Search

B-box zinc-finger transcription factor (BBX) plays important regulatory roles in plant secondary metabolism. Here, we identified 21 BBXs that could be further categorized into five subfamilies from Cerasus humilis. Two segmentally duplicated Subfamily IV members, ChBBX6 and ChBBX18, were found to share high homology with reported anthocyanin-related BBXs and express highly in fruits with high anthocyanins but low carotenoids contents. Their transient overexpression in apple and C. humilis fruits both led to significantly increased anthocyanins accumulation and significantly upregulated expression of anthocyanins-related genes. However, their overexpression resulted in decreased carotenoids accumulation and greatly upregulated the expression of carotenoids-related genes especially degradation-related genes. Additionally, their overexpression both greatly improved the ABA content in C. humilis fruits. Through yeast one-hybrid and dual-luciferase reporter assays, we found that both ChBBX6 and ChBBX18 could bind to and activate the promoters of chalcone synthase (ChCHS), flavanone 3-hydroxylase (ChF3H), and 9-cis-epoxycarotenoid dioxygenase 5 (ChNCED5). Our study demonstrates that ChBBX6 and ChBBX18 are positive regulators of anthocyanins biosynthesis and carotenoids degradation and can provide basis for understanding the roles of BBX genes in C. humilis., Competing Interests: Declaration of competing interest The authors declare no conflicts of interests., (Copyright © 2024. Published by Elsevier B.V.)

Pepper (Capsicum annuum L.) is widely consumed vegetables worldwide, and F 1 hybrids are highly sought after in the pepper seed industry. However, studies on gene mutations affecting the color of cotyledon are rare, and the same is true for peppers. In this study, a segregating population was developed by crossing the pepper accession 21C1344 with purple cotyledon and accession 21C912 with green cotyledon. Initially, a target genomic region was identified by screening polymorphic SSR markers distributed across 12 chromosomes. Subsequently, polymorphic markers were developed based on resequencing data from the two parental lines, and genetic linkage analysis was performed. This approach ultimately identified Capana10g001433 (CaMYB113) as the candidate gene responsible for the purple cotyledons. The gene mutation type in 21C912 represents a new mutation type distinct from the reported missense mutation types, and this mutation affects the biosynthesis of anthocyanins. Virus-induced gene silencing (VIGS) of CaMYB113 substantially decreased anthocyanin accumulation in the cotyledons. Subsequent overexpression of CaMYB113 resulted in purple callus and leaves of pepper, and changed the expression levels of downstream genes involved in anthocyanin synthesis. Yeast one-hybrid and dual-luciferase transient expression assays demonstrated the binding of CaMYB113 to anthocyanin biosynthesis-related genes, thereby regulating anthocyanin accumulation in pepper cotyledons., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Anthocyanidins and anthocyanins are one subclass of flavonoids in plants with diverse biological functions and have health-promoting effects. Dihydroflavonol 4-reductase (DFR) is one of the important enzymes involved in the biosynthesis of anthocyanidins and other flavonoids. Here, a new MOF-based nano-immobilized DFR enzyme acting as a nano-biocatalyst for the production of anthocyanidins in vitro was designed. We prepared UiO-66-NH 2 MOF nano-carrier and recombinant DFR enzyme from genetic engineering. DFR@UiO-66-NH 2 nano-immobilized enzyme was constructed based on covalent bonding under the optimum immobilization conditions of the enzyme/carrier ratio of 250 mg/g, 37 °C, pH 6.5 and fixation time of 10 min. DFR@UiO-66-NH 2 was characterized and its catalytic function for the synthesis of anthocyanidins in vitro was testified using UPLC-QQQ-MS analysis. Compared with free DFR enzyme, the enzymatic reaction catalyzed by DFR@UiO-66-NH 2 was more easily for manipulation in a wide range of reaction temperatures and pH values. DFR@UiO-66-NH 2 had better thermal stability, enhanced adaptability, longer-term storage, outstanding tolerances to the influences of several organic reagents and Zn 2+ , Cu 2+ and Fe 2+ ions, and relatively good reusability. This work developed a new MOF-based nano-immobilized biocatalyst that had a good prospect of application in the green synthesis of anthocyanins in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study investigates the transition of Rosa canina L. petals from pink to white, driven by genetic and biochemical factors. It characterizes the expression of ten key genes involved in anthocyanin and flavonoid biosynthesis across five developmental stages, correlating gene expression with flavonoid and anthocyanin concentrations and colorimetric changes. Initially, the petals exhibit a rich flavonoid profile, dominated by Rutin and Kaempferol derivatives. The peak anthocyanin concentration, corresponding to the deepest color saturation, occurs in the subsequent stage. Advanced chromatographic analyses identify key flavonoids persisting into the final white petal stage. Notably, the ANS gene shows a dramatic 137.82-fold increase in expression at the final stage, indicating its crucial role in petal color maturation despite the absence of visible pigmentation. The study provides a comprehensive characterization of the genetic and biochemical mechanisms underlying petal pigmentation, suggesting that reduced anthocyanin synthesis and increased flavonol concentration led to white petals. It also highlights the roles of other genes such as PAL, CCD1, FLS, CHI, CHS, UFGT, F3H, DFR, and RhMYB1, indicating that post-translational modifications and other regulatory mechanisms may influence anthocyanin stability and degradation., Competing Interests: Declaration of competing interest All the authors listed have agreed with the submission and declared no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Canna indica, which produce conspicuous and colorful flowers, are widely appreciated as ornamental plants. We used Pacific Biosciences sequencing (PacBio) and chromosome conformation capture (Hi-C) genome scaffolding to build a high-quality chromosome-scale genome assembly of C. indica and the genome assembly was 821Mb with a contig N50 of 48Mb assembled into nine chromosomes. The genome of C. indica was predicted to contain 31,130 genes and 30,816 genes were functionally annotated. Genome annotation identified 522 Mb (63.59 %) as repetitive sequences. Genome evolution analysis showed that whole-genome duplication occurred 53.4 million years ago. Transcriptome analysis revealed that petal coloration was linked with the expression of genes encoding enzymes involved in anthocyanin biosynthesis, carotenoid biosynthesis, and the methylerythritol phosphate (MEP) pathway. Furthermore, modules of co-expressed genes and hub genes were identified via weighted gene co-expression network analysis. These results suggested that, in Canna indica, deep red petal coloration was regulated by CHS2 and yellow petal coloration was associated with expression of ARF6 and NAC14. Considered together, the current study revealed a high-quality reference genome which may provide new insights into the molecular basis of flower coloration in Canna indica and help enhance the conservation and breeding of ornamental plants in general., Competing Interests: Declaration of competing interest Wen'e Zhang reports administrative support was provided by Guizhou Education Department of China., (Copyright © 2023. Published by Elsevier B.V.)

