), HY5 proteins directly upregulate R2R3-MYB activators and anthocyanin biosynthesis genes and are negatively regulated by COP1 in the dark. Therefore, the role of the COP1-HY5 module in light-induced anthocyanin accumulation is likely highly conserved among fruits and vegetative organs, although the direct impact of high temperatures on the COP1-HY5 module has not yet been evaluated in species other than Arabidopsis.

B-box (BBX) transcription factors, also known as CONSTANS-like (COL) transcription factors, physically interact with HY5 to regulate the expression of target genes (Fig. 1). Therefore, BBXs play key roles in promoting photomorphogenesis and anthocyanin biosynthesis. In Arabidopsis, AtBBX20, AtBBX21, and AtBBX22, which show functional redundancy, activate target genes, whereas when AtBBX24, AtBBX25, and AtBBX32 interact with HY5, they repress the expression of the genes targeted by HY5 (Bursch et al., 2020; Gangappa and Botto, 2014; Xu et al., 2018). Moreover, BBX proteins that are involved in the regulation of anthocyanin biosynthesis have been identified in apples, pears, and grapevines (An et al., 2019, 2020; Bai et al., 2019a, b; Fang et al., 2019b; Liu et al., 2023). Among the BBX proteins, MdCOL11 in apples, a homolog of AtBBX22, directly stimulates MdMYBA/MdMYB1/MdMYB10 transcription. Notably, UV-B radiation enhances MdCOL11 expression, whereas high temperatures suppress its expression (Bai et al., 2014). Moreover, MdCOL4 in apples, a homolog of the AtBBX24 repressor, downregulates the expression of the ANS, UFGT, and MdMYBA/MdMYB1/MdMYB10 genes via the formation of an MdCOL4-MdHY5 dimer. The expression of the MdCOL4 gene is regulated by heat shock transcription factors (HSF) and induced by high temperatures; therefore, MdCOL4 is implicated in high-temperature-induced anthocyanin suppression (Fang et al., 2019a). Based on these findings, we conclude that light and high temperatures influence anthocyanin biosynthesis by regulating the expression of target genes via common signaling pathways in a wide range of fruits and vegetative organs.

Upstream factors that affect the R2R3-MYB activator in flowers

In flowers, an intriguing report on rose (Rosa hybrida) elucidated the mechanism underlying light-induced anthocyanin biosynthesis. The levels of RhHY5 protein increase under light, but decrease under dark conditions. RhHY5 activates the expression of RhCHS, RhF3'H, RhANS, RhGT1 (UFGT), and an RhMYB114a activator, and suppresses that of the RhMYB3b repressor in the petals of ‘Burgundy Iceberg’ (Yan et al., 2023). However, it remains to be evaluated whether high temperatures influence RhHY5 function. To the best of our knowledge, this is the first report to demonstrate the role of HY5 in light-induced anthocyanin accumulation in flowers. However, knowledge regarding the upstream factors regulating the transcription of R2R3-MYB regulators for anthocyanin biosynthesis and how light and temperature signals are transduced to the regulators is limited in flowers other than roses compared with those in vegetative organs and fruits. Why are upstream factors unclear in flowers? Light exerts strong effects on fruit pigmentation, as anthocyanin-free fruits are often produced when grown in the dark (Bai et al., 2019b; Fang et al., 2019a; Jiang et al., 2016; Li et al., 2020c). Unlike in fruits and vegetative organs, light exerts relatively small effects on anthocyanin pigmentation in flowers, as colored flowers usually develop under completely dark conditions, although bud-blush pigmentation is strongly light-dependent. In addition, the expression of R2R3-MYB activators that create the bud-blush pattern, such as PURPLE HAZE in Petunia hybrida and LrMYB15 in Lilium regale, is light-dependent (Albert et al., 2011, 2014; Yamagishi, 2016). Therefore, it is believed that the COP1-HY5 module is not responsible for the regulation of R2R3-MYB regulators in many flowers. In the rose ‘Burgundy Iceberg’, five SG6 R2R3-MYB activators are included, of which RhMYB114a is influenced by light (Yan et al., 2023), whereas RhMYB113a is the major activator in rose flowers and is highly expressed in the petals of ‘Burgundy Iceberg’. The expression of the RhMYB113a gene is unaffected by dark treatment, so dark-grown petals continue to show considerable accumulation of anthocyanins (Yan et al., 2023). As a single plant species often possesses a couple of R2R3-MYB activators that create varied pigmentation patterns, RhMYB113a and RhMYB114a are probably responsible for pigmentation in the entire petal region and bud-blush patterning, respectively, in rose flowers. However, further research is necessary to confirm this hypothesis. Based on these findings, we conclude that the main upstream factors regulating R2R3-MYB activators in flowers differ from those in fruits and vegetative organs and that temperature signals are transduced using COP1-HY5 module-independent pathways. Further research to identify such upstream factors and elucidate the related signaling pathways in flowers is needed to find solutions to overcome the problem of color fading caused by high ambient temperatures.

Color fading in some fruits is caused by the active degradation of anthocyanin pigments

Other mechanisms for color fading, in which transcriptional regulation by R2R3-MYBs is not involved, have been clarified. In grapevines, unlike in other species, the transcription of anthocyanin biosynthesis genes and their regulatory genes in berries is not always affected by high temperatures, and anthocyanin degradation is relatively strongly linked to the suppression of anthocyanin accumulation under high temperatures (Mori et al., 2007; Pastore et al., 2017). Class III peroxidases are responsible for the active degradation of anthocyanins in vacuoles (Zipor et al., 2015), and their activity is significantly increased at high temperatures in grapevine berries (Movahed et al., 2016). Similarly, an increase in class III peroxidase activity is correlated with anthocyanin degradation at high temperatures in plum (Prunus salicina) fruits (Niu et al., 2017). Up-regulation of class III peroxidase under high temperatures is also involved in color fading in crabapple fruits (Malus profusion) in addition to the suppression of anthocyanin biosynthesis caused by downregulation of the MpMYB10 activator and upregulation of the MpMYB15 repressor (Rehman et al., 2017). We believe the involvement of class III peroxidase in active degradation of anthocyanin pigments under high temperature has not been reported in flower petals.

