Anthocyanin Biosynthetic Genes Research Articles

The Expression of Key Ethylene and Anthocyanin Biosynthetic Genes of 'Honeycrisp' Apples Subjected to the Combined Use of Reflective Groundcovers and Aminoethoxyvinylglycine in the Mid-Atlantic US.

The decreased profitability of important apple cultivars, such as 'Honeycrisp', results from the poor red skin coloration and high fruit drop in the mid-Atlantic US. Apple red skin coloration is determined by the anthocyanin concentration. Reflective groundcovers promote red skin coloration, whereas aminoethoxyvinylglycine (AVG) decreases the ethylene production and fruit drop, thus reducing the coloration. Although our previous study showed that combinations of these practices impact the fruit quality and color, research is lacking regarding their effects at the gene and metabolite levels. In this work, for two years, we compared the differences in the internal ethylene concentration (IEC), red skin coloration, fruit drop, transcript accumulation of key ethylene and anthocyanin biosynthetic pathway-related genes, and total anthocyanin concentration of 'Honeycrisp' apples. The fruit was treated with combinations of reflective groundcover (Extenday) and AVG (130 mg L-1) and was assessed throughout ripening. Extenday-only-treated fruit displayed the highest upregulation of ethylene and anthocyanin biosynthetic-related genes and of total anthocyanins, exceeding 50% blush, while boosting the IEC. In contrast, AVG significantly decreased the expression of key ethylene and anthocyanin biosynthetic-related genes and total anthocyanins, thus preventing apples from reaching 50% blush, while also decreasing the IEC and fruit drop. The combination of Extenday x AVG fine-tuned the transcript accumulation of ethylene and anthocyanin biosynthetic-related genes as well as the total anthocyanins, allowing the 'Honeycrisp' fruit to exceed 50% blush, while increasing the IEC moderately and reducing the fruit drop (as compared to Extenday-only and control), thus enhancing the fruit economic value.

FaMYB6-like negatively regulates FaMYB10-induced anthocyanin accumulation during strawberry fruit ripening

Anthocyanins are one of the essential compounds affecting the quality of strawberry fruit. Several R2R3-MYB transcription factors have been well characterized as the anthocyanin activators in strawberry. However, we still lack valuable information on how R2R3-MYB repressors regulate strawberry anthocyanin accumulation. Here, we cloned a novel R2R3-MYB gene, FaMYB6-like, and characterized its repressive role during anthocyanin accumulation. FaMYB6-like encodes a nuclear localization protein, which was clustered into the FaMYB1-like type repressor clade in phylogenetic analysis. FaMYB6-like showed high expressions in anthocyanin-accumulating fruit and low transcript abundance in flowers, vegetative tissues, and early development stages of fruit. Transient overexpression of FaMYB6L-like repressed the expression of anthocyanin biosynthetic and transport genes, and reduced anthocyanin content in postharvest strawberry fruit. Transgenic tobacco lines overexpressing FaMYB6-like displayed a reduction in petal pigmentation. Overexpression of FaMYB6L-like in white-fleshed strawberry inhibited anthocyanin accumulation induced by FaMYB10. Protein interaction and promoter activation assays revealed that FaMYB6-like repressed the activation of target gene promoters by FaMYB10-FabHLH3 activation complex via its interaction with FabHLH3. Additionally, FaMYB6-like promoter activity can be activated by FaMYB10-FabHLH3 complex. Taken together, our study revealed that FaMYB6-like acted as a transcriptional repressor involved in an ‘activator-and-repressor’ feedback regulation mechanism for maintaining the balance of anthocyanin concentrations.

Polymorphisms in two key anthocyanic genes of clivia (Clivia miniata L.) reveal evidence of selection and possible association with flower pigmentation.

