Regulation Of Anthocyanin Accumulation Research Articles

Graph pangenome reveals the regulation of malate content in blood-fleshed peach by NAC transcription factors

BackgroundFruit acidity and color are important quality attributes in peaches. Although there are some exceptions, blood-fleshed peaches typically have a sour taste. However, little is known about the genetic variations linking organic acid and color regulation in peaches.ResultsHere, we report a peach graph-based pangenome constructed from sixteen individual genome assemblies, capturing abundant structural variations and 82.3 Mb of sequences absent in the reference genome. Pangenome analysis reveals a long terminal repeat retrotransposon insertion in the promoter of the NAC transcription factor (TF) PpBL in blood-fleshed peaches, which enhances PpBL expression. Genome-wide association study identifies a significant association between PpBL and malate content. Silencing PpBL in peach fruit and ectopic overexpression of PpBL in tomatoes confirm that PpBL is a positive regulator of malate accumulation. Furthermore, we demonstrate that PpBL works synergistically with another NAC TF, PpNAC1, to activate the transcription of the aluminum-activated malate transporter PpALMT4, leading to increased malate content.ConclusionsThese findings, along with previous research showing that PpBL and PpNAC1 also regulate anthocyanin accumulation, explain the red coloration and sour taste in blood-fleshed peach fruits.

ArMYB89 and ArCOP1 interaction modulates anthocyanin biosynthesis in Acer rubrum leaves under low-temperature conditions.

Acer rubrum, a famous ornamental tree, produces bright red-coloured leaves because of the temperature decline from summer to autumn. This process's molecular mechanism is elusive, so we have investigated how anthocyanin biosynthesis is induced in A. rubrum leaves under low temperatures. The results of low-temperature treatment under light and dark conditions showed that the low-temperature promoted anthocyanin accumulation in A. rubrum is light-dependent. The transcriptome analysis showed that ArMYB89 was significantly highly expressed in leaves of A. rubrum growing under low temperatures with light conditions. The findings from the Dap-seq analysis, yeast one hybridisation, electrophoretic mobility shift assay and luciferase reporter assay indicated that the ArMYB89 transcription factor binds directly to the promoter of ArUGT52 and stimulates its transcription. The co-expression of ArUGT52 with ArMYB89 significantly induced anthocyanin levels under low temperatures with light conditions. Enzyme activity analysis showed that ArUGT52 could convert Cyanidins and Pelargonidins into Cyanidin-3-O-glucoside and Pelargonidin 3-glucoside, which are considered the main anthocyanins in red colour leaves of A. rubrum. The results of yeast two hybridisation, pulldown assay and bimolecular fluorescence complementation experiment showed an interaction between COP1 and ArMYB89, while in vivo and in vitro protein ubiquitination assay demonstrated that ArCOP1 ubiquitinates ArMYB89. Notably, co-expression of ArCOP1 with ArMYB89 significantly reduced anthocyanin levels, while the virus-induced gene silencing of ArCOP1 significantly induced anthocyanin levels under low temperatures with light conditions. In conclusion, this work revealed the molecular mechanism regulating anthocyanin accumulation in the A. rubrum leaves under low temperatures.

Analysis of Transcriptional and Metabolic Differences in the Petal Color Change Response to High-Temperature Stress in Various Chrysanthemum Genotypes

Flower color is one of the most important ornamental traits of chrysanthemums. Previous studies have shown that high temperatures can cause the petals of some chrysanthemum varieties to fade; however, the molecular mechanisms behind this phenomenon remain poorly understood. This study examines the mechanisms of color change in purple chrysanthemums under high-temperature stress using combined metabolomic and transcriptomic analyses. Four chrysanthemum varieties—two heat-stable (‘Zi Feng Che’ and ‘Chrystal Regal’) and two heat-sensitive (‘Zi Hong Tuo Gui’ and ‘Zi Lian’)—were analyzed. High-temperature conditions (35 °C) significantly downregulated key anthocyanins in heat-sensitive varieties, particularly cyanidin-3-O-(3″,6″-O-dimalonyl)glucoside and pelargonidin-3-O-(3″,6″-O-dimalonyl)glucoside. Transcriptome analysis revealed differential gene expression involved in anthocyanin biosynthesis and degradation, with significant enrichment in the MAPK signaling, phenylpropanoid biosynthesis, flavonoid biosynthesis, and anthocyanin biosynthesis pathways. The study highlighted the differential expression of CHS, DFR, ANS, GT1, 3AT, and UGT75C1 genes in anthocyanin synthesis between heat-sensitive and heat-tolerant varieties. Compared to heat-stable varieties, the petals of heat-sensitive varieties exhibited greater differential expression of heat-responsive transcription factors, including HSFs, ERFs, MYBs, and WRKYs. Genes that show a significant negative correlation with the downregulated anthocyanins, including Cse_sc012959.1_g030.1 (βG), Cse_sc001798.1_g020.1 (MYB), Cse_sc006944.1_g010.1 (MYB), and Cse_sc000572.1_g090.1 (HSF), might regulate anthocyanin accumulation in chrysanthemums in response to high-temperature stress. These results provide guidance for the cultivation management and variety selection of chrysanthemums under high-temperature conditions. Additionally, they lay the foundation for elucidating the molecular mechanisms of flower color stability under heat stress and for breeding new heat-tolerant varieties.