Anthocyanin Pigmentation in Certain Floricultural Crops is High-temperature-tolerant

High temperatures enhance the anthocyanin pigmentation in flowers of Asiatic hybrid lilies

Although high atmospheric temperatures often suppress anthocyanin accumulation in flowers and fruits, exceptional cases in which anthocyanin content is not affected, or is instead accelerated, by high temperatures have been reported. In Asiatic hybrid lilies (Lilium spp.), the anthocyanin color became deeper in most of the tepal parts after incubation at 35°C than at 20°C (Yamagishi, 2022b). The expression of the lily LhMYB12 activator, as well as of the genes involved in anthocyanin biosynthesis, was upregulated by high temperatures (the results for an Asiatic hybrid lily cultivar ‘Lollypop’ are shown in Fig. 2), despite the suppression of R2R3-MYB activators by high ambient temperatures being a major occurrence reported in other species.

Fig. 2

Anthocyanin contents (mmol·g⁻¹FW) and relative expression levels of LhMYB12, LhbHLH2, CHSa, F3H, DFR, and ANS genes in the tepals of the Asiatic hybrid lily ‘Lollypop’ cultured at 20°C or 35°C. Tepal segments were collected from the upper half of inner tepals a day before anthesis (flower developmental stage 4), and anthocyanins and RNA were extracted. Anthocyanin levels and gene expression were estimated by photospectrometry measurement and quantitative RT-PCR (the ACTIN gene was used as reference), respectively. Superscript symbols * and ** indicate significant differences at 5% and 1% levels, respectively, after the t-test. Other methods are the same as those described by Yamagishi (2022b). Abbreviations: FW, fresh weight; RT-PCR, reverse transcription polymerase chain reaction.

Asiatic and Oriental hybrid lilies are genetically distantly related to each other, as these hybrids are developed using wild plant species belonging to different sections of the genus Lilium. These hybrids have opposite responses to high temperatures. Anthocyanin coloration in Oriental hybrid lily flowers is severely inhibited, and the expression of the LhMYB12 activator is suppressed by high temperatures (Lai et al., 2011). What is the reason for this difference in LhMYB12 expression between the two lily hybrids? An important clue is that the sequences of the LhMYB12 promoter are significantly different between Asiatic and Oriental hybrid lilies, although those downstream of the start codon, including two introns and the 3' untranslated region, are similar (Yamagishi, 2021). Therefore, putative cis-acting elements in promoter regions and temperature-responsive trans-acting elements that interact with the putative cis-acting elements must differ (Yamagishi, 2022b). When such cis- and trans-acting elements are identified we can apply the findings to mechanistic evaluations in other cultivated plants.

Anthocyanin pigmentation in Cymbidium flowers is high-temperature tolerant

Notable findings have also been reported in Cymbidium flowers, composed of tepal, labellum, and columnar organs. Anthocyanin contents within the tepals are suppressed severely by high temperatures, but those in the labellum and column remain unaffected by temperature conditions, and are similar at high and low temperatures. Expression levels of the genes involved in anthocyanin biosynthesis and most of the regulatory genes in the labellum are reduced by high temperatures, but those at high temperatures are the same as, or even higher than, those in the tepals at low temperatures. Thus, the tepals, labellum, and column exhibit different responses to high temperatures in Cymbidium (Nakatsuka et al., 2019). If we clarify the cause of the difference between the tepals and labellum, we can obtain clues to help avoid heat-related disorders. The phenomena observed in Asiatic hybrid lilies and Cymbidium species demonstrate that plants display varied responses to climate change and shed new light on pigmentation regulation under various environmental conditions.

Conclusions

Elucidating the molecular mechanisms underlying high ambient temperature-mediated suppression of anthocyanin biosynthesis is an important research theme because atmospheric temperatures will continue to rise. Downregulation of R2R3-MYB activators and upregulation of R2R3-MYB repressors and R3-MYB negative regulators have been observed in many flowers and fruits under high-temperature conditions. Expression of R2R3-MYB activators in fruits and vegetative organs is influenced by light and temperature stimuli and is likely to be under the control of the COP1-HY5 module and BBX transcription factors. Both the COP1-HY5 module and BBX transcription factors were initially characterized as being involved in light signal transduction pathways and later were reported to be involved in the mediation of temperature stimulus. These upstream factors reduce the expression levels of R2R3-MYB activators and increase those of R3-MYB negative regulators under high-temperature conditions. However, our knowledge of upstream factors that regulate R2R3-MYB activators in flowers is insufficient, and this limits our understanding of how high temperatures inhibit anthocyanin accumulation in flowers. One possibility is that the COP1-HY5 module is unlikely to be involved in the regulation in flowers. Therefore, our research aimed to identify novel upstream factors and signaling pathways that regulate R2R3-MYB activators, which in turn regulate the expression of the genes involved in anthocyanin biosynthesis in flowers.

In addition, it has been reported in the labellum and column of Cymbidium flowers, and the tepals of Asiatic hybrid lily flowers, that anthocyanin content is not affected or is instead accelerated by high temperatures. Such plant materials will help elucidate the underlying mechanisms and be useful as parental materials for crossbreeding. Investigating such phenomena in other plant species that exhibit high-temperature-tolerant anthocyanin pigmentation would also be valuable.