Members of the genus Clivia show considerable variation in flower pigmentation and morphology. Such variation is affected by mutations that emerge in candidate flower development genes over time. Besides population history, mutations can further illuminate the effects of demographic events in populations in addition to population genetic parameters including selection, recombination, and linkage disequilibrium (LD). The current study aimed to find sequence variants in 2 anthocyanin biosynthetic genes (DFR and bHLH) of Clivia miniata and use the data to assess population genetic factors from a random collection of orange/red- and yellow-flowered specimens. Overall, average nucleotide diversity in the 2 anthocyanin genes was moderate (π = 0.00646), whereas haplotypes differed significantly (Hd ≥ 0.9). Gene evolution was seemingly driven by mutations (CmiDFR) or recombinations (CmibHLH001). LD decayed swiftly within the analyzed gene regions and supported the feasibility of assessing trait-variant associations via the association/linkage mapping approach. In the end, most associations were found to be spurious, but 1 haplotype in CmibHLH001 showed a promising correlation to the orange/red flower phenotype in Clivia specimens. In all, the present study is the first to measure gene-level diversity in C. miniata-data that had never been reported so far. Furthermore, the study also identified allelic and haplotypic variants that may be beneficial in future association genetic studies of Clivia. Such studies, however, consider large diverse populations to control for statistical bias intrinsic to the analysis of small datasets.

Transcriptomic and metabolic analysis unveils the mechanism behind leaf color development in Disanthus cercidifolius var. longipes.

Introduction: Leaf coloration in Disanthus cercidifolius var. longipes results from the interplay of various pigments undergoing complex catalytic reactions. Methods: We aimed to elucidate the mechanisms of pigment biosynthesis affecting leaf color transition in D. cercidifolius var. longipes by analyzing variations in pigment accumulation and levels of gene expression. Results: We identified 468, 577, and 215 differential metabolites in green leaves (GL), gradual-color-changing leaves (GCCL), and red leaves (RL), respectively, with 94 metabolites shared across all comparisons. Metabolite accumulation patterns were similar among GL, GCCL, and RL, with flavonoids being the main differential metabolites. Delphinidin, malvidin, and petunidin derivatives were mostly accumulated in GCCL, whereas cyanidin, pelargonidin, and peonidin derivatives accumulated in RL. Transcriptome sequencing was used to identify differentially expressed genes. The expression of anthocyanin biosynthetic pathway genes was associated with anthocyanin accumulation patterns. Discussion: Our findings reveal that the content of delphinidin, malvidin, petunidin, and carotenoids collectively determines the gradual transition of leaf color from green in spring and summer to green, purple, and orange-yellow in early autumn, whereas the content of cyanidin, peonidin, pelargonidin, and carotenoids together causes the autumnal transition to red or orange-red colors as leaves of D. cercidifolius var. longipes age.

Overexpression of a BR inactivating enzyme gene GhPAG1 impacts eggplant fruit development and anthocyanin accumulation mainly by altering hormone homeostasis

Brassinosteroids (BRs) function importantly in plant growth and development, but the roles in regulating fruit development and anthocyanin pigmentation remain unclear. Eggplant (Solanum melongena L.) is an important Solanaceae vegetable crop rich in anthocyanins. The fruit size and coloration are important agronomic traits for eggplant breeding. In this study, transgenic eggplant exhibiting endogenous BRs deficiency was created by overexpressing a heterologous BRs-inactivating enzyme gene GhPAG1 driven by CaMV 35 S promoter. 35 S::GhPAG1 eggplant exhibited severe dwarfism, reduced fruit size, and less anthocyanin accumulation. Microscopic observation showed that the cell size of 35 S::GhPAG1 eggplant was significantly reduced compared to WT. Furthermore, the levels of IAA, ME-IAA, and active JAs (JA, JA-ILE, and H2JA) all decreased in 35 S::GhPAG1 eggplant fruit. RNA-Seq analyses showed a decrease in the expression of genes involved in cell elongation, auxin signaling, and JA signaling. Besides, overexpression of GhPAG1 significantly downregulated anthocyanin biosynthetic genes and associated transcription regulators. Altogether, these results strongly suggest that endogenous brassinosteroid deficiency arising from GhPAG1 overexpression impacts eggplant fruit development and anthocyanin coloration mainly by altering hormone homeostasis.

ABA functions in low phosphate-induced anthocyanin accumulation through the transcription factor ABI5 in Arabidopsis.

