Anthocyanin Biosynthesis In Plants Research Articles

ERF transcription factors govern anthocyanin biosynthesis in litchi pericarp by modulating the expression of anthocyanin biosynthesis genes

Anthocyanins are the most prevalent phytochemicals that color the fruits. Ethylene promotes fruit ripening and modulates anthocyanin biosynthesis in plants, however, the underlying molecular mechanism in litchi is unclear. Herein, the slow-reddening cultivar ‘Feizixiao’ (FXZ) and swift-reddening cultivar ‘Guiwei’ (GW) were selected to study the regulation of anthocyanin biosynthesis in litchi. Total anthocyanin contents were found to be higher in ‘GW’ and lower in ‘FZX’. We then comprehensively investigated ethylene response factor (ERF) subfamily genes and their expression patterns in different tissues and fruit developmental stages. Using transcriptome data, eight LcERF genes were identified as putative key genes associated with litchi pericarp coloration and anthocyanin accumulation. To test whether these genes are expressed under ethephon treatment, we applied 100 and 200 µL L−1 ethephon 4 weeks before harvest. Ethephon treatment dramatically increased anthocyanin accumulation as well as the expression of targeted ERF genes. LcERF1/22/25/37 were significantly upregulated in response to ethephon treatment, while LcERF64 was significantly downregulated. Anthocyanin content and expression of anthocyanin-related genes were considerably reduced in LcERF1/22/25/37-silenced fruits, but the opposite trend was observed in LcERF64-silenced fruits. Yeast-one-hybrid and dual luciferase assays revealed that the tested ERFs bind to the promoters of key anthocyanin biosynthesis genes and alter their expression. These findings suggested that ethylene promotes anthocyanin accumulation and consequently fruit coloration by stimulating the positive anthocyanin biosynthesis regulators LcERF1/22/25/37, or by suppressing the negative regulator LcERF64, which regulates the expression LcDFR and LcMYB1 by directly binding to their promoters.

Reactive oxygen species act as the key signaling molecules mediating light-induced anthocyanin biosynthesis in Eucalyptus

Light plays a pivotal role in regulating anthocyanin biosynthesis in plants, and the early light-responsive signals that initiate anthocyanin biosynthesis remain to be elucidated. In this study, we showed that the anthocyanin biosynthesis in Eucalyptus is hypersensitive to increased light intensity. The combined transcriptomic and metabolomic analyses were conducted on Eucalyptus leaves after moderate (ML; 100 μmol m−2 s−1) and high (HL; 300 μmol m−2 s−1) light intensity treatments. The results identified 1940, 1096, 1173, and 2756 differentially expressed genes at 6, 12, 24, and 36 h after HL treatment, respectively. The metabolomic results revealed the primary anthocyanin types, and other differentially accumulated flavonoids and phenylpropane intermediates that were produced in response to HL, which well aligned with the transcriptome results. Moreover, biochemical analysis showed that HL inhibited peroxidase activity and increased the ROS level in Eucalyptus leaves. ROS depletion through co-application of the antioxidants rutin, uric acid, and melatonin significantly reduced, and even abolished, anthocyanin biosynthesis induced by HL treatment. Additionally, exogenous application of hydrogen peroxide efficiently induced anthocyanin biosynthesis within 24 h, even under ML conditions, suggesting that ROS played a major role in activating anthocyanin biosynthesis. A HL-responsive MYB transcription factor EgrMYB113 was identified to play an important role in regulating anthocyanin biosynthesis by targeting multiple anthocyanin biosynthesis genes. Additionally, the results demonstrated that gibberellic acid and sugar signaling contributed to HL-induced anthocyanin biosynthesis. Conclusively, these results suggested that HL triggers multiple signaling pathways to induce anthocyanin biosynthesis, with ROS acting as indispensable mediators in Eucalyptus.