Plant U-box E3 ligase PpPUB59 regulates anthocyanin accumulation by ubiquitinating PpBBX24 in ‘Zaosu’ pear and its red bud mutation

Ubiquitination is the specific modification of target proteins in cells by ubiquitin molecules, which is under the action of a series of special enzymes such as ubiquitin-activating enzymes, binding, and ligase enzymes. Ubiquitination plays an essential role in anthocyanin accumulation in plants. There are few studies on the coloring of pear peel by ubiquitin ligase E3. In this study, an E3 ubiquitin ligase protein PpPUB59 with seven WD40 repeats was cloned. And the function of PpPUB59 on the ubiquitination and protein stability of PpBBX24 and Ppbbx24-del, and the possible action mechanism in the anthocyanin accumulation of ‘Red Zaosu’ was studied. Our results showed that the WD40 repeats were verified to be the key domain interacting with the VP domain of BBX protein. PpPUB59 could degrade PpBBX24 in vitro by interacting with the VP domain but could not degrade the mutant PpBBX24-del without the VP domain. Dual luciferase assay showed that Ppbbx24-del could activate the PpCHS promoter, while PpPUB59 did not interfere with this activation; PpBBX24 could not activate the promotor of PpCHS but could suppress the activation of PpHY5; when the PpPUB59 was co-expressed with PpBBX24 and PpHY5, the activation roles of PpHY5 in the promotor of PpCHS was not recovered. BiFC and yeast two-hybrid experiments showed that PpPUB59 could also interact with PpHY5, which may make it ubiquitinated and degraded by 26S proteasome. In conclusion, PpPUB59 played an essential role in pear anthocyanin accumulation by ubiquitinating the associated transcription factors. These findings clarified the mutant mechanism of the ‘Red Zaosu’ pear at the post-translational modification level and enriched the regulation theory of the pear anthocyanin accumulation.

Ppbbx24-del mutant positively regulates light-induced anthocyanin accumulation in the ‘Red Zaosu’ pear (Pyrus pyrifolia White Pear Group)

Red fruit peel is one of the most valuable economic traits in pear and is mainly determined by anthocyanins. Many pear cultivars with a red peel originated from bud sports; however, little is known about the genetic mechanisms underlying this trait. We have previously identified a mutant PpBBX24 containing a 14-nucleotide deletion in the coding region (Ppbbx24-del) as the only known variant associated with the red coloration of the mutant ‘Red Zaosu’ pear (Pyrus pyrifolia White Pear Group). Herein, we analyzed the role of the mutant gene in red coloration and its mechanism of action. The results showed that light promoted red <pg=>peel coloration in ‘Red Zaosu’ pear, and Ppbbx24-del had positive effects on light-induced anthocyanin biosynthesis, while normal PpBBX24 had the opposite effects. Transient and stable <pg=>transformation experiments confirmed that Ppbbx24-del could promote anthocyanin accumulation in pear fruit peels, calli, and tobacco flowers. Due to the loss of NLS and VP domains, Ppbbx24-del co-localized in the nucleus and cytoplasm, whereas PpBBX24 localized only in the nucleus. Real-time PCR and transcriptome analyses indicated that PpMYB10 and PpHY5 is highly expressed in ‘Red Zaosu’ pear. In yeast one-hybrid and dual luciferase assays, Ppbbx24-del and PpHY5 independently promoted the expression of PpCHS, PpCHI, and PpMYB10 by binding to their promoters; however, PpBBX24 did not affect the expression of these genes. Additionally, we found that Ppbbx24-del and PpHY5 had additive effects on the expression of PpCHS, PpCHI, and PpMYB10, as they promote the expression of anthocyanin synthesis genes separately. The co-expression of PpBBX24 and PpHY5 inhibited the activation of downstream genes by PpHY5, and this was attributed to the interaction between the two loci. In conclusion, our results clarify the molecular mechanism by which mutant Ppbbx24-del and PpBBX24 exert opposite effects in the regulation of anthocyanin accumulation in pear. These findings lay an important theoretical foundation for the use of Ppbbx24-del to create red pear cultivars.