Literature Cited

•  Aharoni,  A.,  C. H. R.  De Vos,  M.  Wein,  Z.  Sun,  R.  Greco,  A.  Kroon,  J. N.  Mol and  A. P.  O’Connell. 2001. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. Plant J. 28: 319–332.
•  Albert,  N. W.,  K. M.  Davies,  D. H.  Lewis,  H.  Zhang,  M.  Montefiori,  C.  Brendolise,  M. R.  Boase,  H.  Ngo,  P. E.  Jameson and  K. E.  Schwinn. 2014. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. Plant Cell 26: 962–980.
•  Albert,  N. W.,  D. H.  Lewis,  H.  Zhang,  K. E.  Schwinn,  P. E.  Jameson and  K. M.  Davies. 2011. Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning. Plant J. 65: 771–784.
•  An,  J.,  F.  Qu,  J.  Yao,  X.  Wang,  C.  You,  X.  Wang and  Y.  Hao. 2017. The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple. Hortic. Res. 4: 17023. DOI: 10.1038/hortres.2017.23.
•  An,  J.,  X.  Wang,  X.  Zhang,  S.  Bi,  C.  You and  Y.  Hao. 2019. MdBBX22 regulates UV-B-induced anthocyanin biosynthesis through regulating the function of MdHY5 and is targeted by MdBT2 for 26S proteasome-mediated degradation. Plant Biotechnol. J. 17: 2231–2233.
•  An,  J.,  X.  Wang,  R. V.  Espley,  K.  Lin-Wang,  S.  Bi,  C.  You and  Y.  Hao. 2020. An apple b-box protein MdBBX37 modulates anthocyanin biosynthesis and hypocotyl elongation synergistically with MdMYBs and MdHY5. Plant Cell Physiol. 61: 130–143.
•  Bai,  S.,  T.  Saito,  C.  Honda,  Y.  Hatsuyama,  A.  Ito and  T.  Moriguchi. 2014. An apple B-box protein, MdCOL11, is involved in UV-B- and temperature-induced anthocyanin biosynthesis. Planta 240: 1051–1062.
•  Bai,  S.,  R.  Tao,  Y.  Tang,  L.  Yin,  Y.  Ma,  J.  Ni,  X.  Yan,  Q.  Yang,  Z.  Wu,  Y.  Zeng and  Y.  Teng. 2019a. BBX16, a B-box protein, positively regulates light-induced anthocyanin accumulation by activating MYB10 in red pear. Plant Biotechnol. J. 17: 1985–1997.
•  Bai,  S.,  R.  Tao,  L.  Yin,  J.  Ni,  Q.  Yang,  X.  Yan,  F.  Yang,  X.  Guo,  H.  Li and  Y.  Teng. 2019b. Two B-box proteins, PpBBX18 and PpBBX21, antagonistically regulate anthocyanin biosynthesis via competitive association with Pyrus pyrifolia ELONGATED HYPOCOTYL 5 in the peel of pear fruit. Plant J. 100: 1208–1223.
•  Bonar,  N.,  M.  Liney,  R.  Zhang,  C.  Austin,  J.  Dessoly,  D.  Davidson,  J.  Stephens,  G.  McDougall,  M.  Taylor,  G. J.  Bryan and  C.  Hornyik. 2018. Potato miR828 is associated with purple tuber skin and flesh color. Front. Plant Sci. 9: 1742. DOI: 10.3389/fpls.2018.01742.
•  Bursch,  K.,  G.  Toledo-Ortiz,  M.  Pireyre,  M.  Lohr,  C.  Braatz and  H.  Johansson. 2020. Identification of BBX proteins as rate-limiting cofactors of HY5. Nat. Plants 6: 921–928.
•  Butelli,  E.,  A.  Garcia-Lor,  C.  Licciardello,  G.  Las Casas,  L.  Hill,  G. R.  Recupero,  M. L.  Keremane,  C.  Ramadugu,  R.  Krueger,  Q.  Xu,  X.  Deng,  A.  Fanciullino,  Y.  Froelicher,  L.  Navarro and  C.  Martin. 2017. Changes in anthocyanin production during domestication of Citrus. Plant Physiol. 173: 2225–2242.
•  Cavallini,  E.,  J. T.  Matus,  L.  Finezzo,  S.  Zenoni,  R.  Loyola,  F.  Guzzo,  R.  Schlechter,  A.  Ageorges,  P.  Arce-Johnson and  G. B.  Tornielli. 2015. The phenylpropanoid pathway is controlled at different branches by a set of R2R3-MYB C2 repressors in grapevine. Plant Physiol. 167: 1448–1470.
•  Chagné,  D.,  K.  Lin-Wang,  R. V.  Espley,  R. K.  Volz,  N. M.  How,  S.  Rouse,  C.  Brendolise,  C.  Carlisle,  S.  Kumar,  N.  De Silva,  D.  Micheletti,  T. K.  Mcghie,  R. N.  Crowhurst,  R.  Storey,  R.  Velasco,  R. P.  Hellens,  S. E.  Gardiner and  A. C.  Allan. 2013. An ancient duplication of apple MYB transcription factors is responsible for novel red fruit-flesh phenotypes. Plant Physiol. 161: 225–239.
•  Chiou,  C. Y. and  K. W.  Yeh. 2008. Differential expression of MYB gene (OgMYB1) determines color patterning in floral tissue of Oncidium Gower Ramsey. Plant Mol Biol. 66: 379–388.
•  Colanero,  S.,  P.  Perata and  S.  Gonzali. 2018. The atroviolacea gene encodes an R3-MYB protein repressing anthocyanin synthesis in tomato plants. Front. Plant Sci. 9: 1–17.
•  Cone,  K. C.,  M. S.  Cocciolone,  A. F.  Burr and  B.  Burr. 1993. Maize anthocyanin regulatory gene pl is a duplicate of c1 that functions in the plant. Plant Cell 5: 1795–1805.
•  Cui,  L.,  J.  Shan,  M.  Shi,  J.  Gao and  H.  Lin. 2014. The miR156-SPL9-DFR pathway coordinates the relationship between development and abiotic stress tolerance in plants. Plant J. 80: 1108–1117.
•  Davies,  K.,  N.  Albert,  Y.  Zhou and  K.  Schwinn. 2018. Functions of flavonoid and betalain pigments in abiotic stress tolerance in plants. Annu. Plant Rev. Online 1: 1–41.