Pistacia chinensis is known for its biodiesel production. Several varieties of this plant have leaves that produce anthocyanin, which is responsible for their reddish coloration in autumn. This reddish hue is what makes them useful as ornamental plants. However, the mechanism of anthocyanin accumulation during autumn leaf coloration remains unclear. R2R3-MYB proteins reportedly regulated anthocyanin biosynthesis in many plant species. Here, we performed a genome-wide analysis and expression profiles of R2R3-MYB transcription factor in Pistacia. A total of 158 R2R3-MYB proteins were identified and grouped into 32 clades. Combining the data from RNA-seq and qRT-PCR, one key gene, EVM0016534, was screened and identified to have the highest correlation with anthocyanin accumulation. It was named PcMYB113 due to its sequence similarity to AtMYB113 and it could bind to the promoter of PcF3H. Furthermore, ectopic expression of PcMYB113 in Arabidopsis promoted the accumulation of anthocyanin in the seed coat, cotyledon, and mature leaves, thus confirming the function of PcMYB113 in anthocyanin biosynthesis. In addition, PcMYB113 had a specifically higher expression in senesced red leaves than in mature green leaves and young red leaves in P. chinensis, thereby suggesting the potential role of PcMYB113 in promoting anthocyanin biosynthesis during autumn leaf coloration. These findings enrich our understanding of the function of R2R3-MYB genes in anthocyanin biosynthesis and autumn leaf coloration., (Copyright © 2021. Published by Elsevier B.V.)