A feedback loop comprising RhMYB114 and RhMYB16 regulates anthocyanin accumulation and tissue acidification in Rosa hybrida

Coloration in rose (Rosa hybrida) petals is primarily determined by anthocyanin accumulation in vacuoles, and vacuolar acidification plays a central role in controlling the accumulation of this pigment. Nevertheless, the regulatory interplay between anthocyanin accumulation and tissue acidification processes remains somewhat unclear. The present study characterized an activator RhMYB114 and a repressor RhMYB16, which functioned synergistically in anthocyanin accumulation and tissue acidification in rose. Transforming tobacco and roses by overexpression, the introduction of RhMYB114 resulted in an increase in anthocyanin levels and a noticeable decrease in pH in the petal cells of both rose and tobacco, whereas RhMYB16 introduction led to inverse effects. To further clarify the underlying the regulatory mechanisms, the yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and dual-luciferase (LUC) were employed. The results showed that RhMYB16 competed with RhMYB114, bound to RhbHLH3 or RhbHLH33, and inhibited its ability to induce the expression of genes related to anthocyanin biosynthesis and acidification. Our findings revealed a feedback mechanism for the regulation of anthocyanin synthesis and tissue acidification involving RhMYB114, which stimulated the transcriptional expression of RhMYB16, whose encoded protein RhMYB16, in turn, negatively regulated the transcriptional expression of RhMYB114. Therefore, this study underscores the pivotal roles of the RhMYB114–RhMYB16 loop in regulating anthocyanin synthesis and tissue acidification, offering insights into metabolic manipulation to enhance the aesthetic appeal of roses.

Genome-wide analysis of glutathione S-transferase genes in four Prunus species and the function of PmGSTF2, activated by PmMYBa1, in regulating anthocyanin accumulation in Prunus mume

Glutathione S-transferases (GSTs) are proteases with multiple physiological functions and play an important role in plant responses to abiotic stresses. Nevertheless, there is a paucity of systematic research on GST genes in Prunus genus. Here, 330 GST genes in four Prunus species were identified for the first time and classified into eight subgroups based on protein sequence and conserved structure, among which Tau subfamily genes had the largest number. The amino acid lengths of GST-encoded proteins in the four species ranged from 66 to 1152 aa, most of which were soluble proteins and located in the cytoplasm and chloroplasts. The GST family was propelled by tandem duplications, yet robust purifying selection constrained its divergence. Conserved motif and domain analysis revealed that the majority of PmGSTs exhibited a highly conserved GST-N structure. The expression pattern of PmGSTs exhibited tissue specificity and spatiotemporal specificity. qRT-PCR validated the transcriptome results and 11 genes were differentially expressed in varieties with different flower and stem colors. In addition, we discovered an anthocyanin-related gene PmGSTF2, which can effectively restore the anthocyanin and proanthocyanidin deficiency-related phenotypes of the Arabidopsis tt19 mutant. Recombinant PmGSTF2 enhanced the water solubility of cyanidin and cyanidin-3-O-glucoside in vitro. Moreover, PmMYBa1 could directly bind to the promoter of PmGSTF2 and activate its expression. The findings revealed that GSTs were preserved in Prunus species and that PmGSTF2 was critical in regulating anthocyanin accumulation.

Phenotypic, genetic, variation, and molecular function of CaMYB113 in pepper (Capsicum annuum L.).

