Anthocyanin Biosynthesis Genes Research Articles

Transcriptome analysis identifies key genes involved in anthocyanin biosynthesis in black and purple fruits (Lycium ruthenicum Murr. L)

Lycium ruthenicum is a characteristic plant resource in Northwest China. The fruit with high anthocyanin content is the main reason for its high nutritional value and economic value, while the regulation of key genes regulating anthocyanin biosynthesis in black fruit is not clear. In this study, high-throughput sequencing technology was used to compare the expression differences of all genes in black and purple fruits. The purple samples obtained 45.13 and 51.49 M Raw reads, while the black fruit samples measured 53.85 and 47.38 M Raw Reads. After splicing and assembly, 162,842 transcripts were obtained after removing redundant and duplicated results. Homology comparison of all predicted proteins found that 36,978 (22.43%) genes in Lycium ruthenicum were homologous to Solanum tuberosum, followed by 24,459 (14.84%) genes were homologous to Capsicum annuum. Overall, 3685 genes were up-regulated in purple fruit, 2837 genes were down-regulated in purple fruit, and the remaining 156320 genes were not differentially expressed in black and purple fruit. Through homology comparison of anthocyanin biosynthesis and metabolism structure genes in Lycium ruthenicum, 63 homologous genes, 4 MYB transcription factors and 5 bHLH transcription factors were screened. LrAN2p (909 bp) in purple fruit was 136 bp more upstream of ‘ATG’ than LrAN2 (774 bp) in black fruit. Three highly expressed copies of bHLH transcription factors AN1b with different lengths were screened in black fruit, but they were almost not expressed in purple fruit. This study possibly identified the major LrAN1b genes regulating the black fruit trait.

An integrated metabolome and transcriptome analysis of the Hibiscus syriacus L. petals reveal the molecular mechanisms of anthocyanin accumulation.

Hibiscus syriacus L. var. Shigyoku is a new double-flowered bluish-purple variety in China that changes color during flower development from bluish-purple to light purple. There is limited information on the anthocyanin accumulation patterns and associated transcriptome signatures in Shigyoku from D1 (bud) to open flower (D3). Here, we employed a combined transcriptome and metabolome approach to understanding the mechanism of this color change. Our results demonstrate that cyanidins, pelargonidins, delphinidins, petunidins, peonidins, and malvidins were differentially accumulated in Shigyoku petals. The anthocyanin biosynthesis started in D1, was significantly upregulated in D2 (semi-open flower), and reduced in D3. However, malvidins, pelargonidins, and peonidins could be associated with the bluish-purple coloration on D2. Their reduced accumulation in D3 imparted the light purple coloration to Shigyoku petals on D3. Significant contributions in the color change could be associated with the expression changes in anthocyanin biosynthesis genes i.e., LARs, ANSs, DFRs, UGT79B1, C3’Hs, 3ATs, and BZ1s. The UFGTs were associated with the higher accumulation of glycosylated anthocyanins in D2 and D3. Furthermore, the changes in the expressions of the MYB and bHLH transcription factors were consistent with the anthocyanin accumulation. Finally, we discussed the possible roles of Jasmonic acid, auxin, and gibberellic acid signaling in regulating the MBW complex. Taken together, we conclude that H. syriacus petal coloration is associated with anthocyanin biosynthesis genes, the MBW complex, and phytohormone signaling.

BnbHLH92a negatively regulates anthocyanin and proanthocyanidin biosynthesis in Brassica napus

Yellow seed trait is a desirable characteristic with potential for increasing seed quality and commercial value in rapeseed, and anthocyanin and proanthocyanidins (PAs) are major seed-coat pigments. Few transcription factors involved in the regulation of anthocyanin and PAs biosynthesis have been characterized in rapeseed. In this study, we identified a transcription factor gene BnbHLH92a (BnaA06T0441000ZS) in rapeseed. Overexpressing BnbHLH92a both in Arabidopsis and in rapeseed reduced levels of anthocyanin and PAs. Correspondingly, the expression profiles of anthocyanin and PA biosynthesis genes (TT3, BAN, TT8, TT18, and TTG1) were shown by quantitative real-time PCR to be inhibited in BnbHLH92a-overexpressing Arabidopsis seeds, indicating that BnbHLH92a represses the anthocyanin and PA biosynthesis pathway in Arabidopsis. BnbHLH92a physically interacts with the BnTTG1 protein and represses the biosynthesis of anthocyanins and PAs in rapeseed. BnbHLH92a also binds directly to the BnTT18 promoter and represses its expression. These results suggest that BnbHLH92a is a novel upstream regulator of flavonoid biosynthesis in B. napus.