Multifaceted Regulation of Anthocyanin Biosynthesis in Plants: A Comprehensive Review

Multifaceted Regulation of Anthocyanin Biosynthesis in Plants: A Comprehensive Review

Integrated transcriptome and metabolome analysis reveals anthocyanin biosynthesis mechanisms in pepper (Capsicum annuum L.) leaves under continuous blue light irradiation

BackgroundDifferent metabolic compounds give pepper leaves and fruits their diverse colors. Anthocyanin accumulation is the main cause of the purple color of pepper leaves. The light environment is a critical factor affecting anthocyanin biosynthesis. It is essential that we understand how to use light to regulate anthocyanin biosynthesis in plants.ResultPepper leaves were significantly blue–purple only in continuous blue light or white light (with a blue light component) irradiation treatments, and the anthocyanin content of pepper leaves increased significantly after continuous blue light irradiation. This green-to-purple phenotype change in pepper leaves was due to the expression of different genes. We found that the anthocyanin synthesis precursor-related genes PAL and 4CL, as well as the structural genes F3H, DFR, ANS, BZ1, and F3’5’H in the anthocyanin synthesis pathway, had high expression under continuous blue light irradiation. Similarly, the expression of transcription factors MYB1R1-like, MYB48, MYB4-like isoform X1, bHLH143-like, and bHLH92-like isoform X3, and circadian rhythm-related genes LHY and COP1, were significantly increased after continuous blue light irradiation. A correlation network analysis revealed that these transcription factors and circadian rhythm-related genes were positively correlated with structural genes in the anthocyanin synthesis pathway. Metabolomic analysis showed that delphinidin-3-O-glucoside and delphinidin-3-O-rutinoside were significantly higher under continuous blue light irradiation relative to other light treatments. We selected 12 genes involved in anthocyanin synthesis in pepper leaves for qRT-PCR analysis, and the accuracy of the RNA-seq results was confirmed.ConclusionsIn this study, we found that blue light and 24-hour irradiation together induced the expression of key genes and the accumulation of metabolites in the anthocyanin synthesis pathway, thus promoting anthocyanin biosynthesis in pepper leaves. These results provide a basis for future study of the mechanisms of light quality and photoperiod in anthocyanin synthesis and metabolism, and our study may serve as a valuable reference for screening light ratios that regulate anthocyanin biosynthesis in plants.

The ncRNAs Involved in the Regulation of Abiotic Stress-Induced Anthocyanin Biosynthesis in Plants.

Plants have evolved complicated defense and adaptive systems to grow in various abiotic stress environments such as drought, cold, and salinity. Anthocyanins belong to the secondary metabolites of flavonoids with strong antioxidant activity in response to various abiotic stress and enhance stress tolerance. Anthocyanin accumulation often accompanies the resistance to abiotic stress in plants to scavenge reactive oxygen species (ROS). Recent research evidence showed that many regulatory pathways such as osmoregulation, antioxidant response, plant hormone response, photosynthesis, and respiration regulation are involved in plant adaption to stress. However, the molecular regulatory mechanisms involved in controlling anthocyanin biosynthesis in relation to abiotic stress response have remained obscure. Here, we summarize the current research progress of specific regulators including small RNAs, and lncRNAs involved in the molecular regulation of abiotic stress-induced anthocyanin biosynthesis. In addition, an integrated regulatory network of anthocyanin biosynthesis controlled by microRNAs (miRNAs), long non-coding RNAs (lncRNAs), transcription factors, and stress response factors is also discussed. Understanding molecular mechanisms of anthocyanin biosynthesis for ROS scavenging in various abiotic stress responses will benefit us for resistance breeding in crop plants.

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.

Genome-wide identification and expression analysis of anthocyanin synthesis-related R2R3-MYB gene family in purple passion fruit (Passiflora edulis)

The myeloblastosis (MYB) transcription factors (TFs) participate in a variety of physiological and biochemical processes, especially in the anthocyanin biosynthesis in plants. However, the comprehensive functional study of MYB TFs, especially in regulating anthocyanin accumulation in passion fruit (Passiflora edulis) remains limited. To gain a better understanding of the evolutionary history of this family, we conducted a genome-wide analysis of MYB TFs in passion fruit, including phylogenetic analysis, gene structure, conserved motifs, chromosomal locations, and collinearity analysis. A total of 254 MYB genes were identified in the passion fruit genome, comprising 128 1R-MYBs, 119 R2R3-MYBs, 5 3R-MYBs and 2 4R-MYBs. Based on the classification of Arabidopsis thaliana MYB (AtMYB) genes, the passion fruit R2R3-MYB (PeMYB) genes were further divided into 37 subgroups, which were dispersed in 9 chromosomes. The analyses of gene structure and protein conserved motif analyses further supports the clustering results of the phylogenetic tree. RT-qPCR validation revealed that the relative expression of PeMYB67 and PeMYB86 increased during fruit development, while PeMYB95 exhibited no significant changes. The expression levels of other eight MYB genes involving PeMYB4, PeMYB13, PeMYB32, PeMYB40, PeMYB78, PeMYB115, PeMYB25 and PeMYB118 manifested declining regulation alongside fruit ripening. They were either positively or negatively correlated with the accumulation of anthocyanins during fruit growth. Overall, this study provides a comprehensive overview of the passion fruit MYB superfamily genes and lays a foundation for future cloning and functional analysis of PeMYB genes related to anthocyanin accumulation in passion fruit.