Pepper (Capsicum annuum L.) is widely consumed vegetables worldwide, and F1 hybrids are highly sought after in the pepper seed industry. However, studies on gene mutations affecting the color of cotyledon are rare, and the same is true for peppers. In this study, a segregating population was developed by crossing the pepper accession 21C1344 with purple cotyledon and accession 21C912 with green cotyledon. Initially, a target genomic region was identified by screening polymorphic SSR markers distributed across 12 chromosomes. Subsequently, polymorphic markers were developed based on resequencing data from the two parental lines, and genetic linkage analysis was performed. This approach ultimately identified Capana10g001433 (CaMYB113) as the candidate gene responsible for the purple cotyledons. The gene mutation type in 21C912 represents a new mutation type distinct from the reported missense mutation types, and this mutation affects the biosynthesis of anthocyanins. Virus-induced gene silencing (VIGS) of CaMYB113 substantially decreased anthocyanin accumulation in the cotyledons. Subsequent overexpression of CaMYB113 resulted in purple callus and leaves of pepper, and changed the expression levels of downstream genes involved in anthocyanin synthesis. Yeast one-hybrid and dual-luciferase transient expression assays demonstrated the binding of CaMYB113 to anthocyanin biosynthesis-related genes, thereby regulating anthocyanin accumulation in pepper cotyledons.

Genome-Wide Identification and Expression Analysis of GST Genes during Light-Induced Anthocyanin Biosynthesis in Mango (Mangifera indica L.).

Anthocyanins are important secondary metabolites contributing to the red coloration of fruits, the biosynthesis of which is significantly affected by light. Glutathione S-transferases (GSTs) play critical roles in the transport of anthocyanins from the cytosol to the vacuole. Despite their importance, GST genes in mango have not been extensively characterized. In this study, 62 mango GST genes were identified and further divided into six subfamilies. MiGSTs displayed high similarity in their exon/intron structure and motif and domain composition within the same subfamilies. The mango genome harbored eleven pairs of segmental gene duplications and ten sets of tandemly duplicated genes. Orthologous analysis identified twenty-nine, seven, thirty-four, and nineteen pairs of orthologous genes among mango MiGST genes and their counterparts in Arabidopsis, rice, citrus, and bayberry, respectively. Tissue-specific expression profiling highlighted tissue-specific expression patterns for MiGST genes. RNA-seq and qPCR analyses revealed elevated expression levels of seven MiGSTs including MiDHAR1, MiGSTU7, MiGSTU13, MiGSTU21, MiGSTF3, MiGSTF8, and MiGSTF9 during light-induced anthocyanin accumulation in mango. This study establishes a comprehensive genetic framework of MiGSTs in mango fruit and their potential roles in regulating anthocyanin accumulation, which is helpful in developing GST-derived molecular markers and speeding up the process of breeding new red-colored mango cultivars.

BoMYBL2b, an R3-MYB transcription factor, inhibits anthocyanin accumulation via directly repressing BoDFR1 gene transcription in kale

Kale is a globally popular edible and ornamental plant with colorful inner leaves. Anthocyanin is one of the most major pigments for kale. However, the underlying regulatory mechanism for the accumulation of anthocyanin in kale has not been well understood to date. Here, we characterized an R3-MYB transcription factor, BoMYBL2b, whose mRNA was missing in the anthocyanin-rich kale inbred line, but abundant in the anthocyanin-less line. Knockdown of BoMYBL2b enhanced the anthocyanin accumulation. Y1H and DLR assays showed that BoMYBL2b directly inhibited the BoDFR1 gene expression by binding to the MRE sites on the ProBoDFR1. Besides, we found that BoMYBL2b did not interact with the bHLH proteins, indicating that the BoMYBL2b might not be part of the MBW complex. BoMYBL2b expression was significantly repressed by BoMYB1R1 under high light and cold stimulus, suggesting the possibility that BoMYB1R1-BoMYBL2b module might act as a cross-node for integrating light and temperature signals to regulate anthocyanin accumulation in kale. These findings provided a theoretical basis for molecular breeding of new kale cultivars and a reference for understanding the regulatory mechanism of anthocyanin accumulation in Brassica.

Regulation of Anthocyanin Accumulation in Tomato Solanum lycopersicum L. by Exogenous Synthetic dsRNA Targeting Different Regions of SlTRY Gene.