Coleoptile Purple Line Regulated by A-P Gene System Is a Valuable Marker Trait for Seed Purity Identification in Hybrid Rice

In plants, a large number of anthocyanin biosynthetic genes encoding enzymes and regulatory genes encoding transcription factors are required for anthocyanin synthesis. Coleoptile purple lines are two purple lines on both sides of coleoptiles after seed germination. However, the molecular mechanism of coleoptile purple line is not clear in rice so far. In this study, two major dominant genes, coleoptile purple line 1 (OsCPL1, also known as OsC1) and coleoptile purple line 2 (OsCPL2), were isolated via map-based cloning, and both of them were required for anthocyanin biosynthesis of coleoptile purple line in rice. The knockout and complementation experiments confirmed that OsC1 was required for purple color in most organs, such as coleoptile line, sheath, auricle, stigma and apiculus, whereas OsCPL2 was just required for coleoptile purple line. OsC1 was predominantly expressed in coleoptiles, flag leaves, and green panicles, and highly expressed in young leaves, whereas OsCPL2 was predominantly expressed in coleoptiles, and extremely lowly expressed in the other tested organs. Loss-of-function of either OsC1 or OsCPL2 resulted in significant reduction of transcript levels of multiple anthocyanin biosynthesis genes in coleoptiles. Coleoptile purple line was further used as a marker trait in hybrid rice. Purity identification in hybrid rice seeds via coleoptile purple line just needed a little water, soil and a small plate and could be completed within 5 d. Molecular marker and field identification analyses indicated that coleoptile purple line was reliable for the hybrid seed purity identification. Our findings disclosed that coleoptile purple line in rice was regulated by two major dominant genes, OsC1 and OsCPL2, and can be used as a simple, rapid, accurate and economic marker trait for seed purity identification in hybrid rice.

Induced mutation in ELONGATED HYPOCOTYL5 abolishes anthocyanin accumulation in the hypocotyl of pepper

The causal gene, CaHY5 of a chemical induced green-hypocotyl mutant was identified by molecular mapping. CaHY5 regulates anthocyanin accumulation by directly binding to the promoter of genes in anthocyanin pathway. Morphological markers at seedling stage are useful indicators for F1 hybrid seeds screening. Pepper is a worldwide vegetable with diverse uses, and F1 hybrids are popular in the pepper industry. Hypocotyl color is a useful marker to identify F1 hybrid seeds. However, most pepper accessions have purple hypocotyl caused by anthocyanin accumulation, while greenhypocotyl pepper accessions are rare. In this study, we identified a greenhypocotyl mutant (e1898) from a pepper ethylmethanesulfonate (EMS) mutant library. By combining bulked segregant RNA-seq (BSR), genome resequencing and recombinant analysis, it was found that CaHY5 is the causal gene of this mutant. Virus-induced gene silencing (VIGS) of CaHY5 resulted in the decrease of anthocyanin accumulation in pepper hypocotyls. RNA-seq data showed that many genes related to anthocyanin biosynthesis and transport decreased significantly in the mutant. Yeast one-hybrid (Y1H) assays showed that CaHY5 can bind to the promoter of CaF3H, CaF3'5'H, CaDFR, CaANS and CaGST, which are important genes in anthocyanin biosynthesis or transport. Our results indicate that CaHY5 directly regulates anthocyanin biosynthesis and transport, thus governing anthocyanin accumulation in pepper hypocotyl. The mutant and gene identified in this work shall be valuable in the purity control of hybrid pepper seeds.