Auxin plays an important role in plant growth and development; for example, it regulates the elongation and division of plant cells, the formation of plantlet's geotropism and phototropism, and the growth of main lateral roots and hypocotyl. IAA gene is associated with auxin and can response to biotic and abiotic stress in plants. However, the regulatory effect of auxin on anthocyanin accumulation has been rarely reported. In this study, we show that auxin inhibites the accumulation of anthocyanin and decreases the expression of genes related to anthocyanin synthesis in calli, leaves, and seedlings of apple. The expression levels of MdIAA family genes were determined, and we found that MdIAA26 significantly responded to auxin, which also induced MdIAA26 degradation. Functional analysis of MdIAA26 showed that overexpressing MdIAA26 in apple calli and Arabidopsis could promote the accumulation of anthocyanin and up-regulate the genes related to anthocyanin synthesis. Furthermore, the MdIAA26-overexpressing Arabidopsis could counteract auxin-induced inhibition on anthocyanin accumulation, which indicates that auxin inhibits the accumulation of anthocyanin in apple by degrading MdIAA26 protein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The glutathione S-transferases (GSTs) are important enzymes of secondary metabolism in plants. In this study, two putative GSTs, GhGSTF1 and GhGSTF2, were identified as anthocyanin-related GSTs by the transcriptome data of the leaves of Gossypium hirsutum L. TM-1 and T586. The quantitative real-time PCR showed that GhGSTF1 and GhGSTF2 were highly expressed in red leaves and stems of Gossypium hirsutum L. T586. Orthologous genes of GhGSTF2 in two Gossypium barbadense L. 3-79 and Xinhai21 contain bases deletion in N-terminal (GbGSTF2a) and C-terminal (GbGSTF2b) respectively. Among which, GhGSTF1 and GhGSTF2 can restore pigmentation in hypocotyls of Arabidopsis thaliana mutant tt19-7 while GbGSTF2a and GbGSTF2b cannot. Furthermore, in vitro assays showed the recombinant GhGSTF1 and GhGSTF2 had Glutathione S-transferase activities. Fluorescence quenching assays showed that Cya could obviously quench the fluorescence of GhGSTF1, GhGSTF2, GbGSTF2a and GbGSTF2b to lower levels as compared to C3G. Moreover, the transient dual-luciferase assays showed that the promoters of GhGSTF1 and GhGSTF2 could be activated by GhPAP1D at different levels. GUS staining assays showed that their promoters have different activities to light. This study indicated that GhGSTF1 and GhGSTF2 play important roles in anthocyanin accumulation and the regulatory mechanism of anthocyanin accumulation in allotetraploid Gossypium are complicated., Competing Interests: Declaration of competing interest The authors confirm that this work is original and has not been published elsewhere, nor it is currently under consideration for publication elsewhere. The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Vacuole is a prominent organelle that often occupies most of the plant cell volume. The vacuolar accumulation of secondary metabolites, also called specialized metabolites, plays important roles in environmental responses such as protecting against insect herbivores and attracting pollinators. The compartmentation of xenobiotics in the vacuole is also essential for adaptation to environmental stresses. These accumulations involve several transport systems, for which some responsible transporter proteins have been reported. Furthermore, studies on biosynthetic enzymes and transporters of secondary metabolites have revealed that vacuoles, which have been recognized for many years as a site for accumulation, also function as a site for biosynthesis of secondary metabolites and are thus actively involved in the entire biosynthetic process. In this review, we briefly summarize recent findings on vacuolar transporters involved in secondary metabolites and xenobiotics, and discuss their roles in plant adaptation to biotic and abiotic stresses, through vacuolar dynamism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bisphenol A (BPA) is an estrogenic endocrine disruptor which disturbs a normal animal development. We generated an indicator plant that senses and provides a clear visual indicator of an estrogen-like compound BPA in the environment. We developed transgenic Arabidopsis lines expressing a construct designed to synthesize anthocyanin (thus showing a red color) in response to BPA. We transformed Arabidopsis with a recombinant vector containing the chimeric estrogen receptor (XVE region), LAP and coding region of PtrMYB119 (transcription factor involved in anthocyanin biosynthesis in poplar and Arabidopsis). Upon binding of the estrogen compound to the ligand-binding domain of E (estrogen receptor) in XVE, the XV domain binds to LAP promoter and triggering the transcription of PtrMYB119 with a subsequent enhancement of anthocyanin biosynthetic gene expression, resulting in anthocyanin synthesis. The leaves of the transgenic Arabidopsis line XVE-PtrMYB119 turned red in the presence of 10 ppm BPA. The transcript level of PtrMYB119 peaked at day 3 of BPA exposure, then decreased to its minimal level at day 5. Similar expression patterns to that of PtrMYB119 were detected for genes encoding the anthocyanin biosynthetic enzymes chalcone synthase, chalcone flavanone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase, and UFGT (UGT78D2). The leaves of transgenic plants did not turn red in response to BPA at concentrations below 10 ppm, but PtrMYB119 expression was induced by BPA at concentrations as low as 1 ppt BPA. Since this transgenic plant turns red in the presence of BPA without any experimental procedures, this line can be easily used by non-scientists., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Jasmonates (JAs) are signals in plant stress responses and development. One of the first observed and prominent responses to JAs is the induction of biosynthesis of different groups of secondary compounds. Among them are nicotine, isoquinolines, glucosinolates, anthocyanins, benzophenanthridine alkaloids, artemisinin, and terpenoid indole alkaloids (TIAs), such as vinblastine. This brief review describes modes of action of JAs in the biosynthesis of anthocyanins, nicotine, TIAs, glucosinolates and artemisinin. After introducing JA biosynthesis, the central role of the SCF COI1 -JAZ co-receptor complex in JA perception and MYB-type and MYC-type transcription factors is described. Brief comments are provided on primary metabolites as precursors of secondary compounds. Pathways for the biosynthesis of anthocyanin, nicotine, TIAs, glucosinolates and artemisinin are described with an emphasis on JA-dependent transcription factors, which activate or repress the expression of essential genes encoding enzymes in the biosynthesis of these secondary compounds. Applied aspects are discussed using the biotechnological formation of artemisinin as an example of JA-induced biosynthesis of secondary compounds in plant cell factories., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Anthocyanins are naturally occurring flavonoids derived from the phenylpropanoid pathway. There is increasing evidence of the preventative and protective roles of anthocyanins against a broad range of pathologies, including different cancer types and metabolic diseases. However, most of the fresh produce available to consumers typically contains only small amounts of anthocyanins, mostly limited to the epidermis of plant organs. Therefore, transgenic and non-transgenic approaches have been proposed to enhance the levels of this phytonutrient in vegetables, fruits, and cereals. Here, were review the current literature on the anthocyanin biosynthesis pathway in model and crop species, including the structural and regulatory genes involved in the differential pigmentation patterns of plant structures. Furthermore, we explore the genetic regulation of anthocyanin biosynthesis and the reasons why it is strongly repressed in specific cell types, in order to create more efficient breeding strategies to boost the biosynthesis and accumulation of anthocyanins in fresh fruits and vegetables., (Copyright © 2018. Published by Elsevier Ltd.)

Nitrogen (N) is a major nutrient of plants but often a limiting factor for plant growth and crop yield. To adapt to N deficiency, plants have evolved adaptive responses including accumulation of anthocyanins. However, it is still unclear whether the accumulated anthocyanins are part of the components of plant tolerance under low N stress. Here, we demonstrate that low N-induced anthocyanins contribute substantially to the low N tolerance of Arabidopsis thaliana. pap1-1, a mutant defective in MYB75 (PAP1), a MYB-type transcription factor that positively regulates anthocyanin biosynthesis in Arabidopsis, was found to have significantly decreased survival rate to low N stress compared to its wild-type plants. Similarly, tt3, a mutant with severe deficiency in dihydroflavonol 4-reductase (DFR), a key enzyme in anthocyanin biosynthesis, also showed much lower survival rate under low N stress. These results indicate that anthocyanins are substantial contributors of plant tolerance to low N stress. Furthermore, a metabolomics analysis using LC-MS revealed changes in flavonoid profile in the pap1-1 and tt3 plants, which established a causal relationship between plant adaptation to low N stress and these compounds including anthocyanins. Our results showed an important role of anthocyanins rather than flavonols in conferring plant tolerance to low N stress., (Copyright © 2018 Elsevier Inc. All rights reserved.)