RNA interference (RNAi) is a regulatory and protective mechanism that plays a crucial role in the growth, development, and control of plant responses to pathogens and abiotic stresses. In spray-induced gene silencing (SIGS), exogenous double-stranded RNAs (dsRNA) are used to efficiently regulate target genes via plant surface treatment. In this study, we aimed to evaluate the effect of specific exogenous dsRNAs on silencing different regions (promoter, protein-coding and intron) of the target SlTRY tomato gene, encoding an R3-type MYB repressor of anthocyanin biosynthesis. We also assessed the impact of targeting different SlTRY regions on the expression of genes involved in anthocyanin and flavonoid biosynthesis. This study demonstrated the critical importance of selecting the appropriate gene target region for dsRNA action. The highest inhibition of the SlTRY gene expression and activation of anthocyanin biosynthesis was achieved by dsRNA complementary to the protein-coding region of SlTRY gene, compared with dsRNAs targeting the SlTRY promoter or intron regions. Silencing the SlTRY gene increased the content of anthocyanins and boosted levels of other substances in the phenylpropanoid pathway, such as caffeoyl putrescine, chlorogenic acid, ferulic acid glucoside, feruloyl quinic acid, and rutin. This study is the first to examine the effects of four different dsRNAs targeting various regions of the SlTRY gene, an important negative regulator of anthocyanin biosynthesis.

WRKY75 regulates anthocyanin accumulation in juvenile citrus tissues.

The online version contains supplementary material available at 10.1007/s11032-024-01490-9.

PpBBX32 and PpZAT5 modulate temperature-dependent and tissue-specific anthocyanin accumulation in peach fruit.

Anthocyanins are important compounds for fruit quality and nutrition. The R2R3 MYB transcription factor PpMYB10.1 is known to be critical for regulating anthocyanin accumulation in peach. However, regulatory factors upstream of PpMYB10.1 which control temperature-dependent, cultivar-contrasted and tissue-specific anthocyanin accumulation remain to be determined. In this study, differential anthocyanin accumulation in the outer flesh near the peel (OF) of peach [Prunus persica (L.) Batsch] was observed between cultivars 'Zhonghuashoutao' and 'Dongxuemi', as well as among different storage temperatures and different fruit tissues of 'Zhonghuashoutao'. By cross-comparisons of RNA-Seq data of samples with differential anthocyanin accumulation, transcription factor genes PpBBX32 and PpZAT5 were identified. These were functionally characterized as two positive regulators for anthocyanin accumulation via transient expression and genetic transformation. Various interaction assays revealed that both PpBBX32 and PpZAT5 can directly activate the PpMYB10.1 promoter and meanwhile interact at protein level as a PpZAT5-PpBBX32-PpMYB10.1 complex. Furthermore, the results of in silico analysis and exogenous application of methyl jasmonate (MeJA) indicated that MeJA favored anthocyanin accumulation, while it was also found that anthocyanin accumulation as well as PpBBX32 and PpZAT5 expression correlated significantly with endogenous JA and JA-Ile in different fruit tissues. In summary, PpBBX32 and PpZAT5 are upstream activators of PpMYB10.1, allowing JAs to take part in temperature-dependent and tissue-specific anthocyanin accumulation by modulating their expression. This work enriches the knowledge of the transcriptional regulatory mechanisms for differential anthocyanin accumulation under internal and external factors.

Transcription factors LvBBX24 and LvbZIP44 coordinated anthocyanin accumulation in response to light in lily petals.

Lily (Lilium spp.), a horticultural crop serving both ornamental and edible functions, derives its coloration primarily from anthocyanins. However, limited studies have been conducted on the accumulation of anthocyanins within lilies. In this study, we cloned a light-induced transcription factor named as LvBBX24 in lilies. Through genetic and biochemical analysis, we determined that LvBBX24 could upregulate the transcription of LvMYB5 and facilitate anthocyanin synthesis. Moreover, we identified that darkness promoted the degradation of LvBBX24 protein. Through screening a yeast library, we identified LvbZIP44 acts as its interacting partner. Genetic testing confirmed that LvbZIP44 also plays a role in promoting lily anthocyanin synthesis. This indicates a potential synergistic regulatory effect between LvBBX24 and LvbZIP44. Our study indicates that LvBBX24 and LvbZIP44 cooperate to regulate anthocyanin accumulation in lily petals. These findings provide compelling evidence supporting the idea that LvBBX24 and LvbZIP44 may form a looped helix surrounding the LvMYB5 promoter region to regulate anthocyanin biosynthesis.

Association analysis of BSA-seq, BSR-seq, and RNA-seq reveals key genes involved in purple leaf formation in a tea population (Camellia sinensis).

