Regulation Of Anthocyanin Biosynthesis Research Articles

Analysis of bZIP gene family in lotus (Nelumbo) and functional study of NnbZIP36 in regulating anthocyanin synthesis

BackgroundThe basic leucine zipper (bZIP) family is a predominant group of transcription factors in plants, involved in regulating plant growth, development, and response to stressors. Additionally, the bZIP gene family has a key role in anthocyanin production. Despite the significant role of bZIP genes in plants, their potential contribution in lotus remains understudied.ResultsA total of 124 bZIP genes (59 NnbZIPs and 65 NlbZIPs) were identified from genomes of two lotus species. These genes were classified into 13 groups according to the grouping principle of the Arabidopsis bZIP gene family. Analysis of promoter cis-acting elements indicated that most bZIP gene family members in lotus are associated with response to abiotic stresses. The promoters of some bZIP genes contain MYB binding sites that regulate anthocyanin synthesis. We examined the anthocyanin content of the petals from three different colored lotus, combined with transcriptome data analysis and qRT-PCR results, showing that the expression trends of NnbZIP36 and the homologous gene NlbZIP38 were significantly correlated with the anthocyanin content in lotus petals. Furthermore, we found that overexpression of NnbZIP36 in Arabidopsis promoted anthocyanin accumulation by upregulating the expression of genes (4CL, CHI, CHS, F3H, F3'H, DFR, ANS and UF3GT) related to anthocyanin synthesis.ConclusionsOur study enhances the understanding of the bZIP gene family in lotus and provides evidence for the role of NnbZIP36 in regulating anthocyanin synthesis. This study also sets the stage for future investigations into the mechanism by which the bZIP gene family regulates anthocyanin biosynthesis in lotus.

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.

Yeast One-Hybrid Screening for Regulators of IbWD40 in Purple-Fleshed Sweet Potato (Ipomoea batatas [L] Lam.).

The transcription regulator IbWD40 is known to be involved in anthocyanin biosynthesis in purple-flesh sweet potato (Ipomoea batatas). However, little is known about the upstream transcription regulators on the promoter of IbWD40. Yeast one-hybrid screening was performed on the storage roots of purple-fleshed sweet potato to identity upstream transcription regulators on the promoter of IbWD40. Luciferase reporter assays and Yeast one-hybrid assays were used to verify these upstream binding proteins interacted with the promoter. Real-time PCR was used to analyze the gene expression of upstream transcription regulators, transcription factors, and structural genes involved in anthocyanin biosynthesis in different root stages of purple-fleshed and white-fleshed sweet potato. IbERF1, IbERF10, IbEBF2, IbPDC, IbPGP19, IbUR5GT, IbDRM, IbPPA and IbERF73 were identified as candidate binding proteins for the promoter of IbWD40. Furthermore, IbERF1, IbERF10 and IbERF73 were identified as upstream transcription regulators on the promoter of IbWD40 involved in anthocyanin biosynthesis. IbERF1, IbERF10 and IbERF73 were identified as transcription regulators on the promoter of IbWD40, which is involved in the regulation of anthocyanin biosynthesis in purple-fleshed sweet potato.

Comparative Transcriptome Analysis of Purple and Green Flowering Chinese Cabbage and Functional Analyses of BrMYB114 Gene.

Flowering Chinese cabbage (Brassica rapa var. parachinensis) is one of the most popular vegetables in the south of China. As an antioxidant, anthocyanin is an important quality trait in vegetables, and the gene related to anthocyanin biosynthesis in purple flowering Chinese cabbage is also important. In this study, two flowering Chinese cabbage with extreme colors in the stem were used as materials for transcriptome analysis. RNA-seq analysis showed that 6811 differentially expressed genes (DEGs) were identified, including 295 transcription factors. Phenylpropanoid biosynthesis, flavone and flavanol biosynthesis, and flavonoid biosynthesis pathways were found to be significantly enriched in the purple flowering Chinese cabbage. A total of 25 DEGs associated with anthocyanin biosynthesis were found at a higher expression in purple flowering Chinese cabbage than in green flowering Chinese cabbage. Bioinformatics analysis shows that BrMYB114 is a candidate gene for the regulation of anthocyanin biosynthesis, and heterologous expression analysis of BrMYB114 in Nicotiana benthamiana indicates that BrMYB114 functions in anthocyanin biosynthesis. Therefore, our findings provide vital evidence for elucidating the molecular mechanism in the purple stem in flowering Chinese cabbage.