ABI5 functions in ABA-mediated anthocyanin accumulation in plant response to low phosphate. Low phosphate (LP)-induced anthocyanin biosynthesis and accumulation play an important role in plant adaptive response to phosphate starvation conditions. However, whether and how the stress phytohormone abscisic acid (ABA) participates in LP-induced anthocyanin accumulation remain elusive. Here, we report that ABA is required for LP-induced anthocyanin accumulation in Arabidopsis thaliana. Disrupting ABA DEFICIENT2 (ABA2), a key ABA-biosynthetic gene, or BETA-GLUCOSIDASE1 (BG1), a major gene implicated in converting conjugated ABA to active ABA, significantly impairs LP-induced anthocyanin accumulation, as LP-induced expression of the anthocyanin-biosynthetic genes Chalcone Synthase (CHS) is dampened in the aba2 and bg1 mutant. In addition, LP-induced anthocyanin accumulation is defective in the mutants of ABA signaling pathway, including ABA receptors, ABA Insensitive2, and the transcription factors ABA Insensitive5 (ABI5), suggesting a role of ABI5 in ABA-mediated upregulation of anthocyanin-biosynthetic genes in plant response to LP. Indeed, LP-induced expression of CHS is repressed in the abi5-7 mutant but further promoted in the ABI5-overexpressing plants compared to the wild-type. Moreover, ABI5 can bind to and transcriptionally activate CHS, and the defectiveness of LP-induced anthocyanin accumulation in abi5-7 can be restored by overexpressing CHS. Collectively, our findings illustrates that ABI5 functions in ABA-mediated LP-induced anthocyanin accumulation in Arabidopsis.

MdbHLH162 connects the gibberellin and jasmonic acid signals to regulate anthocyanin biosynthesis in apple.

Anthocyanins are secondary metabolites induced by environmental stimuli and developmental signals. The positive regulators of anthocyanin biosynthesis have been reported, whereas the anthocyanin repressors have been neglected. Although the signal transduction pathways of gibberellin (GA) and jasmonic acid (JA) and their regulation of anthocyanin biosynthesis have been investigated, the cross-talk between GA and JA and the antagonistic mechanism of regulating anthocyanin biosynthesis remain to be investigated. In this study, we identified the anthocyanin repressor MdbHLH162 in apple and revealed its molecular mechanism of regulating anthocyanin biosynthesis by integrating the GA and JA signals. MdbHLH162 exerted passive repression by interacting with MdbHLH3 and MdbHLH33, which are two recognized positive regulators of anthocyanin biosynthesis. MdbHLH162 negatively regulated anthocyanin biosynthesis by disrupting the formation of the anthocyanin-activated MdMYB1-MdbHLH3/33 complexes and weakening transcriptional activation of the anthocyanin biosynthetic genes MdDFR and MdUF3GT by MdbHLH3 and MdbHLH33. The GA repressor MdRGL2a antagonized MdbHLH162-mediated inhibition of anthocyanins by sequestering MdbHLH162 from the MdbHLH162-MdbHLH3/33 complex. The JA repressors MdJAZ1 and MdJAZ2 interfered with the antagonistic regulation of MdbHLH162 by MdRGL2a by titrating the formation of the MdRGL2a-MdbHLH162 complex. Our findings reveal that MdbHLH162 integrates the GA and JA signals to negatively regulate anthocyanin biosynthesis. This study provides new information for discovering more anthocyanin biosynthesis repressors and explores the cross-talk between hormone signals.

A stress-responsive R2R3-MYB transcription factor, EpMYB1 is involved in the regulation of anthocyanin biosynthesis in purple coneflower