•  Ding,  B.,  E. L.  Patterson,  S. V.  Holalu,  J.  Li,  G. A.  Johnson,  L. E.  Stanley,  A. B.  Greenlee,  F.  Peng,  H. D.  Bradshaw Jr.,  M. L.  Blinov,  B. K.  Blackman and  Y.  Yuan. 2020. Two MYB proteins in a self-organizing activator-inhibitor system produce spotted pigmentation patterns. Curr. Biol. 30: 802–814.
•  Dubos,  C.,  J.  Le Gourrierec,  A.  Baudry,  G.  Huep,  E.  Lanet,  I.  Debeaujon,  J. M.  Routaboul,  A.  Alboresi,  B.  Weisshaar and  L.  Lepiniec. 2008. MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana. Plant J. 55: 940–953.
•  Espley,  R. V.,  R. P.  Hellens,  J.  Putterill,  D. E.  Stevenson,  S.  Kutty-Amma and  A. C.  Allan. 2007. Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. Plant J. 49: 414–427.
•  Fang,  H.,  Y.  Dong,  X.  Yue,  X.  Chen,  N.  He,  J.  Hu,  S.  Jiang,  H.  Xu,  Y.  Wang,  M.  Su,  J.  Zhang,  Z.  Zhang,  N.  Wang and  X.  Chen. 2019a. MdCOL4 interaction mediates crosstalk between UV-B and high temperature to control fruit coloration in apple. Plant Cell Physiol. 60: 1055–1066.
•  Fang,  H.,  Y.  Dong,  X.  Yue,  J.  Hu,  S.  Jiang,  H.  Xu,  Y.  Wang,  M.  Su,  J.  Zhang,  Z.  Zhang,  N.  Wang and  X.  Chen. 2019b. The B-box zinc finger protein MdBBX20 integrates anthocyanin accumulation in response to ultraviolet radiation and low temperature. Plant Cell Environ. 42: 2090–2104.
•  Ferreyra,  M. L. F.,  P.  Serra and  P.  Casati. 2021. Recent advances on the roles of flavonoids as plant protective molecules after UV and high light exposure. Physiol. Plant. 173: 736–749.
•  Gangappa,  S. N. and  J. F.  Botto. 2014. The BBX family of plant transcription factors. Trends Plant Sci. 19: 460–470.
•  Gangappa,  S. N. and  J. F.  Botto. 2016. The multifaceted roles of HY5 in plant growth and development. Mol. Plant 9: 1353–1365.
•  Gates,  D. J.,  B. J. S. C.  Olson,  T. E.  Clemente and  S. D.  Smith. 2018. A novel R3 MYB transcriptional repressor associated with the loss of floral pigmentation in Iochroma. New Phytol. 217: 1346–1356.
•  Gonzalez,  A.,  M.  Zhao,  J. M.  Leavitt and  A. M.  Lloyd. 2008. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J. 53: 814–827.
•  Gou,  J.,  F. F.  Felippes,  C.  Liu,  D.  Weigel and  J.  Wang. 2011. Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. Plant Cell 23: 1512–1522.
•  Hsu,  C.,  Y.  Chen,  W.  Tsai,  W.  Chen and  H.  Chen. 2015. Three R2R3-MYB transcription factors regulate distinct floral pigmentation patterning in Phalaenopsis spp. Plant Physiol. 168: 175–191.
•  Huang,  D.,  Z.  Tang,  J.  Fu,  Y.  Yuan,  X.  Deng and  Q.  Xu. 2020. CsMYB3 and CsRuby1 form an ‘activator-and-repressor’ loop for the regulation of anthocyanin biosynthesis in citrus. Plant Cell Physiol. 61: 318–330.
•  Jiang,  M.,  L.  Ren,  H.  Lian,  Y.  Liu and  H.  Chen. 2016. Novel insight into the mechanism underlying light-controlled anthocyanin accumulation in eggplant (Solanum melongena L.). Plant Sci. 249: 46–58.
•  Jin,  H.,  E.  Cominelli,  P.  Bailey,  A.  Parr,  F.  Mehrtens,  J.  Jones,  C.  Tonelli,  B.  Weisshaar and  C.  Martin. 2000. Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. EMBO J. 19: 6150–6161.
•  Kim,  S.,  G.  Hwang,  S.  Lee,  J.  Zhu,  I.  Paik,  T. T.  Nguyen,  J.  Kim and  E.  Oh. 2017. High ambient temperature represses anthocyanin biosynthesis through degradation of HY5. Front. Plant Sci. 8: 1787. DOI: 10.3389/fpls.2017.01787.
•  Kobayashi,  S.,  N.  Goto-Yamamoto and  H.  Hirochika. 2004. Retrotransposon-induced mutations in grape skin color. Science 304: 982. DOI: 10.1126/science.1095011.
•  LaFountain,  A. M. and  Y.  Yuan. 2021. Repressors of anthocyanin biosynthesis. New Phytol. 231: 933–949.
•  Lafferty,  D. J.,  R. V.  Espley,  C. H.  Deng,  C. S.  Günther,  B.  Plunkett,  J. L.  Turner,  L.  Jaakola,  K.  Karppinen,  A. C.  Allan and  N. W.  Albert. 2022. Hierarchical regulation of MYBPA1 by anthocyanin- and proanthocyanidin-related MYB proteins is conserved in Vaccinium species. J. Exp. Bot. 73: 1344–1356.
•  Lai,  Y.,  M.  Yamagishi and  T.  Suzuki. 2011. Elevated temperature inhibits anthocyanin biosynthesis in the tepals of an Oriental hybrid lily via the suppression of LhMYB12 transcription. Sci Hortic. 132: 59–65.
•  Landi,  M.,  M.  Tattini and  K. S.  Gould. 2015. Multiple functional roles of anthocyanins in plant-environment interactions. Environ. Exp. Bot. 119: 4–17.
•  Li,  X.,  Y.  Hou,  X.  Xie,  H.  Li,  X.  Li,  Y.  Zhu,  L.  Zhai,  C.  Zhang and  S.  Bian. 2020a. A blueberry MIR156a–SPL12 module coordinates the accumulation of chlorophylls and anthocyanins during fruit ripening. J. Exp. Bot. 71: 5976–5989.