Study of <i>Myb114</i> Gene Polymorphism in the Cole Crops (<i>Brassica oleracea</i> L.) in Connection with Anthocyanin Biosynthesis Regulation Based on Comparison with the MYB Factors of Vegetable Nightshades (Solanaceae)

In order to study a regulation mechanism for one of the most ancient processes of anthocyanin biosynthesis in plants based on comparison of R2R3-MYB genes in Solanaceae (S. lycopersicum: An1, An2; S. melongena: Myb1; C. annuum: Myb113-like1 и Myb113-like2) and Brassicaeae crops, a search for orthologous sequences in Brassica oleracea was performed. A sequence encoding MYB114 TF was found to be the closest in a nucleotide structure to the previously investigated genes in Solanaceae. Primers were selected and a comparative analysis of Myb114 gene sequences was performed in B. oleracea varieties (white cabbage, red cabbage, kale, cauliflower, broccoli, and kohlrabi) with a wide intraspecies diversity by the anthocyanin accumulation in leaves and various food organs: heads, curds and stems. Alignment of Myb114 sequences revealed a number of gene polymorphisms that closely correlate with high anthocyanin accumulation in B. oleracea leaves, including 3 SNPs in exon regions and a 271 bp deletion in the 1st intron. The identified SNPs lead to the replacement of two amino acids located in the region of DNA-binding domains, which leads to a change in the binding efficiency of this transcription factor with the promoters of structural biosynthesis genes and a decrease in their expression level in the forms without anthocyanin accumulation in leaves compared with the forms with their accumulation in vegetative plant parts. A molecular marker MYB114.2 has been proposed that may be used for the identification of headed cabbages and kales with high/low anthocyanin accumulation. By the amino acid structure and phenotypic manifestation (regulation of anthocyanin accumulation in leaves), Myb114 B. oleracea is the closest to Ant2 tomato, Myb2 eggplant and Myb113-like2 pepper alleles. A high degree of conservatism of the SANT DNA-binding domains of MYB114 TF of B. oleracea and R2R3-MYB TF of nightshades was established.

Integrated transcriptional and metabolomics signature pattern of pigmented wheat to insight the seed pigmentation and other associated features

Anthocyanin biosynthesis in plants is complex, especially in a polyploid monocot wheat plant. Using whole-genome sequencing, transcriptomics, and LC-MS/MS, we investigated anthocyanin pigmentation patterns in (black, blue, and purple) colored wheat seeds. According to differential gene expression profiling, 2AS-MYC, 7DL-MYB, and WD40 regulatory genes control purple pericarp coloration, 4DL-MYC, 2AS-MYC, 7DL-MYB, WD40 control blue aleurone coloration, and 4DL-MYC, 7DL-MYB, WD40 controls black aleurone color. We hypothesized that at least one MYC and MYB isoform is sufficient to regulate the anthocyanin synthesis in pericarp or aleurone. Transcriptomics and metabolomics revealed that the purple pericarp trait is associated with acylated anthocyanins compared to blue aleurone. Based upon the reduced expressions of the genes belonging to the 4D, SSR molecular marker mapping, variant calling using genome sequencing, and IGV browser gene structure visualization, it was inferred that the advanced black and blue wheat lines were substitution lines (4E{4D}), with very small recombinations. Pericarp anthocyanin pigmentation is controlled by a mutation in chromosome 2AS of purple wheat, and environmental variations influence pigmented pericarp trait. The expression patterns of anthocyanin structural and other genes varied in different colored wheat, corroborating differences in agronomical metrics. Ovate seed shape trait in black and blue wheat dragged with 4E chromosome.