High-quality perilla leaves are purple on upper and lower surfaces and have a good aroma. The Japanese Pharmacopoeia specifies the content of essential oils in perilla leaves but not that of anthocyanins. Several reports have described the chemical species of anthocyanins in red perilla, but a complete analysis of anthocyanins in perilla has not been reported. In this study, the anthocyanins in the leaves of cultivated and wild species of perilla and those in commercially available perilla herbs were studied. Red perilla and most P. citriodora strains accumulate cyanidin derivatives that differ in the acyl group on the glucose moiety at the 3-O- and 5-O-positions of the anthocyanins. Several strains of P. citriodora contain cyanidin derivatives that are different from those in red perilla and most P. citriodora species. Green perilla and wild species other than P. citriodora do not contain foliar anthocyanins. The anthocyanins in commercially available perilla herbs and natural dyes made from red perilla were in agreement with those in fresh red perilla leaves and most P. citriodora samples. The amounts and types of anthocyanins were not associated with place of cultivation, although some changes occurred due to degradation during storage. These results provide clues regarding the biosynthesis of anthocyanins in perilla and the evolution of red perilla. The characteristics and stability of anthocyanins are discussed., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

GLABRA3 (GL3), a bHLH transcription factor, has previously proved to be involved in anthocyanin biosynthesis and trichome formation in Arabidopsis, however, its downstream targeted genes are still largely unknown. Here, we found that GL3 was widely present in Arabidopsis vegetative and reproductive organs. New downstream targeted genes of GL3 for anthocyanin biosynthesis and trichome formation were identified in young shoots and expanding true leaves by RNA sequencing. GL3-mediated gene expression was tissue specific in the two biological processes. This study provides new clues to further understand the GL3-mediated regulatory network of anthocyanin biosynthesis and trichome formation in Arabidopsis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Unlabelled: A comparative proteomic approach was carried out to investigate anthocyanin biosynthesis in the tuberous roots of yellow sweet potato (YSP) and purple sweet potato (PSP) cultivars. More than 800 proteins were reproducibly detected through two-dimensional electrophoresis (2-DE), of which 50 proteins with 39 more and 11 less accumulated in PSP were identified through matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF-MS). Most of the analyzed proteins are annotated to be involved in starch metabolism and glycolysis. The more abundant starch phosphorylase (SP) and phosphoglucomutase (PGM) in PSP promoted the synthesis of precursors for anthocyanin synthesis. The results implied that starch degradation provided abundant substrates for anthocyanin biosynthesis in tuberous roots of PSP. 24kDa vacuolar protein (VP24) is related to anthocyanin transport and accumulation in vacuoles. Vacuole-associated annexin protein, VCaB42, is correlated with tonoplast biogenesis. Synergistic action of the two proteins is probably involved in the microautophagy and the intravacuolar trapping of anthocyanins. Interestingly, both VCaB42 and VP24 were more accumulated in PSP, suggesting that anthocyanins generated in the cytosol were transported into and became stored in the vacuoles of PSP. The present study provides new insights into the mechanism of tuberous root-specific anthocyanin accumulation in PSP., Biological Significance: Sweet potato ranks as the seventh most important crop worldwide. Purple sweet potato, a special sweet potato cultivar, has been extensively investigated because large amounts of anthocyanin accumulate in its tuberous roots. Anthocyanin is well known for its free radical-scavenging activity and beneficial effects on human health. Its biosynthetic pathway has been well characterized in model plants. Although large-scale systematic studies have been performed to identify the proteins present in sweet potato, information on the regulation of anthocyanin synthesis in sweet potato is insufficient. Our proteome study demonstrated that starch degradation may contribute to anthocyanin accumulation in purple sweet potato. To our knowledge, this study is the first to propose that starch degradation may provide precursors of anthocyanin biosynthesis in sweet potato., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Flavonoids are responsible of different fruit sensorial properties. In Japanese plum (Prunus salicina L.) these compounds are variable in both type and quantity during the different stages of fruit growth and maturation. Here we present the first study which determines the expression profile of structural genes of the flavonoid pathway and accumulation profiles of total phenols, proanthocyanidins and anthocyanins during fruit development stages in contrasting cultivars in Japanese plum. The biosynthesis of these compounds is differentially regulated in different tissues and cultivars. Our result showed that all pigmented tissues increased the expression of the leucoanthocyanidin dioxygenase (LDOX) gene, while all tissues without anthocyanin accumulation presented a minimal expression of LDOX. In addition, the regulation of putative transcription factors PsMYB10 and PsMYB1 were correlated positively and negatively with the pigmented tissues respectively, suggesting a critical and coordinated mechanism involved in the change of the fruit color., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Transcriptional activation of anthocyanin biosynthesis genes in vegetative tissues of monocotyledonous plants is mediated by cooperative activity of one component from each of the following two transcription factor families: MYB encoded by PURPLE PLANT1/COLORED ALEURONE1 (PL1/C1), and basic helix-loop-helix (bHLH) encoded by RED/BOOSTER (R1/B1). In the present study, putative PL cDNA was cloned from the wheat (Triticum aestivum) cultivar Iksan370, which preferentially expresses anthocyanins in coleoptiles. Phylogenetic tree analysis of deduced amino acid sequences showed that a putative TaPL1 is highly homologous to barley (Hordeum vulgare) HvPL1, but is distinct from wheat TaC1. Transgenic Arabidopsis thaliana stably expressing putative TaPL1 accumulated anthocyanin pigments in leaves and up-regulated structural genes involved in both early and late anthocyanin biosynthesis steps. TaPL1 transcript levels in Iksan370 were more prominent in vegetative tissues such as young coleoptiles than in reproductive tissues such as spikelets. TaPL1 expression was significantly up-regulated by environmental stresses including cold, salt, and light, which are known to induce anthocyanin accumulation. These combined results suggest that TaPL1 is an active positive regulator of anthocyanin biosynthesis in wheat coleoptiles., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Anthocyanins and proanthocyanidins (PAs), flavonoid-derived metabolites with different physiological roles, are produced by plants in a coordinated manner during fruit development by the action of transcription factors (TFs). These regulatory proteins have either an activating or repressing effect over structural genes from the biosynthetic pathway under their control. FaMYB1, a TF belonging to the R2R3-MYB family and isolated from commercial strawberry fruit (Fragaria×ananassa), was reported as a transcriptional repressor and its heterologous over-expression in tobacco flowers suppressed flavonoid-derived compound accumulation. FcMYB1, an ortholog of FaMYB1 isolated from the white Chilean strawberry (Fragaria chiloensis ssp. chiloensis f. chiloensis), showed higher transcript levels in white (F. chiloensis) than in red (F.