•  Li,  Y.,  X.  Shan,  R.  Gao,  T.  Han,  J.  Zhang,  Y.  Wang,  S.  Kimani,  L.  Wang and  X.  Gao. 2020b. MYB repressors and MBW activation complex collaborate to fine-tune flower coloration in Freesia hybrida. Commun. Biol. 3: 1–14.
•  Li,  Y.,  P.  Xu,  G.  Chen,  J.  Wu,  Z.  Liu and  H.  Lian. 2020c. FvbHLH9 functions as a positive regulator of anthocyanin biosynthesis by forming a HY5–bHLH9 transcription complex in strawberry fruits. Plant Cell Physiol. 61: 826–837.
•  Lin-Wang,  K.,  D.  Micheletti,  J.  Palmer,  R.  Volz,  L.  Lozano,  R.  Espley,  R. P.  Hellens,  D.  Chagnè,  D. D.  Rowan,  M.  Troggio,  I.  Iglesias and  A. C.  Allan. 2011. High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex. Plant Cell Environ. 34: 1176–1190.
•  Liu,  C.,  C.  Chi,  L.  Jin,  J.  Zhu,  J.  Yu and  Y.  Zhou. 2018. The bZip transcription factor HY5 mediates CRY1a-induced anthocyanin biosynthesis in tomato. Plant Cell Environ. 41: 1762–1775.
•  Liu,  Y.,  K.  Lin-Wang,  R. V.  Espley,  L.  Wang,  Y.  Li,  Z.  Liu,  P.  Zhou,  L.  Zeng,  X.  Zhang,  J.  Zhang and  A. C.  Allan. 2019. StMYB44 negatively regulates anthocyanin biosynthesis at high temperatures in tuber flesh of potato. J. Exp. Bot. 70: 3809–3824.
•  Liu,  Y.,  K.  Lin-Wang,  R. V.  Espley,  L.  Wang,  H.  Yang,  B.  Yu,  A.  Dare,  E.  Varkonyi-Gasic,  J.  Wang,  J.  Zhang,  D.  Wang and  A. C.  Allan. 2016. Functional diversification of the potato R2R3 MYB anthocyanin activators AN1, MYBA1, and MYB113 and their interaction with basic helix-loop-helix cofactors. J. Exp. Bot. 67: 2159–2176.
•  Liu,  W.,  H.  Mu,  L.  Yuan,  Y.  Li,  Y.  Li,  S.  Li,  C.  Ren,  W.  Duan,  P.  Fan,  Z.  Dai,  Y.  Zhou,  Z.  Liang,  S.  Li and  L.  Wang. 2023. VvBBX44 and VvMYBA1 form a regulatory feedback loop to balance anthocyanin biosynthesis in grape. Hortic. Res. 10: uhad176. DOI: 10.1093/hr/uhad176.
•  Lloyd,  A.,  A.  Brockman,  L.  Aguirre,  A.  Campbell,  A.  Bean,  A.  Cantero and  A.  Gonzalez. 2017. Advances in the MYB–bHLH–WD repeat (MBW) pigment regulatory model: addition of a WRKY factor and co-option of an anthocyanin MYB for betalain regulation. Plant Cell Physiol. 58: 1431–1441.
•  Ma,  D. and  C. P.  Constabel. 2019. MYB repressors as regulators of phenylpropanoid metabolism in plants. Trends Plant Sci. 24: 275–289.
•  Ma,  D.,  M.  Reichelt,  K.  Yoshida,  J.  Gershenzon and  C. P.  Constabel. 2018. Two R2R3‐MYB proteins are broad repressors of flavonoid and phenylpropanoid metabolism in poplar. Plant J. 96: 949–965.
•  Man,  Y.,  Y.  Wang,  Z.  Li,  Z.  Jiang,  H.  Yang,  J.  Gong,  S.  He,  S.  Wu,  Z.  Yang,  J.  Zheng and  Z.  Wang. 2015. High-temperature inhibition of biosynthesis and transportation of anthocyanins results in the poor red coloration in red-fleshed Actinidia chinensis. Physiol. Plant. 153: 565–583.
•  Matsui,  K.,  Y.  Umemura and  M.  Ohme-Takagi. 2008. AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis. Plant J. 55: 954–967.
•  Matsushita,  K.,  A.  Sakayori and  T.  Ikeda. 2016. The effect of high air temperature on anthocyanin concentration and the expressions of its biosynthetic genes in strawberry ‘Sachinoka’. Environ. Control Biol. 54: 101–107.
•  Mori,  K.,  N.  Goto-Yamamoto,  M.  Kitayama and  K.  Hashizume. 2007. Loss of anthocyanins in red-wine grape under high temperature. J. Exp. Bot. 58: 1935–1945.
•  Movahed,  N.,  C.  Pastore,  A.  Cellini,  G.  Allegro,  G.  Valentini,  S.  Zenoni,  E.  Cavallini,  E.  D’Incà,  G. B.  Tornielli and  I.  Filippetti. 2016. The grapevine VviPrx31 peroxidase as a candidate gene involved in anthocyanin degradation in ripening berries under high temperature. J. Plant Res. 129: 513–526.
•  Naik,  J.,  P.  Misra,  P. K.  Trivedi and  A.  Pandey. 2022. Molecular components associated with the regulation of flavonoid biosynthesis. Plant Sci. 317: 111196. DOI: 10.1016/j.plantsci.2022.111196.
•  Naing,  A. H. and  C. K.  Kim. 2021. Abiotic stress-induced anthocyanins in plants: their role in tolerance to abiotic stresses. Physiol. Plant. 172: 1711–1723.
•  Nakatsuka,  T.,  T.  Suzuki,  K.  Harada,  Y.  Kobayashi,  H.  Dohra and  H.  Ohno. 2019. Floral organ- and temperature-dependent regulation of anthocyanin biosynthesis in Cymbidium hybrid flowers. Plant Sci. 287: 110173. DOI: 10.1016/j.plantsci.2019.110173.
•  Nakatsuka,  T.,  E.  Yamada,  M.  Saito,  K.  Fujita and  M.  Nishihara. 2013. Heterologous expression of gentian MYB1R transcription factors suppresses anthocyanin pigmentation in tobacco flowers. Plant Cell Rep. 32: 1925–1937.