Mitotically heritable epigenetic modifications of CmMYB6 control anthocyanin biosynthesis in chrysanthemum

Flower color, which is determined by various chemical pigments, is a vital trait for ornamental plants, in which anthocyanin is a major component. However, the epigenetic regulation of anthocyanin biosynthesis remains poorly understood. During chrysanthemum cultivation, we found a heterochromatic chrysanthemum accession (YP) whose progeny generated by asexual reproduction contained both yellow-flowered (YP-Y) and pink-flowered (YP-P) plants. In this study, we aimed to elucidate the epigenetic mechanisms of different flower colors in the YP plant progeny. Metabolome and transcriptome analyses revealed that the difference in flower color between YP-Y and YP-P was caused by expression variation of the anthocyanin biosynthesis gene CmMYB6. Bisulfite sequencing revealed that methylation at the CmMYB6 promoter, especially in the CHH context, was higher in YP-Y than YP-P. After demethylation of the CmMYB6 promoter using the dCas9-TET1cd system, the flower color returned from yellow to pink. Furthermore, the methylation status of the CmMYB6 promoter was higher in YP-Y over three consecutive generations, indicating that this methylation status was heritable mitotically. Finally, investigation of other chrysanthemum cultivars showed that the methylation of CmMYB6 decreased gradually with the increase in anthocyanin content. These results lay an epigenetic foundation for the improvement of flower color in horticultural plants.

The chromosome-scale genome sequence of Triadica sebifera provides insight into fatty acids and anthocyanin biosynthesis

The Chinese tallow tree (Triadica sebifera) can produce oil with high content of unsaturated fatty acids in seeds and shows attractive leaf color in autumn and winter. Here, the 739 Mb chromosome-scale genome sequence of the Chinese tallow tree was assembled and it reveals the Chinese tallow tree is a tetraploid. Numerous genes related to nutrition assimilation, energy utilization, biosynthesis of secondary metabolites and resistance significantly expanded or are specific to the Chinese tallow tree. These genes would enable the Chinese tallow tree to obtain high adaptability. More genes in fatty acids biosynthesis in its genome, especially for unsaturated fatty acids biosynthesis, and higher expression of these genes in seeds would be attributed to its high content of unsaturated fatty acids. Cyanidin 3-O-glucoside was identified as the major component of anthocyanin in red leaves. All structural genes in anthocyanin biosynthesis show significantly higher expression in red leaves than in green leaves. Transcription factors, seven MYB and one bHLH, were predicted to regulate these anthocyanin biosynthesis genes. Collectively, we provided insight into the polyploidization, high adaptability and biosynthesis of the high content of unsaturated fatty acids in seeds and anthocyanin in leaves for the Chinese tallow tree.

Purple stem Brassica napus exhibits higher photosynthetic efficiency, antioxidant potential and anthocyanin biosynthesis related genes expression against drought stress.

Purple-stem Brassica napus (B. napus) is a phenotype with unique color because of its high anthocyanins content. Anthocyanins are naturally occurring plant pigments that have antioxidants activity and play important role in plant defense against abiotic and biotic stresses. In the present study, drought induced effects on plants were investigated in hydroponically grown seedlings of green stem (GS) and purple stem (PS) genotypes of B. napus. The results of this study showed that the major function of anthocyanins accumulation during drought was to enhance the antioxidant capability and stress tolerance in B. napus plants. Our results showed that drought significantly inhibited the plant growth in terms of decreased biomass accumulation in both genotypes, although marked decline was observed in GS genotype. The reduction in photosynthetic attributes was more noticeable in the GS genotype, whereas the PS genotype showed better performance under drought stress. Under stressful conditions, both the genotype showed excessive accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as higher levels of antioxidant enzymes activities. Under drought conditions, the GS genotype showed apparent damages on chloroplast deformation like in thylakoid membrane and grana structural distortion and fewer starch grains and bigger plastoglobuli. Moreover, during drought stress, the PS genotype exhibited maximum expression levels of anthocyanins biosynthesis genes and antioxidant enzymes accompanied by higher stress tolerance relative to GS genotype. Based on these findings, it can be concluded that GS genotype found more sensitive to drought stress than the PS genotype. Furthermore this research paper also provides practical guidance for plant biologists who are developing stress-tolerant crops by using anthocyanin biosynthesis or regulatory genes.