Purple coneflower (Echinacea purpurea (L.) Menoch), a North American native and world-popular medicinal and horticultural plant, has been named after its beautiful purple petals due to the accumulation of anthocyanins. However, so far the biosynthesis of anthocyanin and its regulation in purple coneflower remain elusive. In this study, the total anthocyanin contents of eight different purple coneflower tissues were measured, and anthocyanin biosynthetic genes were identified from the multi-tissue transcriptome. Using co-expression, phylogenetic screening, and transient functional verification, EpMYB1 was identified as a potential anthocyanin biosynthesis regulator. EpMYB1 is an R2R3-MYB TF and co-expressed with anthocyanin biosynthetic genes. When EpMYB1 was over-expressed in purple coneflower calli, it positively promoted anthocyanin biosynthesis by upregulating the anthocyanin-specific biosynthetic genes. Dual-luciferase reporter assay indicated that EpMYB1 could significantly activate anthocyanin biosynthetic gene expression by directly binding to the promoters. Moreover, EpMYB1 is a stress-responsive TF involved in the ultraviolet-induced anthocyanin biosynthesis in purple coneflower. The present study identifies an MYB TF that participated in anthocyanin biosynthesis and bridged the gap between environmental stress and anthocyanin biosynthesis in purple coneflower. It provides valuable insights into the anthocyanin biosynthesis in purple coneflower and will significantly improve the development of purple coneflower as an ornament plant.

MiR156b-targeted VvSBP8/13 functions downstream of the abscisic acid signal to regulate anthocyanins biosynthesis in grapevine fruit under drought.

Anthocyanins are the primary color components of grapevine berries and wines. In cultivation practices, a moderate water deficit can promote anthocyanin accumulation in red grape skins. Our previous study showed that abscisic acid (ABA) plays a key role in this process. Herein, we identified a microRNA, vv-miR156b, that is generated in grapevine berries in response to drought stress, along with increasing anthocyanin content and biosynthetic structural gene transcripts. In contrast, vv-miR156b short tandem target mimic (STTM) function-loss callus exhibits the opposite phenotype. Results from in vivo and in vitro experiments revealed that the ABA-signaling-regulated transcription factor VvAREB2 binds directly to the ABA-responsive element (ABRE) of the MIR156b promoter and activates miR156b expression. Furthermore, two miR156b downstream targets, VvSBP8 and VvSBP13, exhibited reduced grape anthocyanin content in their overexpressors but there was a contrary result in their CRISPR-edited lines, the decrease in anthocyanin content was rescued in miR156b and SBP8/13 double overexpressors. We further demonstrated that both VvSBP8 and VvSBP13, encoding transcriptional repressors, displayed sufficient ability to interact with VvMYC1 and VvMYBA1, thereby interfering with MYB-bHLH-WD (MBW) repeat transcriptional complex formation, resulting in the repression of anthocyanin biosynthesis. Our findings demonstrate a direct functional relationship between ABA signaling and the miR156-SBP-MBW complex regulatory module in driving drought-induced anthocyanin accumulation in grape berries.

CstMYB1R1, a REVEILLE-8-like transcription factor, regulates diurnal clock-specific anthocyanin biosynthesis and response to abiotic stress in Crocus sativus L.

CstMYB1R1 acts as a positive regulator of Crocus anthocyanin biosynthesis and abiotic stress tolerance which was experimentally demonstrated through molecular analysis and over-expression studies in Crocus and Nicotiana. Regulatory mechanics of flavonoid/anthocyanin biosynthesis in Crocus floral tissues along the diurnal clock has not been studied to date. MYB proteins represent the most dominant, functionally diverse and versatile type of plant transcription factors which regulate key metabolic and physiological processes in planta. Transcriptome analysis revealed that MYB family is the most dominant transcription factor family in C. sativus. Considering this, a MYB-related REVEILLE-8 type transcription factor, CstMYB1R1, was explored for its possible role in regulating Crocus flavonoid and anthocyanin biosynthetic pathway. CstMYB1R1 was highly expressed in Crocus floral tissues, particularly tepals and its expression was shown to peak at dawn and dusk time points. Anthocyanin accumulation also peaked at dawn and dusk and was minimum at night. Moreover, the diurnal expression pattern of CstMYB1R1 was shown to highly correlate with Crocus ANS/LDOX gene expression among the late anthocyanin pathway genes. CstMYB1R1 was shown to be nuclear localized and transcriptionally active. CstMYB1R1 over-expression in Crocus tepals enhanced anthocyanin levels and upregulated transcripts of Crocus flavonoid and anthocyanin biosynthetic pathway genes. Yeast one hybrid (Y1H) and GUS reporter assay confirmed that CstMYB1R1 interacts with the promoter of Crocus LDOX gene to directly regulate its transcription. In addition, the expression of CstMYB1R1 in Nicotiana plants significantly enhanced flavonoid and anthocyanin levels and improved their abiotic stress tolerance. The present study, thus, confirmed positive role of CstMYB1R1 in regulating Crocus anthocyanin biosynthetic pathway in a diurnal clock-specific fashion together with its involvement in the regulation of abiotic stress response.