•  Nemie-Feyissa,  D.,  S. M.  Olafsdottir,  B.  Heidari and  C.  Lillo. 2014. Nitrogen depletion and small R3-MYB transcription factors affecting anthocyanin accumulation in Arabidopsis leaves. Phytochem. 98: 34–40.
•  Niu,  J.,  G.  Zhang,  W.  Zhang,  V.  Goltsev,  S.  Sun,  J.  Wang,  P.  Li and  F.  Ma. 2017. Anthocyanin concentration depends on the counterbalance between its synthesis and degradation in plum fruit at high temperature. Sci. Rep. 7: 7684. DOI: 10.1038/s41598-017-07896-0.
•  Nozaki,  K.,  C.  Muramoto,  T.  Takamura and  S.  Fukai. 2006. Effects of growing season and temperature on flower color in apricot and picotee flower genotypes of spray chrysanthemum. Hort. Res. (Japan) 5: 123–128.
•  Park,  Y.,  H.  Lee,  J.  Ha,  J. Y.  Kim and  C.  Park. 2017. COP1 conveys warm temperature information to hypocotyl thermomorphogenesis. New Phytol. 215: 269–280.
•  Pastore,  C.,  S.  Dal Santo,  S.  Zenoni,  N.  Movahed,  G.  Allegro,  G.  Valentini,  I.  Filippetti and  G. B.  Tornielli. 2017. Whole plant temperature manipulation affects flavonoid metabolism and the transcriptome of grapevine berries. Front. Plant Sci. 8: 929. DOI: 10.3389/fpls.2017.00929.
•  Peng,  T.,  T.  Saito,  C.  Honda,  Y.  Ban,  S.  Kondo,  J.  Liu,  Y.  Hatsuyama and  T.  Moriguchi. 2013. Screening of UV-B-induced genes from apple peels by SSH: possible involvement of MdCOP1-mediated signaling cascade genes in anthocyanin accumulation. Physiol. Plant. 148: 432–444.
•  Peng,  Y.,  A. H.  Thrimawithana,  J. M.  Cooney,  D. J.  Jensen,  R. V.  Espley and  A. C.  Allan. 2020. The proanthocyanin-related transcription factors MYBC1 and WRKY44 regulate branch points in the kiwifruit anthocyanin pathway. Sci. Rep. 10: 14161. DOI: 10.1038/s41598-020-70977-0.
•  Podolec,  R. and  R.  Ulm. 2018. Photoreceptor-mediated regulation of the COP1/SPA E3 ubiquitin ligase. Curr. Opin. Plant Biol. 45: 18–25.
•  Qiu,  Z.,  H.  Wang,  D.  Li,  B.  Yu,  Q.  Hui,  S.  Yan,  Z.  Huang,  X.  Cui and  B.  Cao. 2019. Identification of candidate HY5-dependent and -independent regulators of anthocyanin biosynthesis in tomato. Plant Cell Physiol. 60: 643–656.
•  Rajagopalan,  R.,  H.  Vaucheret,  J.  Trejo and  D. P.  Bartel. 2006. A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev. 20: 3407–3425.
•  Rehman,  R. N. U.,  Y.  You,  L.  Zhang,  B. D.  Goudia,  A. R.  Khan,  P.  Li and  F.  Ma. 2017. High temperature induced anthocyanin inhibition and active degradation in Malus profusion. Front. Plant Sci. 8: 1401. DOI: 10.3389/fpls.2017.01401.
•  Rowan,  D. D.,  M.  Cao,  K.  Lin-Wang,  J. M.  Cooney,  D. J.  Jensen,  P. T.  Austin,  M. B.  Hunt,  C.  Norling,  R. P.  Hellens,  R. J.  Schaffer and  A. C.  Allan. 2009. Environmental regulation of leaf colour in red 35S:PAP1 Arabidopsis thaliana. New Phytol. 182: 102–115.
•  Ryu,  S.,  J. H.  Han,  J. G.  Cho,  J. H.  Jeong,  S. K.  Lee and  H. J.  Lee. 2020. High temperature at veraison inhibits anthocyanin biosynthesis in berry skins during ripening in ‘Kyoho’ grapevines. Plant Physiol. Biochem. 157: 219–228.
•  Sakai,  M.,  M.  Yamagishi and  K.  Matsuyama. 2019. Repression of anthocyanin biosynthesis by R3-MYB transcription factors in lily (Lilium spp.). Plant Cell Rep. 38: 609–622.
•  Shang,  Y.,  J.  Venail,  S.  Mackay,  P. C.  Bailey,  K. E.  Schwinn,  P. E.  Jameson,  C. R.  Martin and  K. M.  Davies. 2011. The molecular basis for venation patterning of pigmentation and its effect on pollinator attraction in flowers of Antirrhinum. New Phytol. 189: 602–615.
•  Shi,  Z.,  X.  Han,  G.  Wang,  J.  Qiu,  L.  Zhou,  S.  Chen,  W.  Fang,  F.  Chen and  J.  Jiang. 2022. Transcriptome analysis reveals chrysanthemum flower discoloration under high-temperature stress. Front. Plant Sci. 13: 1003635. DOI: 10.3389/fpls.2022.1003635.
•  Shin,  J.,  E.  Park and  G.  Choi. 2007. PIF3 regulates anthocyanin biosynthesis in an HY5-dependent manner with both factors directly binding anthocyanin biosynthetic gene promoters in Arabidopsis. Plant J. 49: 981–994.
•  Shin,  D.,  M.  Choi,  K.  Kim,  G.  Bang,  M.  Cho,  S.  Choi,  G.  Choi and  Y.  Park. 2013. HY5 regulates anthocyanin biosynthesis by inducing the transcriptional activation of the MYB75/PAP1 transcription factor in Arabidopsis. FEBS Lett. 587: 1543–1547.
•  Stracke,  R.,  M.  Werber and  B.  Weisshaar. 2001. The R2R3-MYB gene family in Arabidopsis thaliana. Curr. Opin. Plant Biol. 4: 447–456.
•  Tan,  Y.,  B.  Wen,  L.  Xu,  X.  Zong,  Y.  Sun,  G.  Wei and  H.  Wei. 2023. High temperature inhibited the accumulation of anthocyanin by promoting ABA catabolism in sweet cherry fruits. Front. Plant Sci. 14: 1079292. DOI: 10.3389/fpls.2023.1079292.