×ananassa cv. Camarosa) fruits. In order to assess its contribution to the discolored phenotype in F. chiloensis, FcMYB1 was transiently down-regulated in planta using an RNAi-based approach. Quantitative real-time PCR on FcMYB1 down-regulated fruits resulted an up-regulation of anthocyanidin synthase (ANS) and a strong repression of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) transcript accumulation. In addition, these fruits showed increased concentrations of anthocyanins and undetectable levels of flavan 3-ols. Altogether, these results indicate a role for FcMYB1 in regulation of the branching-point of the anthocyanin/PA biosynthesis determining the discolored phenotype of the white Chilean strawberry fruit., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Monomeric anthocyanins and polymeric proanthocyanidins (condensed tannins) contribute to important plant traits such as flower and fruit pigmentation, fruit astringency, disease resistance and forage quality. Recent advances in our understanding of the transcriptional control mechanisms that regulate anthocyanin and condensed tannin formation in plants suggest new approaches for the engineering of quality traits associated with these molecules. In particular, MYB family transcription factors are emerging as central players in the coordinated activation of sets of genes specific for the anthocyanin and tannin pathways. Mutations in these genes underlie potentially valuable crop traits, and ectopic over- or under-expression of MYB transcription factors provides routes for engineering of these complex pathways., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Sugars enhance light signaling-induced anthocyanin accumulation in Arabidopsis seedlings via differential regulation of several positive and negative transcription factors. Ca(2+) plays a role as a second messenger in sugar signaling in grape and wheat. However, whether anthocyanin pigmentation is modulated by changes in intracellular Ca(2+) level in Arabidopsis is not known. Here, we used a pharmaceutical approach that Ca(2+) antagonists strongly interfered with sucrose uptake and anthocyanin accumulation by downregulating the expression of sucrose transporter 1 (SUC1) and transcriptional regulatory factors, such as PAP1. Time course analysis of the effect of Ca(2+) antagonists showed the early inhibition of sucrose-induced sugar uptake leading to decreased anthocyanin accumulation, indicating that Ca(2+) signals play a role in sugar uptake rather than in anthocyanin biosynthesis. An early increase in cytosolic Ca(2+) level in Arabidopsis roots in response to sucrose feeding was significantly inhibited by Ca(2+) antagonists. Taken together, these results indicate that sucrose-induced sugar uptake in Arabidopsis is modulated by changes in endogenous Ca(2+) levels, which in turn regulate anthocyanin accumulation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The effects of two synthetic elicitor indanoyl-isoleucine (In-Ile), N-linolenoyl-L-glutamine (Lin-Gln) and one biotic elicitor insect saliva (from Manduca sexta larvae) on plant cell cultures with respect to the induction of secondary metabolite production were investigated. Stimulated production of secondary metabolites, particularly anthocyanins in plant cells and phenolic acids in culture medium, was studied by using suspension culture of Vitis vinifera L. cv. Gamay Fréaux as a model system. In the treatments with In-Ile, the production of anthocyanins was enhanced 2.6-fold. In-Ile, Lin-Gln and saliva significantly elevated the accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol. The used elicitors did not suppress cell growth. Secondary metabolites were differently responsive to elicitation. 3-O-glucosyl-resveratrol was the predominant phenolic acid in V. vinifera cell culture, and its production was significantly stimulated by saliva, with 7.0-fold of the control level 24 h after treatment. The production of 4-(3,5-dihydroxy-phenyl)-phenol was significantly stimulated by In-Ile with 6.4-fold of the control level 24 h after treatment., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Difference in fruit pigmentation observed between two botanical forms of Fragaria chiloensis ssp. chiloensis (form chiloensis and form patagonica) was studied through transcriptional and chemical approaches. The proportion of different anthocyanins was demonstrated to be characteristic of each botanical form, with pelargonidin 3-glucoside being the most abundant in f. patagonica fruit and cyaniding 3-glucoside as the major one in f. chiloensis fruit. Partial gene sequences of the phenylpropanoid and flavonoid biosynthesis pathways were isolated from the native Chilean strawberry fruits, and used to design gene-specific primers in order to perform transcriptional analyses by qRT-PCR. These genes showed spatial, developmental, and genotypic associated patterns. The red fruit of f. patagonica exhibited higher transcript levels of anthocyanin-related genes and higher levels of anthocyanins compared to the barely pigmented fruit of f. chiloensis. The anthocyanin accumulation in F. chiloensis ssp. chiloensis fruits was concomitant with the particular progress of the transcriptional activity of genes involved in the biosynthesis of flavonoid pigments. The differences in anthocyanin contents, both in terms of type and quantity, between the two botanical forms of F. chiloensis ssp. chiloensis were coincident with the differential transcriptional patterns found in the anthocyanin-related genes., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The seed coats of black soybean (Glycine max (L.) Merr.) accumulate red (cyanidin-), blue (delphinidin-), purple (petunidin-), and orange (pelargonidin-based) anthocyanins almost exclusively as 3-O-glucosides; however, the responsible enzyme has not been identified. In this study, the full-length cDNA which encodes the enzyme that catalyzes the final step in anthocyanin biosynthesis, namely UDP-glucose:flavonoid 3-O-glucosyltransferase (UGT78K1), was isolated from the seed coat tissue of black soybean using rapid amplification of cDNA ends (RACE). Of the 28 flavonoid substrates tested, the purified recombinant protein glucosylated only anthocyanidins and flavonols, and demonstrated strict 3-OH regiospecificity. Galactose could also be transferred with relatively low activity to the 3-position of cyanidin or delphinidin in vitro. These findings are consistent with previous reports of mainly 3-O-glucosylated and minor amounts of 3-O-galactosylated anthocyanins in the seed coat of black soybean. The recombinant enzyme exhibited pronounced substrate inhibition by cyanidin at 100 microM acceptor concentration. Transfer of UGT78K1 into the Arabidopsis T-DNA mutant (ugt78d2) deficient in anthocyanidin and flavonol 3-O-glucosyltransferase activity, restored the accumulation of anthocyanins and flavonols, suggesting the in vivo function of the enzyme as a flavonoid 3-O-glucosyltransferase. Genomic and phylogenetic analyses suggest the existence of three additional soybean sequences with high similarity to UGT78K1. RT-PCR confirmed the co-expression of one of these genes (Glyma08g07130) with UGT78K1 in the seed coat of black soybean, suggesting possible functional redundancies in anthocyanin biosynthesis in this tissue., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Flavonols and conditionally also anthocyanins, aside from flavonols, are the predominant polyphenols accumulated in various tissues of the model plant Arabidopsis thaliana L. In vitro experiments suggested that the dioxygenases involved in their biosynthesis, flavonol synthase and anthocyanidin synthase, are "multifunctional" enzymes showing distinct side activities. The in vivo relevance of the additional activities attributed to these enzymes, however, has remained obscure. In this review we summarize the most recent results and present final proof of the complementing activities of these synthases for flavonol and anthocyanidin formation in the model plant A. thaliana. The impact of their modification on the biosynthetic pathway and the pattern of flavonoids in different plant tissues are discussed., (2010 Elsevier Ltd. All rights reserved.)