Uncovering Hierarchical Regulation among MYB-bHLH-WD40 Proteins and Manipulating Anthocyanin Pigmentation in Rice.

Anthocyanins accumulate in various organs of rice, and the regulatory genes involved in pigmentation of specific organs, such as pericarp, hull, leaf, apiculus, and stigma have been elucidated. However, the corresponding gene for rice culm pigmentation has not been clarified. The well-known MYB-bHLH-WD40 (MBW) complex plays vital role in regulating the anthocyanin biosynthesis pathway in plants. However, the core members of MBW and the hierarchical regulation between these members are not fully elucidated in rice. Here, by map-based cloning, we identified the culm-specific pigmentation gene S1 whose alleles are also known for hull/pericarp pigmentation. We also clarified that one WD40 protein encoding gene, WA1, is indispensable for anthocyanin biosynthesis in rice. In the cascading regulation among MBW members, S1 (bHLH) acts as the master gene by activating the expression of C1 (MYB), and then C1 activates the expression of WA1 (WD40), which is unique in plant species. This enables MBW members to be coordinated in a common way to efficiently regulate anthocyanin biosynthesis genes. Based on these studies, we explored the minimal gene set required for anthocyanin biosynthesis in rice. These findings will help us design new rice varieties with anthocyanin accumulation in specific organs as needed.

Genetic and multi-omics analyses reveal BnaA07.PAP2In-184-317 as the key gene conferring anthocyanin-based color in Brassica napus flowers.

The molecular mechanisms underlying anthocyanin-based flower coloration remain unknown in Brassica napus. To identify the key genes and metabolites associated with apricot and pink flower colors, metabolome, BSA-seq, and RNA-seq analyses were conducted on apricot-, pink-, yellow-, and white-flowered F2B. napus. Yellow carotenoids and red anthocyanins were abundant in apricot petals, while colorless carotenoids and red anthocyanins accumulated in pink petals. Most carotenoid genes were not differentially regulated between apricot and yellow or between pink and white petals. Three regulator genes, BnaMYBL2, BnaA07.PAP2, and BnaTT8, and structural genes in anthocyanin biosynthesis were dramatically enhanced in apricot and pink petals in comparison with yellow and white petals. Map-based cloning revealed that BnaA07.PAP2 is responsible for anthocyanin-based flower color and encodes a nucleus-localized protein predominantly expressed in apricot and pink flowers. Two insertions in the promoter region are responsible for the transcriptional activation of BnaA07.PAP2 in flowers. Introducing the BnaA07.PAP2In-184-317 allele broadly activated the expression of anthocyanin-related genes and promoted anthocyanin accumulation in flowers, yielding color change from yellow to apricot. These findings illustrate the genetic basis of anthocyanin-based flower coloration and provide a valuable genetic resource for breeding varieties with novel flower colors in B. napus.

Mitigating chilling injury of pomegranate fruit skin

Pomegranate cv. Wonderful fruit are susceptible to chilling injury of the peel (CIp) when stored at 7 °C in modified-atmosphere bags for more than 3 months. The damage, manifested as superficial browning, appears first on the fruit skin, i.e., the outer colored layer of the peel. We previously suggested that susceptibility to CIp is associated with downregulation of PHENYLALANINE AMMONIA-LYASE (PAL) in storage, whereas POLYPHENOL OXIDASE (PPO) expression is upregulated. To further explore the role of PPO and PAL in CIp disorder, we monitored their gene expression and enzyme activity in fruit skin collected at three phases of fruit development: premature fruit 7 days before harvest when the fruit is susceptible to CIp disorder; during commercial harvest when fruit skin is aging; and during the following 4 months of storage. In addition, prestorage washing of the fruit with CaCl2 and citric acid solutions, previously shown to reduce CIp incidence in pomegranate and other fruit and to manipulate the PAL-to-PPO ratio, was applied. We determined total phenol content and antioxidative capacity of fruit skin during cold storage, and analyzed the expression of antioxidation-related genes CATALASE, SUPEROXIDE DISMUTASE, and GLUTATHIONE REDUCTASE, and key anthocyanin-biosynthesis genes CHALCONE SYNTHASE, CHALCONE ISOMERASE, and DIHYDROFLAVONOL 4-REDUCTASE. Results suggested that increased skin susceptibility to CIp is related to high total phenol content, relatively high PAL activity/gene expression, and low anthocyanin levels. Although PPO activity and gene expression increased in cold storage, this pattern was observed in all tissues tested, irrespective of their susceptibility to CIp development. Similarly, no association was evident between antioxidation-related genes’ expression levels and CIp susceptibility. On the other hand, pre-storage treatments with CaCl2 and citric acid mainly decreased CIp incidence associated with increased antioxidation-related gene expression. Hence, the mechanism governing mitigation of CIp development by prestorage washing with CaCl2 and citric acid differs from that observed in native CIp-resistant pomegranate skin.