Purple tea, rich in anthocyanins, has a variety of health benefits and is attracting global interest. However, the regulation mechanism of anthocyanin in purple tea populations has not been extensively studied. In this experiment, RNA-seq, BSA-seq, and BSR-seq were performed using 30 individuals with extreme colors (dark-purple and green) in an F 1 population of 'Zijuan' and 'Jinxuan'. The results show that 459 genes were differentially expressed in purple and green leaves, among which genes involved in the anthocyanin synthesis and transport pathway, such as CHS, F3H, ANS, MYB75, GST, MATE, and ABCC, were highly expressed in purple leaves. Moreover, there were multiple SNP/InDel variation sites on chromosomes 2 and 14 of the tea plant, as identified by BSA-seq. The integrated analysis identified two highly expressed genes (CsANS and CsMYB75) with SNP/InDel site variations in the purple tea plants. By silencing leaves, we proved that CsMYB75 could positively regulate anthocyanin accumulation and expression of related structural genes in tea plants. A 181-bp InDel in the CsMYB75 promoter was also found to be co-segregating with leaf color. The results of this study provide a theoretical reference for the molecular mechanism of anthocyanin accumulation in purple tea plants and contribute to the creation of new tea cultivars with high anthocyanin content.

Receptor-like kinase ERECTA negatively regulates anthocyanin accumulation in grape

Receptor-like kinase (ERECTA, ER) is essential for mediating growth, development, and stress response signaling pathway in plants. In this study, we investigated the effect of VvER on anthocyanin synthesis as a regulatory factor in transgenic grape callus in response to chilling stress. Results showed that overexpression of VvER reduced the expression of transcription factors VvMYBA1, VvMYB5b, VvMYC2, and VvWDR1, as well as the structural genes VvCHS, VvCHI, VvDFR, VvLDOX, and VvUFGT, and inhibited the anthocyanins synthesis of grape callus at 25℃. VvER reduced proline content and antioxidant enzymes activities of superoxide dismutase (SOD) and peroxidase (POD), and inhibited the expression of anthocyanin synthesis genes to reduce the cold resistance of grape callus. In transgenic Arabidopsis, overexpression of VvER promoted the elongation of Arabidopsis rosettes and sprigs. Under strong light treatment, VvER inhibited the accumulation of anthocyanins in Arabidopsis; Transient expression in strawberry fruit showed that VvER inhibited the synthesis of anthocyanin in strawberry fruit by inhibiting the expression of FaCHI, FaCHS, FaDFR and FaUFGT under low temperature treatment at 10°C, but not under the normal temperature of 25℃. Using Yeast two-hybrid, we found that VvER interacted with transcription factor proteins including VvMYBA1, VvMYB5b and VvWDR1. Furthermore, VvER led to the repression of VvUFGT promoter activity and decreased the anthocyanin biosynthesis genes expression by downregulation MBW complex activity. Totally, VvER could inhibit anthocyanin biosynthesis and involve in the grape plant susceptible to cold stress for grape cultivation in northern China.

A novel three-layer module BoMYB1R1-BoMYB4b/BoMIEL1-BoDFR1 regulates anthocyanin accumulation in kale.

Anthocyanin is an important pigment responsible for plant coloration and beneficial to human health. Kale (Brassica oleracea var. acephala), a primary cool-season flowers and vegetables, is an ideal material to study anthocyanin biosynthesis and regulation mechanisms due to its anthocyanin-rich leaves. However, the underlying molecular mechanism of anthocyanin accumulation in kale remains poorly understood. Previously, we demonstrated that BoDFR1 is a key gene controlling anthocyanin biosynthesis in kale. Here, we discovered a 369-bp InDel variation in the BoDFR1 promoter between the two kale inbred lines with different pink coloration, which resulted in reduced transcriptional activity of the BoDFR1 gene in the light-pink line. With the 369-bp insertion as a bait, an R2R3-MYB repressor BoMYB4b was identified using the yeast one-hybrid screening. Knockdown of the BoMYB4b gene led to increased BoDFR1 expression and anthocyanin accumulation. An E3 ubiquitin ligase, BoMIEL1, was found to mediate the degradation of BoMYB4b, thereby promoting anthocyanin biosynthesis. Furthermore, the expression level of BoMYB4b was significantly reduced by light signals, which was attributed to the direct repression of the light-signaling factor BoMYB1R1 on the BoMYB4b promoter. Our study revealed that a novel regulatory module comprising BoMYB1R1, BoMIEL1, BoMYB4b, and BoDFR1 finely regulates anthocyanin accumulation in kale. The findings aim to establish a scientific foundation for genetic improvement of leaf color traits in kale, meanwhile, providing a reference for plant coloration studies.