The single MYB-type protein, CAPRICE has long been thought to be a positive regulator of root hair development in Arabidopsis thaliana. To further probe the role of AtCPC and how conserved this role is across plant species, the AtCPC gene was constitutively overexpressed in tobacco (Nicotiana tabacum). Here, we show that flowers of transformants overexpressing AtCPC displayed an unexpected defection in pigmentation. A reduction in the level of anthocyanin was detected and the severity of abnormal flavonoid phenotype correlates well with the increased expression level of AtCPC gene. We also found that transcripts of three late biosynthetic genes were substantially down-regulated. Furthermore, AtCPC could interact with other known anthocyanin regulators. Our data suggest that in a heterologous host, AtCPC acts as a repressor of anthocyanin production, from which a new evidence for better understanding of the transcriptional regulation of flavonoid biosynthesis and, an implication for the development of new varieties of tobacco and other commercially important ornamental plants are provided.

This study assessed the impact of fruit temperature on the phenolic metabolism of grape berries (Vitis vinifera L. cv. Merlot) grown under field conditions with controlled exposure to sunlight. Individual cluster temperatures were manipulated in situ. Diurnal temperature fluctuation was damped by daytime cooling and nighttime heating of clusters. Daytime-only and nighttime-only temperature controls were applied for comparison. Berry temperatures were recorded continuously to compare the chemical data. Samples collected at véraison indicated that damping the diurnal temperature fluctuation advanced the onset of ripening. Those berries were larger (double-damped: 0.753+/-0.015gberry(-1) vs control: 0.512+/-0.034gberry(-1)) and more colored than all others. Development of phenolic metabolites was followed by two reversed-phase high performance liquid chromatography methods and gel permeation chromatography. These methods provided information on anthocyanins, proanthocyanidins, flavonols, flavan-3-ol monomers, and polymeric material. Damping the diurnal temperature fluctuation reduced proanthocyanidin mean degree of polymerization (double-damped: 21.8+/-1.0 vs control: 28.0+/-1.7). Proanthocyanidin accumulation at véraison was linearly related to heat summation over the developmental period with nighttime heating yielding the highest concentration and daytime cooling yielding the lowest (night-heat: 1.46+/-0.13mgberry(-1) vs day-cool: 0.97+/-0.09mgberry(-1)). Damping the diurnal temperature fluctuation had a marked effect on the rate of fruit development whereas total heat summation had more of an effect on phenolic metabolism alone. The results provide insight on the direct effect of temperature on phenolic metabolism.

Modern biotechnology has developed powerful tools for genetic engineering and flower colours are an excellent object to study possibilities and limitations of engineering strategies. Osteospermum hybrida became a popular ornamental plant within the last 20 years. Many cultivars display rose to lilac flower colours mainly based on delphinidin-derived anthocyanins. The predominant synthesis of delphinidin derivatives is referred to a strong endogenous flavonoid 3',5'-hydroxylase (F3'5'H) activity. Furthermore, since dihydroflavonol 4-reductase (DFR) of Osteospermum does not convert dihydrokaempferol (DHK) to leucopelargonidin, synthesis of pelargonidin-based anthocyanins is naturally not realised. In order to redirect anthocyanin biosynthesis in Osteospermum towards pelargonidin derivatives, we introduced cDNAs coding for DFRs which efficiently convert DHK to LPg. But neither the expression of Gerbera hybrida DFR nor of Fragaria x ananassa DFR - the latter is characterised by an unusual high substrate preference for DHK - altered anthocyanin composition in flowers of transgenic plants. However, chemical inhibition of F3'5'H activity in ray florets of dfr transgenic plants resulted in the accumulation of pelargonidin derivatives. Accordingly, retransformation of a transgenic plant expressing Gerbera DFR with a construct for RNAi-mediated suppression of F3'5'H activity resulted in double transgenic plants accumulating predominantly pelargonidin derivatives in flowers.

Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) catalyzes the reduction of dihydroflavonols to leucoanthocyanins, a key "late" step in the biosynthesis of anthocyanins. In this study we showed that a strong reduction in DFR expression occurs in the non-red orange cultivar (Navel and Ovale) compared to that of the red orange (Tarocco) suggesting that the enzyme could be involved in the lack of production of anthocyanins. Therefore, we isolated and compared the cDNAs, the genomic clones, as well as the promoter regions of blood and blond orange dfrs. Our data revealed that the cDNA sequences of pigmented and non-pigmented orange DFRs were 100% homologous and contained a 1017 bp open reading frame which encodes a protein of 338 amino acid residues, corresponding to a molecular mass of 38010.76 Da, with a theoretical pI of 5.96. Moreover, we found that there were no significant differences in non-coding regions (introns and 5' upstream region) of dfr sequences. Southern blot analysis of genomic DNA indicated that dfr was present as a single copy gene in both cultivars. From these findings the low expression level of blond orange dfr, which might play a role in the phenotypic change from blood to blond orange, is thought to be the result of a likely mutation in a regulatory gene controlling the expression of dfr. In addition, here we reported the successful expression of orange DFR cDNAs leading to an active DFR enzyme which converts dihydroquercetin to leucoanthocyanidin, thus confirming the involvement of the isolated genes in the biosynthesis of anthocyanins. Moreover, as far as we know, this is the first report concerning the in vitro expression of DFR from fruit flesh whose biochemical properties might be very different from those of other plant organ DFRs.

In this paper we analysed the genome stability of various Arabidopsis thaliana plant organs using a transgenic recombination system. The system was based on two copies of non-functional GUS (lines #651 and #11) or LUC (line #15D8) reporter genes serving as a recombination substrate. Both reporter assays showed that recombination in flowers or stems were rare events. Most of the recombination sectors were found in leaves and roots, with leaves having over 2-fold greater number of the recombination events per single cell genome as compared to roots. The recombination events per single genome were 9.7-fold more frequent on the lateral half of the leaves than on the medial halves. This correlated with a 2.5-fold higher metabolic activity in the energy source (lateral) versus energy sink (medial) of leaves. Higher metabolic activity was paralleled by a higher anthocyanin production in lateral halves. The level of double strand break (DSB) occurrence was also different among plant organs; the highest level was observed in roots and the lowest in leaves. High level of DSBs strongly positively correlated with the activity of the key repair enzymes, AtKU70 and AtRAD51. The ratio of AtRAD51 to AtKU70 expression was the highest in leaves, supporting the more active involvement of homologous recombination pathway in the repair of DSBs in this organ. Western blot analysis confirmed the real time PCR expression data for AtKU70 gene.

We isolated the chalcone reductase (pl-chr) gene of Pueraria montana var. lobata by using a PCR strategy from cDNA pools of storage roots. A high level of expression of RNA was found in both stems and roots. The genomic Southern blot result suggests that pl-chr exists as a member of a small gene family. By introducing a pl-chr gene under the control of the 35S CaMV promoter into the pink-flowering Xanthi line of Nicotiana tabacum, the flower color was changed from pink to white-to-pink. The contents of anthocyanin in the flowers of the transgenic lines were dramatically decreased by 40%, but the total UV absorption compounds remained unchanged. The production of liquiritigenin in pl-chr overexpressed transgenic tobacco lines was confirmed by HPLC and MS analysis. The introduction of pl-chr gene provides a method to redirect the flavonoid pathway into 5'-deoxyflavonoid production in non-legume crops, in order to manipulate the phenylpropanoid pathway for isoflavonoid production.

A stable cell suspension line of Catharanthus roseus producing anthocyanin was obtained. In this strain it was found that approximately 30% of cells regularly accumulated these metabolites and that anthocyanin accumulation occurred between the second half of log phase and the stationary phase of the culture growth cycle. The anthocyanins in the suspension cultures were compared with those biosynthetized in the flowers both of regenerated by somatic embryogenesis and field-grown plants. Six anthocyanins were identified in all the examined samples, three 3-O-glucosides and three 3-O-(6-O-p-coumaroyl) glucosides of petunidin, malvidin and hirsutidin. The hirsutidin coumaroyl glucoside has not been reported previously, and was predominat in all samples. The anthocyanin relative content was similar for cell suspensions and flowers from regenerated plants but different from field-grown plant flowers; instead, the total content was almost the same for the two flower types and higher compared to suspension culture content.

Dextrorotatory 1-amino-3',4'-dichlorobenzylphosphonic acid was found to be a potent inhibitor of the plant enzyme phenylalanine ammonia-lyase both in vitro and in vivo from among the ring-substituted 1-aminobenzylphosphonic acids and other analogues of phenylglycine. A structure activity relationship analysis of the results obtained permits predictions on the geometry of the pocket of the enzyme and is a basis in the strategy of better inhibitor synthesis.