The Coordinated Action of MYB Activators and Repressors Controls Proanthocyanidin and Anthocyanin Biosynthesis in Vaccinium.

Vaccinium berries are regarded as “superfoods” owing to their high concentrations of anthocyanins, flavonoid metabolites that provide pigmentation and positively affect human health. Anthocyanin localization differs between the fruit of cultivated highbush blueberry (V. corymbosum) and wild bilberry (V. myrtillus), with the latter having deep red flesh coloration. Analysis of comparative transcriptomics across a developmental series of blueberry and bilberry fruit skin and flesh identified candidate anthocyanin regulators responsible for this distinction. This included multiple activator and repressor transcription factors (TFs) that correlated strongly with anthocyanin production and had minimal expression in blueberry (non-pigmented) flesh. R2R3 MYB TFs appeared key to the presence and absence of anthocyanin-based pigmentation; MYBA1 and MYBPA1.1 co-activated the pathway while MYBC2.1 repressed it. Transient overexpression of MYBA1 in Nicotiana benthamiana strongly induced anthocyanins, but this was substantially reduced when co-infiltrated with MYBC2.1. Co-infiltration of MYBC2.1 with MYBA1 also reduced activation of DFR and UFGT, key anthocyanin biosynthesis genes, in promoter activation studies. We demonstrated that these TFs operate within a regulatory hierarchy where MYBA1 activated the promoters of MYBC2.1 and bHLH2. Stable overexpression of VcMYBA1 in blueberry elevated anthocyanin content in transgenic plants, indicating that MYBA1 is sufficient to upregulate the TF module and activate the pathway. Our findings identify TF activators and repressors that are hierarchically regulated by SG6 MYBA1, and fine-tune anthocyanin production in Vaccinium. The lack of this TF module in blueberry flesh results in an absence of anthocyanins.

The apple BTB protein MdBT2 positively regulates MdCOP1 abundance to repress anthocyanin biosynthesis.

The ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) plays a central role in light-induced anthocyanin biosynthesis. However, the upstream regulatory factors of COP1 remain poorly understood, particularly in horticultural plants. Here, we identified an MdCOP1-interacting protein, BROAD-COMPLEX, TRAMTRACK AND BRIC A BRAC2 (MdBT2), in apple (Malus domestica). MdBT2 is a BTB protein that directly interacts with and stabilizes MdCOP1 by inhibiting self-ubiquitination. Fluorescence observation and cell fractionation assays showed that MdBT2 increased the abundance of MdCOP1 in the nucleus. Moreover, a series of phenotypic analyses indicated that MdBT2 promoted MdCOP1-mediated ubiquitination and degradation of the MdMYB1 transcription factor, inhibiting the expression of anthocyanin biosynthesis genes and anthocyanin accumulation. Overall, our findings reveal a molecular mechanism by which MdBT2 positively regulates MdCOP1, providing insight into MdCOP1-mediated anthocyanin biosynthesis.