Cytokinin promotes anthocyanin biosynthesis via regulating sugar accumulation and MYB113 expression in Eucalyptus.

Anthocyanins are flavonoid-like substances that play important roles in plants' adaptation to various environmental stresses. In this research, we discovered that cytokinin (CK) alone could effectively induce the anthocyanin biosynthesis in Eucalyptus and many other perennial woody plant species, but not in tobacco and Arabidopsis, suggesting a diverse role of CK in regulating anthocyanin biosynthesis in different species. Transcriptomic and metabolomic strategies were used to further clarify the specific role of CK in regulating anthocyanin biosynthesis in Eucalyptus. The results showed that 801 and 2241 genes were differentially regulated at 6 and 24h, respectively, after CK treatment. Pathway analysis showed that most of the differentially expressed genes were categorized into pathways related to cellular metabolism or transport of metabolites, including amino acids and sugars. The metabolomic results well supported the transcriptome data, which showed that most of the differentially regulated metabolites were related to the metabolism of sugar, amino acids and flavonoids. Moreover, CK treatment significantly induced the accumulation of sucrose in the CK-treated leaves, while sugar starvation mimicked by either defoliation or shading treatment of the basal leaves significantly reduced the sugar increase of the CK-treated leaves and thus inhibited CK-induced anthocyanin biosynthesis. The results of in vitro experiment also suggested that CK-induced anthocyanin in Eucalyptus was sugar-dependent. Furthermore, we identified an early CK-responsive transcription factor MYB113 in Eucalyptus, the expression of which was significantly upregulated by CK treatment in Eucalyptus, but was inhibited in Arabidopsis. Importantly, the overexpression of EgrMYB113 in the Eucalyptus hairy roots was associated with significant anthocyanin accumulation and upregulation of most of the anthocyanin biosynthetic genes. In conclusion, our study demonstrates a key role of CK in the regulation of anthocyanin biosynthesis in Eucalyptus, providing a molecular basis for further understanding the regulatory mechanism and diversity of hormone-regulated anthocyanin biosynthesis in different plant species.

A transient expression tool box for anthocyanin biosynthesis in Nicotiana benthamiana.

Transient expression in Nicotiana benthamiana offers a robust platform for the rapid production of complex secondary metabolites. It has proven highly effective in helping identify genes associated with pathways responsible for synthesizing various valuable natural compounds. While this approach has seen considerable success, it has yet to be applied to uncovering genes involved in anthocyanin biosynthetic pathways. This is because only a single anthocyanin, delphinidin 3-O-rutinoside, can be produced in N. benthamiana by activation of anthocyanin biosynthesis using transcription factors. The production of other anthocyanins would necessitate the suppression of certain endogenous flavonoid biosynthesis genes while transiently expressing others. In this work, we present a series of tools for the reconstitution of anthocyanin biosynthetic pathways in N. benthamiana leaves. These tools include constructs for the expression or silencing of anthocyanin biosynthetic genes and a mutant N. benthamiana line generated using CRISPR. By infiltration of defined sets of constructs, the basic anthocyanins pelargonidin 3-O-glucoside, cyanidin 3-O-glucoside and delphinidin 3-O-glucoside could be obtained in high amounts in a few days. Additionally, co-infiltration of supplementary pathway genes enabled the synthesis of more complex anthocyanins. These tools should be useful to identify genes involved in the biosynthesis of complex anthocyanins. They also make it possible to produce novel anthocyanins not found in nature. As an example, we reconstituted the pathway for biosynthesis of Arabidopsis anthocyanin A5, a cyanidin derivative and achieved the biosynthesis of the pelargonidin and delphinidin variants of A5, pelargonidin A5 and delphinidin A5.