VvBBX44 and VvMYBA1 form a regulatory feedback loop to balance anthocyanin biosynthesis in grape.

Anthocyanins are essential for the quality of perennial horticultural crops, such as grapes. In grapes, ELONGATED HYPOCOTYL 5 (HY5) and MYBA1 are two critical transcription factors that regulate anthocyanin biosynthesis. Our previous work has shown that Vitis vinifera B-box protein 44 (VvBBX44) inhibits anthocyanin synthesis and represses VvHY5 expression in grape calli. However, the regulatory mechanism underlying this regulation was unclear. In this study, we found that loss of VvBBX44 function resulted in increased anthocyanin accumulation in grapevine callus. VvBBX44 directly represses VvMYBA1, which activates VvBBX44. VvMYBA1, but not VvBBX44, directly modulates the expression of grape UDP flavonoid 3-O-glucosyltransferase (VvUFGT). We demonstrated that VvBBX44 represses the transcriptional activation of VvUFGT and VvBBX44 induced by VvMYBA1. However, VvBBX44 and VvMYBA1 did not physically interact in yeast. The application of exogenous anthocyanin stimulated VvBBX44 expression in grapevine suspension cells and tobacco leaves. These findings suggest that VvBBX44 and VvMYBA1 form a transcriptional feedback loop to prevent overaccumulation of anthocyanin and reduce metabolic costs. Our work sheds light on the complex regulatory network that controls anthocyanin biosynthesis in grapevine.

E3 ubiquitin ligases SINA4 and SINA11 regulate anthocyanin biosynthesis by targeting the IAA29-ARF5-1-ERF3 module in apple.

Auxin/indole-3-acetic acid (AUX/IAA) and auxin response factor (ARF) proteins are important components of the auxin signalling pathway, but their ubiquitination modification and the mechanism of auxin-mediated anthocyanin biosynthesis remain elusive. Here, the ARF MdARF5-1 was identified as a negative regulator of anthocyanin biosynthesis in apple, and it integrates auxin and ethylene signals by inhibiting the expression of the ethylene response factor MdERF3. The auxin repressor MdIAA29 decreased the inhibitory effect of MdARF5-1 on anthocyanin biosynthesis by attenuating the transcriptional inhibition of MdERF3 by MdARF5-1. In addition, the E3 ubiquitin ligases MdSINA4 and MdSINA11 played negative and positive regulatory roles in anthocyanin biosynthesis by targeting MdIAA29 and MdARF5-1 for ubiquitination degradation, respectively. MdSINA4 destabilized MdSINA11 to regulate anthocyanin accumulation in response to auxin signalling. In sum, our data revealed the crosstalk between auxin and ethylene signals mediated by the IAA29-ARF5-1-ERF3 module and provide new insights into the ubiquitination modification of the auxin signalling pathway.

Light-Induced TaHY5-7A and TaBBX-3B Physically Interact to Promote PURPLE PERICARP-MYB 1 Expression in Purple-Grained Wheat.

Purple-grained wheat (Triticum aestivum L.) is an important germplasm source in crop breeding. Anthocyanin biosynthesis in the pericarps of purple-grained wheat is largely light-dependent; however, the regulatory mechanisms underlying light-induced anthocyanin accumulation in the wheat pericarp remain unknown. Here we determined that anthocyanins rapidly accumulate in the pericarps of the purple-grained wheat cultivar Heixiaomai 76 (H76) at 16 days after pollination under light treatment. Using transcriptome sequencing, differential gene expression analysis, and phylogenetic analysis, we identified two key genes involved in light signaling in wheat: ELONGATED HYPOCOTYL 5-7A (TaHY5-7A) and B-BOX-3B (TaBBX-3B). TaHY5-7A and TaBBX-3B were highly expressed in purple-grained wheat pericarps. The heterologous expression of TaHY5-7A partially restored the phenotype of the Arabidopsis (Arabidopsis thaliana) hy5 mutant, resulting in increased anthocyanin accumulation and a shortened hypocotyl. The heterologous expression of TaBBX-3B in wild-type Arabidopsis had similar effects. TaHY5-7A and TaBBX-3B were nucleus-localized, consistent with a function in transcription regulation. However, TaHY5-7A, which lacks a transactivation domain, was not sufficient to activate the expression of PURPLE PERICARP-MYB 1 (TaPpm1), the key anthocyanin biosynthesis regulator in purple pericarps of wheat. TaHY5-7A physically interacted with TaBBX-3B in yeast two-hybrid and bimolecular fluorescence complementation assays. Additionally, TaHY5-7A, together with TaBBX-3B, greatly enhanced the promoter activity of TaPpm1 in a dual luciferase assay. Overall, our results suggest that TaHY5-7A and TaBBX-3B collaboratively activate TaPpm1 expression to promote light-induced anthocyanin biosynthesis in purple-pericarp wheat.