Comparative RNA-Seq analysis to understand anthocyanin biosynthesis and regulations in Curcuma alismatifolia

Abstract Anthocyanins play a critical role in flower colour pattern formation, and their biosynthesis is typically regulated by transcription factors (TFs). Curcuma alismatifolia is a well-known ornamental plant with colourful flowers. However, little is known about the genes that regulate anthocyanin accumulation in C. alismatifolia. In the present study, high-quality RNA was extracted from three flowering stages of ‘Dutch Red’ and the blossoming stage of ‘Chocolate’. In all, 576.45 Mb clean data and 159,687 de-redundant sequences were captured. The Kyoto Encyclopedia of Genes and Genomes analysis showed that the pathways of phenylpropanoid biosynthesis, flavonoid biosynthesis, flavone and flavonol biosynthesis, and terpenoid backbone biosynthesis were the most enriched. Thirty unique isoforms were annotated as encoding enzymes or TFs involved in anthocyanin biosynthesis. Further analysis showed that the up-regulation of anthocyanin biosynthesis genes was associated with the red colour formation of ‘Dutch Red’, and their expression was induced at the initial flowering stage. The gene flavonoid 3′, 5′-hydroxylase, a key enzyme in the formation of delphinidin-based anthocyanins, reduced expression in ‘Chocolate’. In addition, we identified totally 14 TFs including 11 MYB proteins and 3 WD proteins, which might play important roles in the regulation of anthocyanin biosynthesis. The quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) results were generally consistent with the high-throughput sequencing results. Together, the results of our study provide a valuable resource for the regulatory mechanism of anthocyanin biosynthesis in C. alismatifolia and for the breeding of Curcuma cultivars with novel and charming flower colours.

Metabolome and transcriptome profiling unveil the mechanisms of light-induced anthocyanin synthesis in rabbiteye blueberry (vaccinium ashei: Reade)

BackgroundBlueberry is one of the most important fruit crops worldwide. Anthocyanin is an important secondary metabolites that affects the appearance and nutritive quality of blueberries. However, few studies have focused on the molecular mechanism underlying anthocyanin accumulation induced by light intensity in blueberries.ResultsThe metabolic analysis revealed that there were 134 significantly changed metabolites in the natural light compared to the control, and flavone, flavonol, and anthocyanins were the most significantly increased. Transcriptome analysis found 6 candidate genes for the anthocyanin synthesis pathway. Quantitative reverse transcription PCR (qRT-PCR) results confirmed changes in the expression levels of genes encoding metabolites involved in the flavonoid synthesis pathways. The flavonoid metabolic flux in the light intensity-treatment increased the accumulation of delphinidin-3-O-arabinoside compared to under the shading-treatment. Furthermore, we performed qRT-PCR analysis of anthocyanin biosynthesis genes and predicted that the gene of VcF3’5’H4 may be a candidate gene for anthocyanin accumulation and is highly expressed in light intensity-treated fruit. Through the co-expression analysis of transcription factors and anthocyanin synthesis pathway genes, we found that the VcbHLH004 gene may regulate VcF3’5’H4, and then we transformed VcbHLH004 heterologously into tomato to verify its function.ConclusionThese results provide novel insights into light intensity regulation of blueberry anthocyanin accumulation and represent a valuable data set to guide future functional studies and blueberry breeding.

RNA-Seq Analysis Identifies Transcription Factors Involved in Anthocyanin Biosynthesis of 'Red Zaosu' Pear Peel and Functional Study of PpPIF8.

Red-skinned pears are favored by people for their attractive appearance and abundance of anthocyanins. However, the molecular basis of anthocyanin biosynthesis in red pears remains elusive. Here, a comprehensive transcriptome analysis was conducted to explore the potential regulatory mechanism of anthocyanin biosynthesis in ‘Red Zaosu’ pear (Pyrus pyrifolia × Pyrus communis). Gene co-expression analysis and transcription factor mining identified 263 transcription factors, which accounted for 6.59% of the total number of transcription factors in the pear genome in two gene modules that are highly correlated with anthocyanin biosynthesis. Clustering, gene network modeling with STRING-DB, and local motif enrichment analysis (CentriMo) analysis suggested that PpPIF8 may play a role in anthocyanin biosynthesis. Furthermore, eight PIFs were identified in the pear genome, of which only PpPIF8 was rapidly induced by light. Functional studies showed that PpPIF8 localizes in the nucleus and is preferentially expressed in the tissue of higher levels of anthocyanin. The overexpression of PpPIF8 in pear peel and pear calli promotes anthocyanin biosynthesis and upregulates the expression of anthocyanin biosynthesis genes. Yeast-one hybrid and transgenic analyses indicated that PpPIF8 binds to the PpCHS promoter to induce PpCHS expression. The positive effect of PpPIF8 on anthocyanin biosynthesis is different from previously identified negative regulators of PyPIF5 and MdPIF7 in pear and apple. Taken together, our data not only provide a comprehensive view of transcription events during the coloration of pear peel, but also resolved the regulatory role of PpPIF8 in the anthocyanin biosynthesis pathway.