Sucrose-delaying flower color fading associated with delaying anthocyanin accumulation decrease in cut chrysanthemum

As fresh ornamental crops, vase life and post-harvested quality of cut flowers have attracted much attention. Flower color fading is the prominent defect in red and purple cut flowers, especially in cut chrysanthemum which have a relative long vase life. Here, the effect of sucrose on change in anthocyanin contents during the vase life of ‘Dante Purple’ cut chrysanthemum was studied. Results showed that 500 mM sucrose as holding solution could significantly delay the decrease in anthocyanin content and maintain the ornamental value for as long as 38 vase days. Moreover, the sucrose also increased the flower diameter, soluble sugar contents and total antioxidant capacity, while decreasing the malondialdehyde contents. Further studies suggested that the transcript levels of anthocyanin biosynthetic genes and transcription factors, CmMYB6 and CmMYB#7, had continuously decreased during the vase life. The changes in these genes expression patterns was retarded by the sucrose treatment, except for CmMYB#7 which is a repressor of anthocyanin biosynthesis gene expression. The decline in relative expression of CmMYB#7 was accelerated by sucrose. These results have supplied clues to study the mechanism whereby sucrose serves as a signal molecule to regulate anthocyanin biosynthesis.

Integrated analysis of the metabolome and transcriptome provides insights into anthocyanin biosynthesis of cashew apple

The cashew apple remains an underutilized agricultural product despite its abundance as a by-product of cashew nut production. Anthocyanins are water-soluble pigments responsible for red, purple, and blue hues in plant tissues and have various health-promoting properties. To investigate the anthocyanin biosynthesis in cashew apples, fruits with varying peel colors from three cultivars were subjected to integrative analyses with metabolomics and transcriptomics. Through a UPLC-ESI-MS/MS-based targeted metabolomics analysis, a total of 26 distinct anthocyanin compounds were identified in the fruits of the three cashew cultivars. Subsequent quantification revealed that Pelargonidin-3-O-galactoside, Petunidin-3-O-arabinoside, and Cyanidin-3-O-galactoside were the primary contributors responsible for the red pigmentation in cashew apple peels. Following transcriptomic analysis showed that the expression levels of anthocyanin biosynthetic genes were predominantly higher in the red cashew apples as compared to the other two cultivars. Moreover, correlation analysis revealed that eight potential transcription factors implicated in the regulation of anthocyanin biosynthesis. Among these, four transcription factors exhibited positive correlations with both anthocyanin contents and anthocyanin biosynthetic gene expression, while the remaining four transcription factors displayed negative correlations. These findings provide a comprehensive understanding of the molecular basis of anthocyanin biosynthesis in cashew apple peels.

Molecular mechanism of different flower color formation of Cymbidium ensifolium.

Cymbidium ensifolium is one of the national orchids in China, which has high ornamental value with changeable flower colors. To understand the formation mechanism of different flower colors of C. ensifolium, this research conducted transcriptome and metabolome analyses on four different colored sepals of C. ensifolium. Metabolome analysis detected 204 flavonoid metabolites, including 17 polyphenols, 27 anthocyanins, 75 flavones, 34 flavonols, 25 flavonoids, 18 flavanones, and 8 isoflavones. Among them, purple-red and red sepals contain a lot of anthocyanins, including cyanidin, pelargonin, and paeoniflorin, while yellow-green and white sepals have less anthocyanins detected, and their metabolites are mainly flavonols, flavanones and flavonoids. Transcriptome sequencing analysis showed that the expression levels of the anthocyanin biosynthetic enzyme genes in red and purple-red sepals were significantly higher than those in white and yellow-green sepals of C. ensifolium. The experimental results showed that CeF3'H2, CeDFR, CeANS, CeF3H and CeUFGT1 may be the key genes involved in anthocyanin production in C. ensifolium sepals, and CeMYB104 has been proved to play an important role in the flower color formation of C. ensifolium. The results of transformation showed that the CeMYB104 is involved in the synthesis of anthocyanins and can form a purple-red color in the white perianth of Phalaenopsis. These findings provide a theoretical reference to understand the formation mechanism of flower color in C. ensifolium.

Integrated Metabolome and Transcriptome Analysis of Petal Anthocyanin Accumulation Mechanism in Gloriosa superba 'Rothschildiana' during Different Flower Development Stages.