Genome-Wide Identification of R2R3-MYB Transcription Factor Family in Tartary Buckwheat (Fagopyrum tataricum) Identifies a Member Involved in Anthocyanin Biosynthesis

Tartary buckwheat (Fagopyrum tataricum Gaertn.) belongs to the family of Polygonaceae and is used as a multi-functional plant. R2R3-MYB transcription factors play a crucial part in plant growth and are involved in many biological processes where they regulate their internal environment. To date, there is no documented systematic research on the R2R3-MYB gene family in Tartary buckwheat. Here, domain features, chromosomal location, motif prediction, gene structure, cis-acting elements, as well as the expression pattern of R2R3-MYB transcription factors were analyzed comprehensively in Tartary buckwheat using a bioinformatic approach. Additionally, one R2R3-MYB gene was verified by transgenic Arabidopsis. Results indicate that a total of 152 R2R3-MYB genes were identified with special R2R3 domains and were distributed on 8 chromosomes of Tartary buckwheat. They were further classified into 25 sub-categories via phylogenetic analysis in terms of the R2R3-MYB gene family classification principles of Arabidopsis thaliana. This classification was further supported by analysis of exon–intron structure, motif, and cis-elements. Tandem and segmental duplication existed among the R2R3-MYB gene family of Tartary buckwheat, and there were 5, 8, 27, and 36 FtR2R3-MYB homologous genes, respectively, when comparing with Zea mays, Oryza sativa, Arabidopsis thaliana, and Solanum melongena by synteny analysis. The expression pattern of FtR2R3-MYB genes in different tissue and under salt stress and different light condition showed that members had tissue-specific expression levels and that these members may play diverse functions in plant growth and adaptation to varying environments. In addition, one of the FtR2R3-MYB gene families, FtMYB43, a homologue of AtTT2, clustered with R2R3-MYB from other plant species, which were reported to be involved in the regulation of anthocyanin or proanthocyanidin biosynthesis. This gene was located in the nucleus, and had transcriptional activation activity, indicating that FtMYB43 may be a positive transcript factor of anthocyanin or proanthocyadin biosynthesis. Moreover, the function of FtMYB43 was further verified to improve the production of anthocyanin in transgenic Arabidopsis by overexpression, and qRT-PCR assay implied that FtMYB43 may regulate the expression levels of most structural genes of the anthocyanin biosynthesis pathway in Arabidopsis seedlings. These results provide more insights into the structure and function of the R2R3-MYB gene family and may accelerate the breeding of ornamental buckwheat cultivars.

Transcription Regulation of Anthocyanins and Proanthocyanidins Accumulation by Bagging in ‘Ruby’ Red Mango: An RNA-seq Study

The biosynthesis of anthocyanins and proanthocyanidins (PAs), components of two main flavonoids in plants, is regulated by environmental factors such as light. We previously found that bagging significantly repressed the biosynthesis of anthocyanins in red ‘Ruby’ mango fruit peel, but induced the accumulation of PAs. However, the molecular mechanism remains unclear. In the current study, transcriptome sequencing was used for screening the essential genes responsible for the opposite accumulation pattern of anthocyanins and PAs by bagging treatment. According to weighted gene co-expression network analysis (WGCNA), structural genes and transcription factors highly positively correlated to anthocyanins and PAs were identified. One flavanone 3-hydroxylase (F3H) and seven structural genes, including one chalcone synthase (CHS), one flavonoid 3’-hydroxylase (F3’H), one anthocyanidin synthesis (ANS), three leucoanthocyanidin reductase (LARs), and one UDP glucose: flavonoid 3-O-glucosyltransferase (UFGT), are crucial for anthocyanin and PA biosynthesis, respectively. In addition to MYB and bHLH, ERF, C2H2, HD-ZIP, and NAC are important transcription factors that participate in the regulation of anthocyanin and PA biosynthesis in ‘Ruby’ mango fruit peel by bagging treatment. Our results are helpful for revealing the transcription regulation mechanism of light-regulated mango anthocyanin and PA biosynthesis, developing new technologies for inducing flavonoid biosynthesis in mangos, and breeding mango cultivars containing high concentrations of flavonoids.