•  Tanaka,  Y.,  N.  Sasaki and  A.  Ohmiya. 2008. Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J. 54: 733–749.
•  Tao,  R.,  S.  Bai,  J.  Ni,  Q.  Yang,  Y.  Zhao and  Y.  Teng. 2018. The blue light signal transduction pathway is involved in anthocyanin accumulation in ‘Red Zaosu’ pear. Planta 248: 37–48.
•  Tirumalai,  V.,  C.  Swetha,  A.  Nair,  A.  Pandit and  P. V.  Shivaprasad. 2019. MiR828 and MiR858 regulate VvMYB114 to promote anthocyanin and flavonol accumulation in grapes. J. Exp. Bot. 70: 4775–4792.
•  Tohge,  T.,  L. P.  de Souza and  A. R.  Fernie. 2017. Current understanding of the pathways of flavonoid biosynthesis in model and crop plants. J. Exp. Bot. 68: 4013–4028.
•  Vaz,  A. P. A.,  R. de C. L.  Figueiredo-Ribeiro and  G. B.  Kerbauy. 2004. Photoperiod and temperature effects on in vitro growth and flowering of P. pusilla, an epiphytic orchid. Plant Physiol. Biochem. 42: 411–415.
•  Wang,  S. and  G.  Chen. 2014. Regulation of cell fate determination by single-repeat R3 MYB transcription factors in Arabidopsis. Front. Plant Sci. 5: 133. DOI: 10.3389/fpls.2014.00133.
•  Wang,  Y.,  Y.  Wang,  Z.  Song and  H.  Zhang. 2016a. Repression of MYBL2 by both microRNA858a and HY5 leads to the activation of anthocyanin biosynthetic pathway in Arabidopsis. Mol. Plant 9: 1395–1405.
•  Wang,  N.,  Z.  Zhang,  S.  Jiang,  H.  Xu,  Y.  Wang,  S.  Feng and  X.  Chen. 2016b. Synergistic effects of light and temperature on anthocyanin biosynthesis in callus cultures of red-fleshed apple (Malus sieversii f. niedzwetzkyana). Plant Cell Tissue Organ Cult. 127: 217–227.
•  Wang,  Y.,  C.  Zhang,  B.  Xu,  J.  Fu,  Y.  Du,  Q.  Fang,  B.  Dong and  H.  Zhao. 2022. Temperature regulation of carotenoid accumulation in the petals of sweet osmanthus via modulating expression of carotenoid biosynthesis and degradation genes. BMC Genomics 23: 418. DOI: 10.1186/s12864-022-08643-0.
•  Wang,  N.,  C.  Qu,  S.  Jiang,  Z.  Chen,  H.  Xu,  H.  Fang,  M.  Su,  J.  Zhang,  Y.  Wang,  W.  Liu,  Z.  Zhang,  N.  Lu and  X.  Chen. 2018. The proanthocyanidin-specific transcription factor MdMYBPA1 initiates anthocyanin synthesis under low-temperature conditions in red-fleshed apples. Plant J. 96: 39–55.
•  Xiang,  L.,  X.  Liu,  H.  Li,  X.  Yin,  D.  Grierson,  F.  Li and  K.  Chen. 2019. CmMYB#7, an R3 MYB transcription factor, acts as a negative regulator of anthocyanin biosynthesis in chrysanthemum. J. Exp. Bot. 70: 3111–3124.
•  Xiao,  Y.,  L.  Chu,  Y.  Zhang,  Y.  Bian,  J.  Xiao and  D.  Xu. 2022. HY5: A pivotal regulator of light-dependent development in higher plants. Front. Plant Sci. 12: 800989. DOI: 10.1093/jxb/erz121.
•  Xie,  X.,  S.  Li,  R.  Zhang,  J.  Zhao,  Y.  Chen,  Q.  Zhao,  Y.  Yao,  C.  You,  X.  Zhang and  Y.  Hao. 2012. The bHLH transcription factor MdbHLH3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples. Plant Cell Environ. 35: 1884–1897.
•  Xu,  W.,  C.  Dubos and  L.  Lepiniec. 2015. Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends Plant Sci. 20: 176–185.
•  Xu,  H.,  N.  Wang,  J.  Liu,  C.  Qu,  Y.  Wang,  S.  Jiang,  N.  Lu,  D.  Wang,  Z.  Zhang and  X.  Chen. 2017. The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes. Plant Mol. Biol. 94: 149–165.
•  Xu,  D.,  Y.  Jiang,  J.  Li,  M.  Holm and  X. W.  Deng. 2018. The B-Box domain protein BBX21 promotes photomorphogenesis. Plant Physiol. 176: 2365–2375.
•  Yan,  Y.,  J.  Zhao,  S.  Lin,  M.  Li,  J.  Liu,  O.  Raymond,  P.  Vergne,  W.  Kong,  Q.  Wu,  X.  Zhang,  M.  Bao,  M.  Bendahmane and  X.  Fu. 2023. Light-mediated anthocyanin biosynthesis in rose petals involves a balanced regulatory module comprising transcription factors RhHY5, RhMYB114a, and RhMYB3b. J. Exp. Bot. 74: 5783–5804.
•  Yamagishi,  M. 2011. Oriental hybrid lily Sorbonne homologue of LhMYB12 regulates anthocyanin biosyntheses in flower tepals and tepal spots. Mol. Breed. 28: 381–389.
•  Yamagishi,  M. 2016. A novel R2R3-MYB transcription factor regulates light-mediated floral and vegetative anthocyanin pigmentation patterns in Lilium regale. Mol. Breed. 36: 3. DOI: 10.1007/s11032-015-0426-y.
•  Yamagishi,  M. 2018. Involvement of a LhMYB18 transcription factor in large anthocyanin spot formation on the flower tepals of the Asiatic hybrid lily (Lilium spp.) cultivar ‘Grand Cru’. Mol. Breed. 38: 60. DOI: 10.1007/s11032-018-0806-1.
•  Yamagishi,  M. 2020a. MYB19LONG is involved in brushmark pattern development in Asiatic hybrid lily (Lilium spp.) flowers. Sci. Hortic. 272: 109570. DOI: 10.1016/j.scienta.2020.109570.