Flower color is a key ornamental trait in plants. The petals of Gloriosa superba 'Rothschildiana' petals undergo a color transformation from yellow to red during their development, but the molecular mechanism of this process remains unexplored. This study examines the anthocyanin profiles and gene expression patterns of 'Rothschildiana' petals across four developmental stages: bud (S1), initial opening (S2), half opening (S3), and full opening stage (S4). A total of 59 anthocyanins were identified with significant increases in cyanidin-3,5-O-diglucoside, cyanidin-3-O-glucoside, pelargonidin-3-O-glucoside, and pelargonidin-3,5-O-diglucoside levels observed during petal maturation. Transcriptome analysis revealed 46 differentially expressed genes implicated in flavonoid and anthocyanin biosynthesis. Additionally, three gene modules were found to be associated with anthocyanin accumulation throughout flower development. Expression levels of genes associated with auxin, abscisic acid, brassinosteroid signaling, and transcription factors such as NACs and WRKYs underwent significant changes and exhibited strong correlations with several flavonoid and anthocyanin biosynthetic genes in these modules. These findings offer novel insights into the molecular underpinnings of flower color variation and lay the groundwork for the improvement of G. superba.

CsTT8 regulates anthocyanin accumulation in blood orange through alternative splicing transcription.

A homologous gene of basic-helix-loop-helix AtTT8 in Arabidopsis thaliana was identified in juice sac cells of pulp tissues from blood orange (Citrus sinensis cv 'Tarocco'), which was designated as CsTT8 in this study. Additionally, the mRNA levels of TT8 with the full-length open reading frame were significantly higher in 'Tarocco' than in mutant fruit lacking pigment in pulp or peel tissues. However, an alternative splicing transcript, Δ15-TT8, with the fourth exon skipped, was also identified from transcripts different in length from that in 'Tarocco'. The mRNA levels of Δ15-TT8 were higher in mutant fruit lacking pigment in pulp or peel tissues than in the wild type. Therefore, the TT8/Δ15-TT8 mRNA level ratio was found to be crucial for sufficient pigment in either pulp or peel tissues. TT8 from blood orange fruit demonstrated the capacity for nucleus localization and binding to other proteins. In contrast, Δ15-TT8, lacking the fourth exon, lost its ability to interact with RUBY1 and to localize at the nucleus. Using a dual luciferase reporter assay and transient overexpression in tobacco, we proved that two regulatory complexes formed by a functional TT8 with different MYB(v-myb avian myeloblastosis viral oncogene homolog)-type partners significantly promoted expression of an anthocyanin biosynthetic gene and a proton pumping gene, leading to anthocyanin and citrate production. Our findings suggest that TT8, rather than dysfunctional Δ15-TT8, is possibly involved in modulating anthocyanin biosynthesis and its transport into vacuoles by proton gradients. However, increased mRNA levels of the dysfunctional alternative splicing transcript may act as a negative feedback to downregulate TT8 expression and limit anthocyanin accumulation in blood oranges.

CstPIF4 Integrates Temperature andCircadian Signals andInteracts with CstMYB16 to Repress Anthocyanins inCrocus.

Crocus sativus has emerged as an important crop because it is the only commercial source of saffron that contains unique apocarotenoids. Saffron is composed of dried stigmas of Crocus flower and constitutes the most priced spice of the world. Crocus floral organs are dominated by different classes of metabolites. While stigmas are characterized by the presence of apocarotenoids, tepals are rich in flavonoids and anthocyanins. Therefore, an intricate regulatory network might play a role in allowing different compounds to dominate in different organs. Work so far done on Crocus is focussed on apocarotenoid metabolism and its regulation. There are no reports describing the regulation of flavonoids and anthocyanins in Crocus tepals. In this context, we identified an R2R3 transcription factor, CstMYB16, which resembles subgroup 4 (SG4) repressors of Arabidopsis. CstMYB16 is nuclear localized and acts as a repressor. Overexpression of CstMYB16 in Crocus downregulated anthocyanin biosynthesis. The C2/EAR motif was responsible for the repressor activity of CstMYB16. CstMYB16 binds to the promoter of the anthocyanin biosynthetic pathway gene (LDOX) and reduces its expression. CstMYB16 also physically interacts with CstPIF4, which in turn is regulated by temperature and circadian clock. Thus, CstPIF4 integrates these signals and forms a repressor complex with CstMYB16, which is involved in the negative regulation of anthocyanin biosynthesis in Crocus. Independent of CstPIF4, CstMYB16 also represses CstPAP1 expression, which is a component of the MYB-bHLH-WD40 (MBW) complex and positively controls anthocyanin biosynthesis. This is the first report on identifying and describing regulators of anthocyanin biosynthesis in Crocus.