FaMYB5 Interacts with FaBBX24 to Regulate Anthocyanin and Proanthocyanidin Biosynthesis in Strawberry (Fragaria × ananassa).

MYB and BBX transcription factors play important roles in flavonoid biosynthesis. Here, we obtained transgenic woodland strawberry with stable overexpression of FaMYB5, demonstrating that FaMYB5 can increase anthocyanin and proanthocyanidin content in roots, stems and leaves of woodland strawberry. In addition, bimolecular fluorescence complementation assays and yeast two-hybridization demonstrated that the N-terminal (1-99aa) of FaBBX24 interacts with FaMYB5. Transient co-expression of FaBBX24 and FaMYB5 in cultivated strawberry 'Xiaobai' showed that co-expression strongly promoted the expression of F3'H, 4CL-2, TT12, AHA10 and ANR and then increased the content of anthocyanin and proanthocyanidin in strawberry fruits. We also determined that FaBBX24 is also a positive regulator of anthocyanin and proanthocyanidin biosynthesis in strawberry. The results reveal a novel mechanism by which the FaMYB5-FaBBX24 module collaboratively regulates anthocyanin and proanthocyanidin in strawberry fruit.

CmMYB3-like negatively regulates anthocyanin biosynthesis and flower color formation during the post-flowering stage in Chrysanthemum morifolium

Color fading caused by a decrease in anthocyanin accumulation during the post-flowering stage significantly affects postharvest quality of chrysanthemum. However, the underlying mechanism by which anthocyanin accumulation decreases during the post-flowering stage still unclear, which greatly restricts design of molecular breeding in chrysanthemum. Here, a chrysanthemum SG7 R2R3 MYB transcription factor (TF), CmMYB3-like, was identified to have a function in regulating anthocyanin biosynthesis during the post-flowering stage. Quantitative real time PCR (qRT-PCR) assays showed that the expression of CmMYB3-like was gradually downregulated when anthocyanin content increased during the flowering stage and was significantly upregulated during the post-flowering stage. Genetic transformation of chrysanthemum and dual-luciferase assays in N. benthamiana leaves showed that CmMYB3-like suppressed anthocyanin accumulation by inhibiting the transcription of CmCHS and CmANS directly and that of CmF3H indirectly. However, overexpression or suppression of CmMYB3-like did not affect the biosynthesis of flavones or flavonols. Genetic transformation of chrysanthemum revealed that the overexpression of CmMYB3-like inhibited anthocyanin accumulation, but its suppression prevented the decrease in anthocyanin accumulation during the post-flowering stage. Our results revealed a crucial role of CmMYB3-like in regulating the color of petals during the post-flowering stage and provided a target gene for molecular design breeding to improve the postharvest quality of chrysanthemum.

Integrative Metabolome and Transcriptome Analyses Reveal the Molecular Mechanism of Yellow-Red Bicolor Formation in Kalanchoe blossfeldiana Petals

The winter pot kalanchoe (Kalanchoe blossfeldiana) is an ornamental plant with succulent leaves and clustered flowers in Crassulaceae, widely used as a potted flower or garden decoration. In nature, the bicolor petal is an interesting phenomenon, and breeders have succeeded in cultivating the winter pot kalanchoe with bicolored petals. However, its potential molecular mechanism of pigmentation is poorly understood. This study collected a yellow-red colored winter pot kalanchoe to investigate the molecular mechanism underlying its bicolor formation using the integrative analyses of metabolome and transcriptome. The metabolome results showed that both flavonoid and carotenoid co-existed in the winter pot kalanchoe petals, whereas only anthocyanin accumulation showed significant differences—about nineteen times higher in the red region than that in the yellow region. The differentially expressed genes were significantly enriched in the anthocyanin biosynthesis pathway, and the expression level of biosynthetic genes, including KbCHS, KbCHI, KbF3H, KbDFR, KbANS and KbGTs, were significantly upregulated in the red region. Moreover, transcription factors potentially regulating anthocyanin biosynthesis were predicted, and KbMYB2 and KbbHLH1 might play important roles in positively regulating anthocyanin biosynthesis in the red region. The findings reported here provide new insights into the understanding of petal bicolor formation mechanisms and will assist cultivar innovation in winter pot kalanchoe.