Identification of anthocyanin-related microRNAs in ornamental kale (Brassica oleracea L. var. acephala) by high throughput sequencing

Anthocyanins have been found to be responsible for the pigmentation of red, purple and blue colors in plants. MicroRNA (miRNA) plays an important role in the regulation of anthocyanin biosynthesis in plants. However, few miRNAs and their corresponding functions related to anthocyanin biosynthesis in ornamental kale (Brassica oleracea var. acephala) have been reported. In this study, small RNA (sRNA) and degradome libraries from inner and outer leaves (IL and OL) of ornamental kale line “Pigeon Red” were constructed, respectively. Totally, 139 known and 14 novel miRNAs were identified by sRNA sequencing, and 304 target genes cleaved by 92 bo-miRNAs were identified by degradome sequencing. Of these, 5 miRNAs and their corresponding targets were potentially involved in the anthocyanin biosynthesis. And 35 miRNAs were differently expressed between IL and OL. Integrated analysis of sRNA-Seq, degradome sequencing and RNA-Seq revealed that bo-miR828 and its targets potentially play a key role in the regulation of anthocyanin biosynthesis in ornamental kale leaves. Finally, an inverse correlation between the expression of bo-miR828 and its targets was revealed by both qRT-PCR and high-throughput sequencing. These results represented a comprehensive expression profiling of miRNAs related to anthocyanin accumulation in B.oleracea var. acephala and provided important clues for understanding the regulatory network of anthocyanin biosynthesis mediated by miRNA in Brassica crops.

Isolation and functional diversification of dihydroflavonol 4-Reductase gene HvDFR from Hosta ventricosa indicate its role in driving anthocyanin accumulation

ABSTRACT Anthocyanins are natural colorants are synthesized in a branch of the flavonoid pathway. Dihydroflavonol-4reductase (DFR) catalyzes dihydroflavonoids into anthocyanins biosynthesis, which is a key regulatory enzyme of anthocyanin biosynthesis in plants. Hosta ventricosa is an ornamental plant with elegant flowers and rich colorful leaves. How the function of HvDFR contributes to the anthocyanins biosynthesis is still unknown. In this study, the DFR homolog was identified from H. ventricosa and sequence analysis showed that HvDFR possessed the conserved NADPH binding and catalytic domains. A phylogenetic analysis showed that HvDFR was close to the clade formed with MaDFR and HoDFR in Asparagaceae. Gene expression analysis revealed that HvDFR was constitutive expressed in all tissues and expressed highly in flower as well as was positively correlated with anthocyanin content. In addition, the subcellular location of HvDFR showed that is in the nucleus and cell membrane. Overexpression of HvDFR in transgenic tobacco lines enhanced the anthocyanins accumulation along with the key genes upregulated, such as F3H, F3ʹH, ANS, and UFGT. Our results indicated a functional activity of the HvDFR, which provide an insight into the regulation of anthocyanins content in H. ventricosa.

A light-responsive transcription factor SmMYB35 enhances anthocyanin biosynthesis in eggplant (Solanum melongena L.)

SmMYB35, a light-responsive R2R3-MYB transcription factor, positively regulates anthocyanin biosynthesis in eggplant by binding to the promoters of SmCHS, SmF3H, SmDFR, and SmANS and enhancing their activities. In addition, SmMYB35 interacts with SmTT8 and SmTTG1 to form a MBW complex, thereby enhancing anthocyanin biosynthesis. Eggplant is a vegetable rich in anthocyanins. SmMYB35, a light-responsive R2R3-MYB transcription factor, was isolated from eggplant and investigated for its biological functions. The results suggested that the expression of SmMYB35 was regulated by SmHY5 through directly binding to G-box in the promoter region, and the overexpression of SmMYB35 could increase the anthocyanin content in the stems and petals of the transgenic eggplants. SmMYB35 could also bind to the promoters of SmCHS, SmF3H, SmDFR, and SmANS and enhance their activities. In addition, SmMYB35 interacted with SmTT8 and SmTTG1 to form a MBW complex which enhanced anthocyanin biosynthesis. Taking together, we firstly verified that SmMYB35 promoted anthocyanin biosynthesis in plants. The results provide new insights into the regulatory effects of SmMYB35 on key anthocyanin biosynthetic genes and advance our understanding of the molecular mechanism of light-induced anthocyanin synthesis in eggplants.