Why Black Flowers? An Extreme Environment and Molecular Perspective of Black Color Accumulation in the Ornamental and Food Crops.

Pollinators are attracted to vibrant flower colors. That is why flower color is the key agent to allow successful fruit set in food or ornamental crops. However, black flower color is the least attractive to pollinators, although a number of plant species produce black flowers. Cyanidin-based anthocyanins are thought to be the key agents to induce black color in the ornamental and fruit crops. R2R3-MYB transcription factors (TFs) play key roles for the tissue-specific accumulation of anthocyanin. MYB1 and MYB11 are the key TFs regulating the expression of anthocyanin biosynthesis genes for black color accumulation. Post-transcriptional silencing of flavone synthase II (FNS) gene is the technological method to stimulate the accumulation of cyanidin-based anthocyanins in black cultivars. Type 1 promoter of DvIVS takes the advantage of FNS silencing to produce large amounts of black anthocyanins. Exogenous ethylene application triggers anthocyanin accumulation in the fruit skin at ripening. Environment cues have been the pivotal regulators to allow differential accumulation of anthocyanins to regulate black color. Heat stress is one of the most important environmental stimulus that regulates concentration gradient of anthocyanins in various plant parts, thereby affecting the color pattern of flowers. Stability of black anthocyanins in the extreme environments can save the damage, especially in fruits, caused by abiotic stress. White flowers without anthocyanin face more damages from abiotic stress than dark color flowers. The intensity and pattern of flower color accumulation determine the overall fruit set, thereby controlling crop yield and human food needs. This review paper presents comprehensive knowledge of black flower regulation as affected by high temperature stress, and the molecular regulators of anthocyanin for black color in ornamental and food crops. It also discusses the black color-pollination interaction pattern affected by heat stress for food and ornamental crops.

Integrated Metabolomic and Transcriptomic Analyses Reveal Novel Insights of Anthocyanin Biosynthesis on Color Formation in Cassava Tuberous Roots.

Yellow roots are of higher nutritional quality and better appearance than white roots in cassava, a crucial tropical and subtropical root crop. In this work, two varieties with yellow and white cassava roots were selected to explore the mechanisms of color formation by using comparative metabolome and transcriptome analyses during seven developmental stages. Compared with the white-rooted cassava, anthocyanins, catechin derivatives, coumarin derivatives, and phenolic acids accumulated at higher levels in yellow-rooted cassava. Anthocyanins were particularly enriched and displayed different accumulation patterns during tuberous root development. This was confirmed by metabolic comparisons between five yellow-rooted and five white-rooted cassava accessions. The integrative metabolomic and transcriptomic analysis further revealed a coordinate regulation of 16 metabolites and 11 co-expression genes participating in anthocyanin biosynthesis, suggesting a vital role of anthocyanin biosynthesis in yellow pigmentation in cassava tuberous roots. In addition, two transcriptional factors, i.e., MeMYB5 and MeMYB42, were also identified to co-express with these anthocyanin biosynthesis genes. These findings expand our knowledge on the role of anthocyanin biosynthesis in cassava root color formation, and offer useful information for the genetic breeding of yellow-rooted cassava in the future.