The E3 ubiquitin ligase SINA1 and the protein kinase BIN2 cooperatively regulate PHR1 in apple anthocyanin biosynthesis.

PHR1 (PHOSPHATE STARVATION RESPONSE1) plays key roles in the phosphate (Pi) starvation response and in Pi-deficiency-induced anthocyanin biosynthesis in plants. However, the post-translational regulation of PHR1 is unclear, and the molecular basis of PHR1-mediated anthocyanin biosynthesis remains elusive. In this study, we determined that MdPHR1 was essential for Pi-deficiency-induced anthocyanin accumulation in apple (Malus × domestica). MdPHR1 interacted with MdWRKY75, a positive regulator of anthocyanin biosynthesis, to enhance the MdWRKY75-activated transcription of MdMYB1, leading to anthocyanin accumulation. In addition, the E3 ubiquitin ligase SEVEN IN ABSENTIA1 (MdSINA1) negatively regulated MdPHR1-promoted anthocyanin biosynthesis via the ubiquitination-mediated degradation of MdPHR1. Moreover, the protein kinase MdBIN2 phosphorylated MdPHR1 and positively regulated MdPHR1-mediated anthocyanin accumulation by attenuating the MdSINA1-mediated ubiquitination degradation of MdPHR1. Taken together, these findings not only demonstrate the regulatory role of MdPHR1 in Pi-starvation induced anthocyanin accumulation, but also provide an insight into the post-translational regulation of PHR1. This article is protected by copyright. All rights reserved.

The PcHY5 methylation is associated with anthocyanin biosynthesis and transport in ‘Max Red Bartlett’ and ‘Bartlett’ pears

The red coloring of pear fruits is mainly caused by anthocyanin accumulation. Red sport, represented by the green pear cultivar ‘Bartlett’ (BL) and the red-skinned derivative ‘Max Red Bartlett’ (MRB), is an ideal material for studying the molecular mechanism of anthocyanin accumulation in pear. Genetic analysis has previously revealed a quantitative trait locus (QTL) associated with red skin color in MRB. However, the key gene in the QTL and the associated regulatory mechanism remain unknown. In the present study, transcriptomic and methylomic analyses were performed using pear skin for comparisons between BL and MRB. These analyses revealed differential PcHY5 DNA methylation levels between the two cultivars; MRB had lower PcHY5 methylation than BL during fruit development, and PcHY5 was more highly expressed in MRB than in BL. These results indicated that PcHY5 is involved in the variations in skin color between BL and MRB. We further used dual luciferase assays to verify that PcHY5 activates the promoters of the anthocyanin biosynthesis and transport genes PcUFGT, PcGST, PcMYB10 and PcMYB114, confirming that PcHY5 not only regulates anthocyanin biosynthesis but also anthocyanin transport. Furthermore, we analyzed a key differentially methylated site between MRB and BL, and found that it was located in an intronic region of PcHY5. The lower methylation levels in this PcHY5 intron in MRB were associated with red fruit color during development, whereas the higher methylation levels at the same site in BL were associated with green fruit color. Based on the differential expression and methylation patterns in PcHY5 and gene functional verification, we hypothesize that PcHY5, which is regulated by methylation levels, affects anthocyanin biosynthesis and transport to cause the variations in skin color between BL and MRB.

PbZAT12, Independent of PbMYB10, Activates Structural Genes to Promote Anthocyanin Biosynthesis in ‘Red Zaosu’ Pear Fruit (Pyrus bretschneideri Rehd.)