The Basic Helix-Loop-Helix Transcription Factor SmbHLH1 Represses Anthocyanin Biosynthesis in Eggplant.

Many basic helix-loop-helix transcription factors (TFs) have been reported to promote anthocyanin biosynthesis in numerous plant species, but little is known about bHLH TFs that inhibit anthocyanin accumulation. In this study, SmbHLH1 from Solanum melongena was identified as a negative regulator of anthocyanin biosynthesis. However, SmbHLH1 showed high identity with SmTT8, which acts as a SmMYB113-dependent positive regulator of anthocyanin-biosynthesis in plants. Overexpression of SmbHLH1 in eggplant caused a dramatic decrease in anthocyanin accumulation. Only the amino acid sequences at the N and C termini of SmbHLH1 differed from the SmTT8 sequence. Expression analysis revealed that the expression pattern of SmbHLH1 was opposite to that of anthocyanin accumulation. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that SmbHLH1 could not interact with SmMYB113. Dual-luciferase assay demonstrated that SmbHLH1 directly repressed the expression of SmDFR and SmANS. Our results demonstrate that the biological function of bHLHs in anthocyanin biosynthesis may have evolved and provide new insight into the molecular functions of orthologous genes from different plant species.

Light Induced Regulation Pathway of Anthocyanin Biosynthesis in Plants

Anthocyanins are natural pigments with antioxidant effects that exist in various fruits and vegetables. The accumulation of anthocyanins is induced by environmental signals and regulated by transcription factors in plants. Numerous evidence has indicated that among the environmental factors, light is one of the most signal regulatory factors involved in the anthocyanin biosynthesis pathway. However, the signal transduction of light and molecular regulation of anthocyanin synthesis remains to be explored. Here, we focus on the research progress of signal transduction factors for positive and negative regulation in light-dependent and light-independent anthocyanin biosynthesis. In particular, we will discuss light-induced regulatory pathways and related specific regulators of anthocyanin biosynthesis in plants. In addition, an integrated regulatory network of anthocyanin biosynthesis controlled by transcription factors is discussed based on the significant progress.

Expression analysis of MYB genes in different apple cultivars with distinct coloration pattern during fruit maturation

Abstract Anthocyanins are water-soluble pigments responsible for red, pink, purple or blue coloration in the flesh and/or skin of apple fruit. Since consumers prefer anthocyanin rich fruits due to their health benefits, anthocyanin content is an important trait for marketability of apples. Synthesis and accumulation of anthocyanins are controlled by environmental and genetic factors such as transcription factors (TFs). Two-repeat (R2R3) MYB TFs are involved in the regulation of anthocyanin biosynthesis in plants. In this study, the expression of the MdMYBA and MdMYB3 genes encoding R2R3 type MYB TFs were analyzed in apple cultivars with fruit skin color from green to dark red at different growth stages. Fruit samples were collected from “Scarlet Spur”, “Galaxy Gala”, Golden Delicious” “Granny Smith” apple cultivars with dark red, light red, yellow and green fruits, respectively, at four different time periods after full-bloom. Total RNA was isolated from the peel of the collected fruits and the expression of the MdMYBA and MdMYB3 genes was analyzed by real-time RT-PCR. The expression of the MdMYBA gene started to increase at 92 DAFB and thereafter in red-skin apples; however, no expression was observed at any time points in yellow or green-skin apple cultivars. On the other hand, the MdMYB3 gene was expressed in red-skin and yellow-skin apple cultivars starting at 55 DAFB, but no expression was observed in green apple cultivar at any time of fruit maturation. The expression levels of the MdMYBA and MdMYB3 genes varied in apple fruits with different skin coloration indicating that they play a role regulation of the anthocyanin biosynthesis in apple cultivars during fruit maturation.