•  Yamagishi,  M. 2020b. Isolation and identification of MYB transcription factors (MYB19Long and MYB19Short) involved in raised spot anthocyanin pigmentation in lilies (Lilium spp.). J. Plant Physiol. 250: 153164. DOI: 10.1016/j.jplph.2020.153164.
•  Yamagishi,  M. 2020c. White with partially pink flower color in Lilium cernuum var. album is caused by transcriptional regulation of anthocyanin biosynthesis genes. Sci. Hortic. 260: 108880. DOI: 10.1016/j.scienta.2019.108880.
•  Yamagishi,  M. 2021. High promoter sequence variation in subgroup 6 members of R2R3-MYB genes is involved in different floral anthocyanin color patterns in Lilium spp. Mol. Genet. Genomics 296: 1005–1015.
•  Yamagishi,  M. 2022a. MicroRNA828/MYB12 module mediated bicolor flower development in Lilium dauricum. Hort. J. 91: 399–407.
•  Yamagishi,  M. 2022b. High temperature enhances anthocyanin coloration in Asiatic hybrid lily flowers via upregulation of the MYB12 positive regulator. Hort. Plant J. 8: 769–776.
•  Yamagishi,  M. and  M.  Sakai. 2020. The microRNA828/MYB12 module mediates bicolor pattern development in Asiatic hybrid lily (Lilium spp.) flowers. Front. Plant Sci. 11: 590791. DOI: 10.3389/fpls.2020.590791.
•  Yamagishi,  M.,  Y.  Shimoyamada,  T.  Nakatsuka and  K.  Masuda. 2010. Two R2R3-MYB genes, homologs of petunia AN2, regulate anthocyanin biosyntheses in flower tepals, tepal spots and leaves of Asiatic hybrid lily. Plant Cell Physiol. 51: 463–474.
•  Yamagishi,  M.,  S.  Toda and  K.  Tasaki. 2014. The novel allele of the LhMYB12 gene is involved in splatter-type spot formation on the flower tepals of Asiatic hybrid lilies (Lilium spp.). New Phytol. 201: 1009–1020.
•  Yamagishi,  M.,  Y.  Yoshida and  M.  Nakayama. 2012. The transcription factor LhMYB12 determines anthocyanin pigmentation in the tepals of Asiatic hybrid lilies (Lilium spp.) and regulates pigment quantity. Mol. Breed. 30: 913–925.
•  Yoshida,  K.,  D.  Ma and  C. P.  Constabel. 2015. The MYB182 protein down-regulates proanthocyanidin and anthocyanin biosynthesis in poplar by repressing both structural and regulatory flavonoid genes. Plant Physiol. 167: 693–710.
•  Yuan,  Y.,  J. M.  Sagawa,  L.  Frost,  J. P.  Vela and  H. D.  Bradshaw Jr. 2014. Transcriptional control of floral anthocyanin pigmentation in monkeyflowers (Mimulus). New Phytol. 204: 1013–1027.
•  Yuan,  Y.,  J. M.  Sagawa,  R. C.  Young,  B. J.  Christensen and  H. D.  Bradshaw. 2013. Genetic dissection of a major anthocyanin QTL contributing to pollinator-mediated reproductive isolation between sister species of Mimulus. Genetics 194: 255–263.
•  Zhang,  S.,  A.  Zhang,  X.  Wu,  Z.  Zhu,  Z.  Yang,  Y.  Zhu and  D.  Zha. 2019. Transcriptome analysis revealed expression of genes related to anthocyanin biosynthesis in eggplant (Solanum melongena L.) under high-temperature stress. BMC Plant Biol. 19: 387. DOI: 10.1186/s12870-019-1960-2.
•  Zhang,  H.,  J.  Gong,  K.  Chen,  W.  Yao,  B.  Zhang,  J.  Wang,  S.  Tian,  H.  Liu,  Y.  Wang,  Y.  Liu and  L.  Du. 2020a. A novel R3 MYB transcriptional repressor, MaMYBx, finely regulates anthocyanin biosynthesis in grape hyacinth. Plant Sci. 298: 110588. DOI: 10.1016/j.plantsci.2020.110588.
•  Zhang,  B.,  H.  Yang,  Y.  Yang,  Z.  Zhu,  Y.  Li,  D.  Qu and  Z.  Zhao. 2020b. Mdm-miR828 participates in the feedback loop to regulate anthocyanin accumulation in apple peel. Front. Plant Sci. 11: 608109. DOI: 10.3389/fpls.2020.608109.
•  Zhao,  X.,  P.  Li,  H.  Zuo,  A.  Peng,  J.  Lin,  P.  Li,  K.  Wang,  Q.  Tang,  M.  Tadege,  Z.  Liu and  J.  Zhao. 2023. CsMYBL2 homologs modulate the light and temperature stress-regulated anthocyanin and catechins biosynthesis in tea plants (Camellia sinensis). Plant J. 115: 1051–1070.
•  Zhou,  H.,  K.  Lin-Wang,  F.  Wang,  R. V.  Espley,  F.  Ren,  J.  Zhao,  C.  Ogutu,  H.  He,  Q.  Jiang,  A. C.  Allan and  Y.  Han. 2019. Activator-type R2R3-MYB genes induce a repressor-type R2R3-MYB gene to balance anthocyanin and proanthocyanidin accumulation. New Phytol. 221: 1919–1934.
•  Zhou,  L.,  Z.  Geng,  Y.  Wang,  Y.  Wang,  S.  Liu,  C.  Chen,  A.  Song,  J.  Jiang,  S.  Chen and  F.  Chen. 2021. A novel transcription factor CmMYB012 inhibits flavone and anthocyanin biosynthesis in response to high temperatures in chrysanthemum. Hortic. Res. 8: 248. DOI: 10.1038/s41438-021-00675-z.
•  Zipor,  G.,  P.  Duarte,  I.  Carqueijeiro,  L.  Shahar,  R.  Ovadia,  P.  Teper-Bamnolker,  D.  Eshel,  Y.  Levin,  A.  Doron-Faigenboim,  M.  Sottomayor and  M.  Oren-Shamir. 2015. In planta anthocyanin degradation by a vacuolar class III peroxidase in Brunfelsia calycina flowers. New Phytol. 205: 653–665.