The red color of the pear peel in red-skinned European pear is due to the accumulation of anthocyanins. Numerous transcription factors play pivotal roles in anthocyanin biosynthesis, with zinc finger proteins frequently implicated in the regulation of this process via MYB10, as documented in earlier studies. In this article, we identified a zinc finger protein gene, named PbZAT12, that does not rely on PbMYB10, to regulate anthocyanin biosynthesis. The PbZAT12 protein was localized within the nucleus and exhibited a positive influence on the accumulation of anthocyanins in the peel of ‘Red Zaosu’ pears. Moreover, overexpression of PbZAT12 resulted in a significant up-regulation of PbDFR, PbANS, and PbUFGT expression levels in pear fruitlets. Y1H assays demonstrated a direct binding ability of PbZAT12 to proPbDFR, proPbANS, and proPbUFGT, which was supported by a dual luciferase assay, indicating its potential to activate the transcriptional activity of these promoters. However, in contrast to its effect on the aforementioned promoters, PbZAT12 did not exhibit an activation of PbMYB10. In summary, our findings suggest that a zinc finger transcription factor, PbZAT12, exerts a positive influence on anthocyanin biosynthesis in pear fruit through direct upregulation of the expression levels of PbDFR, PbANS, and PbUFGT.

The PcERF5 promotes anthocyanin biosynthesis in red-fleshed pear (Pyrus communis) through both activating and interacting with PcMYB transcription factors

As there is a strong interest in red-skinned pears, the molecular mechanism of anthocyanin regulation in red-skinned pears has been widely investigated; however, little is known about the molecular mechanism of anthocyanin regulation in red-fleshed pears due to limited availability of such germplasm, primarily found in European pears (Pyrus communis). In this study, based on transcriptomic analysis in red-fleshed and white-fleshed pears, we identified an ethylene response factor (ERF) from P. communis, PcERF5, of which expression level in fruit flesh was significantly correlated with anthocyanin content. We then verified the function of PcERF5 in regulating anthocyanin accumulation by genetic transformation in both pear skin and apple calli. PcERF5 regulated anthocyanin biosynthesis by different regulatory pathways. On the one hand, PcERF5 can activate the transcription of flavonoid biosynthetic genes (PcDFR, PcANS and PcUFGT) and two key transcription factors encoding genes PcMYB10 and PcMYB114. On the other hand, PcERF5 interacted with PcMYB10 to form the ERF5-MYB10 protein complex that enhanced the transcriptional activation of PcERF5 on its target genes. Our results suggested that PcERF5 functioned as a transcriptional activator in regulating anthocyanin biosynthesis, which provides new insights into the regulatory mechanism of anthocyanin biosynthesis. This new knowledge will provide guidance for molecular breeding of red-fleshed pear.

Light-mediated anthocyanin biosynthesis in rose petals involves a balanced regulatory module comprising transcription factors RhHY5, RhMYB114a, and RhMYB3b.

Roses are significant botanical species with both ornamental and economic value, displaying diverse floral traits, particularly an extensive array of petal colors. The red pigmentation of rose petals is predominantly attributed to anthocyanin accumulation. However, the underlying regulatory mechanism of anthocyanin biosynthesis in roses remains elusive. This study presents a novel light-responsive regulatory module governing anthocyanin biosynthesis in rose petals, which involves the transcription factors RhHY5, RhMYB114a, and RhMYB3b. Under light conditions (1000-1500 μmol m-2 s-1), RhHY5 represses RhMYB3b expression and induces RhMYB114a expression, positively regulating anthocyanin biosynthesis in rose petals. Notably, activation of anthocyanin structural genes probably involves an interaction and synergy between RhHY5 and the MYB114a-bHLH3-WD40 complex. Additionally, RhMYB3b is activated by RhMYB114a to prevent excessive accumulation of anthocyanin. Conversely, under low light conditions (<10 μmol m-2 s-1), the degradation of RhHY5 leads to down-regulation of RhMYB114a and up-regulation of RhMYB3b, which in turn inhibits the expression of both RhMYB114a and anthocyanin structural genes. Additionally, RhMYB3b competes with RhMYB114a for binding to RhbHLH3 and the promoters of anthocyanin-related structural genes. Overall, our study uncovers a complex light-mediated regulatory network that governs anthocyanin biosynthesis in rose petals, providing new insights into the molecular mechanisms underlying petal color formation in rose.