Functional MYB transcription factor gene HtMYB2 is associated with anthocyanin biosynthesis in Helianthus tuberosus L

BackgroundTuber color is an important trait for Helianthus tuberosus L. (Jerusalem artichoke). Usually, purple tubers with high anthocyanin content are more nutritious than white tuber. But, the molecular mechanism underlying it is unknown.ResultsIn the current study, high-throughput RNA-sequencing was used to compare the transcriptomes between plants with tubers with red or white epidermis. Compared with the white-skinned tubers of cultivar QY3, anthocyanin biosynthesis structural genes had greater expression in the red-skinned tubers of cultivar QY1, indicating that the anthocyanin biosynthesis pathway was activated in ‘QY1’; quantitative PCR confirmed this difference in expression. HtMYB2 (Unigene44371_All) was the only MYB transcription factor, homologous to the MYB transcription factor regulating anthocyanin biosynthesis, expressed in the red tuber epidermis of ‘QY1’. The anthocyanin concentration in the root, stem, leaf, flower, and tuber epidermis of ‘QY1’ was higher than in ‘QY3’, especially tuber epidermis. Correspondingly, HtMYB2 had greater expression in these tissues of ‘QY1’ than in ‘QY3’. The expression of HtMYB2 was associated with anthocyanin accumulation in the different tissues. Overexpression of HtMYB2 activated the anthocyanin biosynthesis pathway, accumulating the pigment in leaves of transgenic tobacco, supporting the model that HtMYB2 regulated anthocyanin biosynthesis. Further experiments found that HtMYB2 had the same coding sequence and genomic sequence in ‘QY1’ and ‘QY3’, but that there were several single nucleotide polymorphisms and one insertion–deletion (indel) mutation of 21 nucleotides in the promoter region between the two alleles. The deletion of three nucleotides “AAA” made the promoter of ‘QY1’ predicted to contain one more possible promoter region. A specific primer, based on the indel, could differentiate between cultivars with red or white tuber epidermis. The genetic variation in HtMYB2 was associated with the tuber skin color in a natural population.ConclusionsRNA-seq can successfully isolate the candidate gene (HTMYB2) controlling anthocyanin biosynthesis in purple epidermis of Jerusalem artichoke tuber. HTMYB2 can regulate anthocyanin biosynthesis in plants and is closely related to the formation of purple phenotype in tubers. This study should be useful in understanding the genetic mechanism underlying different tuber skin colors and in breeding new H. tuberosus cultivars with different tuber skin colors.

Functional and correlation analyses of dihydroflavonol-4-reductase genes indicate their roles in regulating anthocyanin changes in Ginkgo biloba

Anthocyanins, a class of flavonoids, are synthesized in a branch of the flavonoid pathway. Dihydroflavonol-4-reductase (DFR) catalyzes the first reaction leading to anthocyanins, and it is considered a key regulatory enzyme of anthocyanin biosynthesis in plants. In this research, we isolated three new DFR genes by searching the whole-genomic sequence data of Ginkgo biloba. The genes have similar structures and are clustered in the same scaffold. Sequence analyses of GbDFR proteins revealed that these GbDFRs have a closer relationship to Arabidopsis thaliana DFRs than previously identified GbDFRs. Expression analyses revealed that GbDFR4 and GbDFR6 were preferentially expressed in leaves and fruit, respectively. Young and mature leaves shared similar gene expression patterns, while fruit harvested in May had significantly higher expression levels of GbDFRs than fruit harvested in June. Transgenic tobacco plants ectopically overexpressing GbDFR genes showed higher DFR enzymatic activity levels, higher anthocyanin contents and darker flowers than the control. Ginkgo seedlings treated with NaCl showed increased total flavonoid and anthocyanin contents, and the expression levels of GbDFR genes were also induced. In contrast, UV-B irradiation increased the total flavonoid content but decreased both the anthocyanin content and GbDFR expression levels. A salicylic acid treatment induced and inhibited the total flavonoid content with and without light, respectively. In contrast, both the GbDFR expression levels and the anthocyanin content were inhibited by the salicylic acid treatment regardless of the light conditions. This research validated the functions of GbDFRs and expands our understanding of the environmental stress-response mechanisms of ginkgo trees.