Genome-wide identification and characterization of bZIP gene family and cloning of candidate genes for anthocyanin biosynthesis in pomegranate (Punica granatum)

BackgroundThe basic leucine zipper (bZIP) transcription factor is one of the most abundant and conserved gene families in eukaryotes. In addition to participating in plant development and growth, bZIP transcription factors play crucial roles in various abiotic stress responses and anthocyanin accumulation. Up to now, analysis of bZIP gene family members in pomegranate (Punica granatum) has not been reported. Three published pomegranate genome sequences provide valuable resources for further gene function analysis.ResultsUsing bioinformatics analysis, 65 PgbZIPs were identified and analyzed from the ‘Taishanhong’ pomegranate genome. We divided them into 13 groups (A, B, C, D, E, F, G, H, I, J, K, M, and S) according to the phylogenetic relationship with those of Arabidopsis, each containing a different number of genes. The regularity of exon/intron number and distribution was consistent with the classification of groups in the evolutionary tree. Transcriptome analysis of different tissues showed that members of the PgbZIP gene family were differentially expressed in different developmental stages and tissues of pomegranate. Among them, we selected PgbZIP16 and PgbZIP34 as candidate genes which affect anthocyanin accumulation. The full-length CDS region of PgbZIP16 and PgbZIP34 were cloned from pomegranate petals by homologous cloning technique, encoding 170 and 174 amino acids, which were 510 bp and 522 bp, respectively. Subcellular localization assays suggested that both PgbZIP16 and PgbZIP34 were nucleus-localized. Real-time quantitative PCR (qPCR) was used to explore the expression of PgbZIP16 and PgbZIP34 in the petals of three kinds of ornamental pomegranates at the full flowering stage. The results demonstrated that the expression of PgbZIP16 in red petals was 5.83 times of that in white petals, while PgbZIP34 was 3.9 times. The results of transient expression in tobacco showed that consistent trends were observed in anthocyanin concentration and expression levels of related genes, which both increased and then decreased. Both PgbZIP16 and PgbZIP34 could promote anthocyanin accumulation in tobacco leaves. We obtained transgenic strains overexpressing PgbZIP16, and the histochemical staining for GUS activity showed that overexpressed PgbZIP16 seedlings were expressed in the stem. Transgenic experiments indicated that overexpression of PgbZIP16 significantly upregulated UF3GT, ANS and DFR genes in Arabidopsis and enhanced anthocyanin accumulation.ConclusionsThe whole genome identification, gene structure, phylogeny, gene cloning, subcellular location and functional verification of the pomegranate bZIP gene family provide a theoretical foundation for the functional study of the PgbZIP gene family and candidate genes for anthocyanin biosynthesis.

Color fading in lotus (Nelumbo nucifera) petals is manipulated both by anthocyanin biosynthesis reduction and active degradation

Flower color is a key trait that determines the ornamental quality of aquatic lotus (Nelumbo nucifera). Color fading significantly decreases the ornamental value of lotus flowers. However, the molecular mechanism underlying lotus petal discoloration remains largely unknown. Here, the anthocyanin content and global transcriptional profiling of lotus petals of cultivar ‘Qiusanse’ in four developmental stages were analyzed. Five anthocyanin components were detected, and the total anthocyanin content decreased as the petal color changed from red to nearly white. Moreover, the malondialdehyde (MDA) content and peroxidase (POD) activity increased during color fading. RNA-seq analysis revealed a total of 4,092 differentially expressed genes (DEGs) between petal developmental stages. Notably, oxidoreductase and hydrolase activity related genes were overrepresented in DEGs. The expression pattern of key anthocyanin biosynthesis genes including, CHS, F3H, ANS, UFGT, and transcription factor regulators, including MYBs, WRKYs and bHLHs were correlated with anthocyanin accumulation. Interestingly, DEGs associated with anthocyanin degradation and vacuolar pH regulation, including peroxidase, proton pumps regulators such as WRKY3 and MYB5-like, were significantly upregulated during the late stages of flowering. This study reveals for the first time the transcriptional dynamics during lotus petal discoloration. Our results suggest the involvement of anthocyanin biosynthesis repressors and degrading genes as well as pH regulators in controlling color fading of lotus petals. The study also provides valuable information and candidate genes for improving the lotus flower color.