Advances in the endogenous and exogenous regulation of anthocyanins–the key to color change in eudicots

Anthocyanins are important water-soluble pigments that are widely found in plants and determine the color of plant organs, which possess significant health and economic value. The anthocyanin biosynthetic pathway is well established and some key regulators controlling this pathway have been identified in a variety of species. In recent years, microRNAs, transcription factors, and external factors, including light, temperature and hormones have emerged as central regulators of a variety of secondary metabolic responses, including anthocyanin metabolism, as both plant self-regulatory factors and environmental signals that stimulate plant development. Many discoveries have begun to suggest gene/signal-gene modules to reveal the signaling pathways involved in anthocyanin biosynthesis during fruit ripening and the links between specific genes. This paper reviews the internal and external factors and associated network mechanisms that regulate anthocyanin biosynthesis and accumulation in eudicots plants. miRNAs have different effects on the biosynthesis of anthocyanins in different species by binding to their target genes to form regulatory modules. The review also focuses on the conclusion that light signals (light quality, photoperiod, and light intensity) further regulate anthocyanin accumulation by binding to their corresponding photoreceptors (UVR8, CRYs, and PHYs) and affecting the binding and stabilization of the central regulators of the light pathway, such as COP1 and the transcription factor HY5. This article aims to provide a reference for future research on the regulation of anthocyanin by effective pathways, and to contribute to the selection of new varieties and the improvement of cultivation facilities.

Development of an inducible excision system of a visual marker Ipomoea batatas Myb gene from the genome of transgenic cells.

In the plant genetic transformation process, single selection by a chemical-resistant marker gene occasionally allows the proliferation of non-transgenic cells, escaping selection pressure. The additional use of a visual marker gene is effective for accurate selection. For instance, R2R3-MYB genes are used for regulating anthocyanin biosynthesis; however, constitutive Myb expression in transgenic plants is not always desirable and may cause developmental abnormalities due to excess anthocyanin accumulation. To overcome the remaining problems in the use of Myb as a visible marker, we developed T-DNA. Ipomoea batatas Myb (IbMyb) and Cre expression cassettes were inserted between two loxP sequences, and the hygromycin phosphotransferase (HPT) and green fluorescent protein (GFP) expression cassettes were located outside of the loxP-IbMyb-Cre-loxP region. In the developed system, IbMyb and Cre were excised from the genomes of transgenic cells using heat-inducible Cre-loxP recombination. Upon heat treatment in a general incubator, green shoots emerged from purple tobacco transgenic calli that were pigmented with IbMyb expression. The excision of IbMyb from the genome of green shoots was confirmed using polymerase chain reaction (PCR) and sequencing. GFP expression was observed in the roots of the obtained green transgenic plants. We report that the system developed here operated successfully in tobacco, showing the potential to provide an easier and cheaper visual selection of transgenic cells in the genetic transformation process.

Enhancement of the anthocyanin contents of Caladium leaves and petioles via metabolic engineering with co-overexpression of AtPAP1 and ZmLc transcription factors.

Metabolic engineering of anthocyanin synthesis is an active research area for pigment breeding and remains a research hotspot involving AtPAP1 and ZmLc transcription factors. Caladium bicolor is a desirable anthocyanin metabolic engineering receptor, with its abundant leaf color and stable genetic transformation system. We transformed C. bicolor with AtPAP1 and ZmLc and successfully obtained transgenic plants. We then used a combination of metabolome, transcriptome, WGCNA and PPI co-expression analyses to identify differentially expressed anthocyanin components and transcripts between wild-type and transgenic lines. Cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside and peonidin-3-O-rutinoside are the main components of anthocyanins in the leaves and petioles of C. bicolor. Exogenous introduction of AtPAP1 and ZmLc resulted in significant changes in pelargonidins, particularly pelargonidin-3-O-glucoside and pelargonidin-3-O-rutinoside in C. bicolor. Furthermore, 5 MYB-TFs, 9 structural genes, and 5 transporters were found to be closely associated with anthocyanin synthesis and transport in C. bicolor. In this study, a network regulatory model of AtPAP1 and ZmLc in the regulation of anthocyanin biosynthesis and transport in C. bicolor was proposed, which provides insights into the color formation mechanisms of C. bicolor, and lays a foundation for the precise regulation of anthocyanin metabolism and biosynthesis for economic plant pigment breeding.