R2R3-MYB transcription factor PhMYB2 positively regulates anthocyanin biosynthesis in Pericallis hybrida

Cineraria (Pericallis hybrida) is an ideal species for anthocyanin accumulation studies in ornamental plants due to its bright flower colors and diverse anthocyanin branching metabolic pathways. However, the regulatory pattern of anthocyanin metabolism was still unclear in previous research on the flower color of cineraria. In order to explore the anthocyanin synthesis mechanism of pure color cultivar of cineraria, we isolated a novel R2R3-MYB gene PhMYB2 based on the previous transcriptome of blue cineraria. PhMYB2 was significantly expressed in carmine and blue cineraria, and its expression profile corresponded with the pattern of anthocyanin accumulation in the ray florets. PhMYB2 could activate the transcription of anthocyanin biosynthetic genes PhF3′H1, PhF3′5′H, PhDFR3, and PhANS when transiently silenced PhMYB2 in cineraria leaves by virus-induced gene silencing (VIGS). Meanwhile, PhMYB2 could interact with PhbHLH17, which belonged to TT8-clade, while PhbHLH17 could interact with a possible R2R3-MYB repressor PhMYB4.Y1H and dual-luciferase assays revealed that PhMYB2 binds and activates the promoter of PhDFR3, and when PhMYB2 interacts with PhbHLH17, the complex can significantly enhance the activity of the PhDFR3 promoter. Furthermore, when PhMYB4 co transformed with PhMYB2 and PhbHLH17, the activity of the PhDFR3 promoter was significantly reduced. Based on this, we speculated that PhMYB4 might fine-tune the anthocyanin synthesis by interacting with PhbHLH17 and competing with PhMYB2. The above results clarified the mechanism of PhMYB2-PhbHLH17 promoting anthocyanin synthesis and improved the anthocyanin regulatory network in cineraria.

Integrated Transcriptomics and Metabolomics Analysis Reveal Anthocyanin Biosynthesis for Petal Color Formation in Catharanthus roseus

Catharanthus roseus exhibits vibrant petals and displays robust resistance to disease and drought, making it highly valuable for ornamental and gardening applications. While the application of C. roseus as a source of anticancer drugs has gained considerable attention in recent years, there has been limited investigation into the regulatory mechanism underlying anthocyanin accumulation in the petals of C. roseus. This study comprehensively analyzed the metabolome and transcriptome of three distinct C. roseus varieties exhibiting different petal colors. Out of the 39 identified flavonoids, 10 anthocyanins exhibited significant variations in accumulation, directly contributing to the diverse coloration of C. roseus petals. Among them, malvidin 3-O-glucoside and petunidin 3-O-glucoside were identified as primary contributors to the purple petal phenotype, while peonidin 3-O-glucoside and delphinidin 3-O-glucoside exhibited the highest contribution rates to the red petals. Additionally, the variation content of cyanidin 3-O-rutinoside, delphinidin 3-O-glucoside, and petunidin 3-O-rutinoside also influenced the color transformation of C. roseus petals. RNA sequencing identified a total of 4173 differentially expressed genes (DEGs), including 1003 overlapping DEGs. A combined transcriptome and metabolome analysis showed that the coordinately regulated anthocyanin biosynthetic genes including chalcone isomerase (CHS), flavonoid 3′-hydroxylase (F3′H), and dihydroflavonol 4-reductase (DFR) played critical roles in the formation of the anthocyanins. MYB and bHLH transcription factors were also found to be significantly correlated with differences in flower color. These results serve as a foundation for future investigations into anthocyanin biosynthesis and regulatory mechanisms in C. roseus.