Identification and expression analysis of BrTT8 during anthocyanin biosynthesis and exposure to abiotic stress in turnip (Brassica rapa subsp. rapa ‘Tsuda’)

Transparent Testa 8 (TT8) is an important transcription factor in regulatory networks controlling development, metabolism and stress responses in plants. However, their molecular mechanism associated with anthocyanin biosynthesis in turnip remains largely unknown. In this study, BrTT8 (HQ337791) was isolated from ‘Tsuda’ turnip. A quantitative PCR analysis found the highest expression of BrTT8 in the red root epidermis of turnip and revealed that BrTT8 expression exhibited tissue specificity. BrTT8 expression in swollen hypocotyls was induced by UV-A light. A yeast two-hybrid assay revealed that BrMYB75 (HQ337792), a member of the set of R2R3-myb/bHLH complexes that regulate anthocyanin biosynthesis in plants, was localized in the nucleus and interacted with BrTT8. The transcript abundance of BrMYB75 in swollen hypocotyls increased after treatment with UV-A light. The cotransformation of BrTT8 and BrMYB75 in tomato led to enhanced anthocyanin biosynthesis. These results suggest that BrTT8, as a key transcription factor, might play a crucial role in UV-A-induced anthocyanin biosynthesis and also reveal that the BrTT8 gene is regulated by abiotic stresses, such as high- and low-temperature, hormone, osmotic and salt stresses.

CBFs Function in Anthocyanin Biosynthesis by Interacting with MYB113 in Eggplant (Solanum melongena L.).

Eggplant is rich in anthocyanins. R2R3-MYB transcription factors play a key role in the anthocyanin pathway. Low temperature is vital abiotic stress that affects the anthocyanin biosynthesis in plants. CBFs (C-repeat binding factors) act as central regulators in cold response. In this study, we found that SmCBF1, SmCBF2 and SmCBF3, via their C-terminal, physically interacted with SmMYB113, a key regulator of anthocyanin biosynthesis in eggplant. SmCBF2 and SmCBF3 upregulated the expression of SmCHS and SmDFR via a SmMYB113-dependent pathway. In addition, the transient expression assays demonstrated that co-infiltrating SmCBFs and SmMYB113 significantly improved the contents of anthocyanin and the expression levels of anthocyanin structural genes in tobacco. When SmTT8, a bHLH partner of SmMYB113, coexpressed with SmCBFs and SmMYB113, the anthocyanin contents were significantly enhanced compared with SmCBFs and SmMYB113. Furthermore, overexpression of SmCBF2 and SmCBF3 could facilitate the anthocyanin accumulation under cold conditions in Arabidopsis. Taken together, these results shed light on the functions of SmCBFs and potential mechanisms of low-temperature-induced anthocyanin biosynthesis in eggplant.

Ectopic expression of the Anthurium andraeanum (Hort.) R2R3-MYB genes AaMYB4 and AaMYB5 enhance the flower color in transgenic tobacco

Anthocyanin coloration is crucially important for ornamental traits in Anthurium andraeanum. However, the molecular mechanisms responsible for anthocyanin accumulation in this important ornamental plant are poorly understood. Here, we isolated and functionally characterized two R2R3-MYB transcription factor genes AaMYB4 and AaMYB5 from A. andraeanum. Analysis of amino acid sequences indicate that both AaMYB4 and AaMYB5 share homology with the C1 subgroup R2R3-MYBs from monocots. AaMYB4 and AaMYB5 proved to be nucleus-localized proteins. Spatial and temporal expression analysis of AaMYB4 and AaMYB5 suggested that AaMYB4 was mainly expressed in the spathe and young leaves, whereas AaMYB5 was mainly expressed in the young leaves and the spadix. Although the expression patterns of AaMYB4 and AaMYB5 did not correlate with anthocyanin accumulation in the spathe and spadix of cultivar Vitara and Topical, they were positively correlated with anthocyanin concentration in the developing leaves of ‘Tropical’ and were coordinately expressed with key anthocyanin biosynthesis pathway genes. The ectopic expression of AaMYB4 and AaMYB5 in tobacco triggered the accumulation of anthocyanins in the corolla limbs of T1 transgenic progeny, and induced the up-regulation of expression of the key enzyme genes in the anthocyanin biosynthesis pathway of tobacco. Overexpression of AaMYB5 in tobacco also markedly up-regulated the expression of the endogenous MYB regulator gene NtAn2 involved in anthocyanin biosynthesis. Our findings suggest that both AaMYB4 and AaMYB5 from A. andraeanum are involved in the regulation of anthocyanin biosynthesis and have potential value in the metabolic engineering of anthocyanin biosynthesis in plants. AaMYB4 and AaMYB5 are R2R3-MYB transcription factors from Anthurium andraeanum. Overexpression of the two genes in tobacco resulted in large anthocyanin accumulation in the corolla limbs.