Identification of Flavonoid 3′-Hydroxylase Genes from Red Chinese Sand Pear (Pyrus pyrifolia Nakai) and Their Regulation of Anthocyanin Accumulation in Fruit Peel

The red Chinese sand pear (Pyrus pyrifolia Nakai) is native to China and exhibits a unique fruit coloration pattern. Flavonoid 3′-hydroxylase (F3′H) catalyzes the hydroxylation of flavonoids, which subsequently determines the components of anthocyanins and the color of plant organs. Two genes encoding flavonoid 3′-hydroxylase (F3′H), PpF3′HI and PpF3′HII, have been identified in red Chinese sand pears. The coding regions for PpF3′HI and PpF3′HII were 1542 and 1536 bp in length, respectively. PpF3′HI shared 95% of its amino acid sequence identity with PpF3′HII, and a highly conserved P450 superfamily domain was found both in PpF3′HI and in PpF3′HII. Phylogenetic analysis showed that PpF3′HI and PpF3′HII clustered with MdF3′HI and MdF3′HII, respectively. PpF3′H genes were highly expressed in anthocyanin-enriched tissues such as young leaves, and transcription of PpF3′H genes corresponded to anthocyanin biosynthesis during the developmental stages, bagging treatment, and postharvest UV-B/visible irradiation treatment. A Y1H assay showed that PpMYB10 and PpHY5 could interact with the −419 bp to 0 bp and −746 bp to −396 bp fragments of the PpF3′HI promoter region, respectively. Understanding the mechanism of flavonoid hydroxylation patterns will, in turn, promote the development of new technologies for modifying flavonoid and anthocyanin composition in fruits.

For a Colorful Life: Recent Advances in Anthocyanin Biosynthesis during Leaf Senescence.

Leaf senescence is the last stage of leaf development, and it is accompanied by a leaf color change. In some species, anthocyanins are accumulated during leaf senescence, which are vital indicators for both ornamental and commercial value. Therefore, it is essential to understand the molecular mechanism of anthocyanin accumulation during leaf senescence, which would provide new insight into autumn coloration and molecular breeding for more colorful plants. Anthocyanin accumulation is a surprisingly complex process, and significant advances have been made in the past decades. In this review, we focused on leaf coloration during senescence. We emphatically discussed several networks linked to genetic, hormonal, environmental, and nutritional factors in regulating anthocyanin accumulation during leaf senescence. This paper aims to provide a regulatory model for leaf coloration and to put forward some prospects for future development.

AcMYB266, a key regulator of the red coloration in pineapple peel: a case of subfunctionalization in tandem duplicated genes.

Red fruit peel is an attractive target for pineapple breeding. Various pineapple accessions with distinct red coloration patterns exist; however, the precise molecular mechanism accounting for these differences remains unknown, which hinders the pineapple breeding process from combining high fruit quality with red peel. In this study, we characterized a transcription factor, AcMYB266, which is preferentially expressed in pineapple peel and positively regulates anthocyanin accumulation. Transgenic pineapple, Arabidopsis, and tobacco plants overexpressing AcMYB266 exhibited significant anthocyanin accumulation. Conversely, transient silencing of this gene led to decreased anthocyanin accumulation in pineapple red bracts. In-depth analysis indicated that variations of AcMYB266 sequences in the promoter instead of the protein-coding region seem to contribute to different red coloration patterns in peels of three representative pineapple varieties. In addition, we found that AcMYB266 was located in a cluster of four MYB genes exclusive to and conserved in Ananas species. Of this cluster, each was proved to regulate anthocyanin synthesis in different pineapple tissues, illustrating an interesting case of gene subfunctionalization after tandem duplication. In summary, we have characterized AcMYB266 as a key regulator of pineapple red fruit peel and identified an MYB cluster whose members were subfunctionalized to specifically regulate the red coloration of different pineapple tissues. The present study will assist in establishing a theoretical mechanism for pineapple breeding for red fruit peel and provide an interesting case for the investigation of gene subfunctionalization in plants.