Stress-tolerant crops are expected to express genes not normally expressed in susceptible crops. We have used desiccation stress coupled with high light intensity to identify groundnut as a relatively tolerant crop. Stress-responsive genes (Gdi, Groundnut Desiccation Induced) were cloned by subtractive hybridisation. The sequence of Gdi-15 shows homology to flavonol 3-O-glucosyltransferases, which are involved in anthocyanin biosynthesis. Gdi-15 transcripts increase markedly in response to stress, suggestive of a role in stress tolerance., (Copyright 2001 Academic Press.)

We have previously reported that sucrose modulates anthocyanin biosynthesis in cell suspension cultures of Vitis vinifera L. The main role of sugar in this response does not seem to be that of general carbohydrate source for the supply of energy. In the present work, a number of pharmacological agents were used to further investigate the components of the signal transduction pathway involved in the induction of anthocyanin biosynthesis by sugar. We found that the phosphorylation of hexose by hexokinase, but not its transport, has to be taken into account for the sucrose signal transduction leading to anthocyanin accumulation. Indeed, 3-O-methylglucose, a glucose analog transported into cells but not phosphorylated by hexokinase, has no effect on anthocyanin production. Mannose mimics the effect of sucrose in grape cells, and mannoheptulose, a specific inhibitor of hexokinase, reduces the accumulation of anthocyanins in response to sucrose. The results with the two latter analogs are discussed. Ca2+ channel blockers, verapamil and LaCl3, which were used to investigate the role of extracellular Ca2+, all inhibited the sugar response. Ca2+ depletion by pretreatment with ethylene glycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) also blocked the sugar response, which was partially recovered when Ca2+ was added exogenously after Ca2+ depletion. The use of two potent calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphtalenesulphonamide (W7) and chlorpromazine, showed that calmodulin is involved in the sugar signal transduction. A protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), and the protein phosphatase inhibitors, endothall and cantharidin, also inhibited the sugar response. The results of the present study suggest the involvement of several components of general signal transduction pathways such as Ca2+, calmodulin, and protein kinases phosphatases in the induction of anthocyanin biosynthesis by sugar.

The use of plant cell cultures for producing isotopically (13C) labelled phenolic substances is reported. Vitis vinifera cells synthesize high levels of anthocyanins when they are cultured in a polyphenol synthesis-inducing medium. Three major anthocyanin monoglucosides found in red wine were identified in grape cells: cyanidin-3-O-beta-glucoside, peonidin-3-O-beta-glucoside, and malvidin-3-O-beta-glucoside. Kinetic study of the intracellular level of phenylalanine and its metabolites showed that it is preferable to add this precursor to grape cell suspensions after the 5th day of culture, i.e. at the beginning of the exponential growth phase. After adding phenylalanine to the culture medium, its uptake was complete and the accumulation of anthocyanins in grape cells was stimulated. Incorporation of [1-13C]-phenylalanine into anthocyanins was measured by means of 13C satellites in the proton NMR spectrum. The maximal rate of 13C enrichment anthocyanins obtained with this technique reached 65%. The production of 13C labelled phenolic compounds was undertaken in order to investigate their absorption and metabolism in humans.

A stable callus line that produces anthocyanins was established from callus derived from a petiole of a Glehnia littoralis seedling and subcultured in the dark. The major anthocyanin which made up about 60% of the total anthocyanins was determined as cyanidin 3-O-(6-O-(6-O-(E)-feruloyl-beta-D-glucopyranosyl) -2-O-beta-D-xylopyranosyl-beta-D-glucopyranoside) by chemical and spectroscopic analyses. Anthocyanin contents in the cells cultured on B5 basal medium containing NAA (1 mg l-1), kinetin (0.01 mg l-1) and 3% sucrose reached 14% (dry wt basis) and the productivity has been sustained for 5 years.

Purple-coloured dandelion (Taraxacum officinale) callus cultures producing anthocyanin pigments were established on a cytokinin-rich medium under the light. When the cells were placed in the dark, only grey cells proliferated. Anthocyanin productivity of these cells was partially restored in the light. The major pigment was identified as cyanidin 3-(6"-malonylglucoside). The lower stem of the original plant contained the same pigment. Chalcone synthase (CHS) activity was detected in the extracts of these purple cells, whereas no activity was observed in grey cells propagated in the dark. When the CHS-active cell-free extract was combined with the extract of Escherichia coli over expressing polyketide reductase (PKR) cDNA of licorice (Glycyrrhiza echinata), isoliquiritigenin (a 6'-deoxychalcone), in addition to naringenin (a 5-hydroxyflavanone), was detected as the reaction product from 4-coumaroyl-CoA, malonyl-CoA and NADPH. This result confirms the catalytic function of the PKR gene product.

The structures of the pigments in the deep purple flowers of a lisianthus line, transformed with a UDP-glucose:flavonoid-3-O-glucosyltransferase cDNA from Antirrhinum majus, have been studied using paper chromatography, HPLC and 1H and 13C NMR spectroscopy involving the use of 1H-1H COSY and TOCSY techniques. The transgenic line is shown to have produced the new anthocyanins delphinidin-3-O-beta-D-(6-O-alpha-L -rhamnopyranosylglucopyranoside)-5-O-beta-D-[6-E (and Z)-p-coumaroylglucopyranoside] and delphinidin -3-O-beta-D-glucopyranoside-5-O-beta-D-(6-O-E-p -coumaroylglucopyranoside) in addition to those found in the untransformed control plants.

Blue-flowering genotypes of Campanula were classified into four phenotypes on the basis of the anthocyanin composition of their flowers. The flowers contained one, two, three or four of the following anthocyanins: bisdeacylplatyconin (7-O-(beta-D-glucopyranosyl)-3-O-(6-O-alpha-L-rhamnopyranosyl-beta-D- glucopyranosyl)delphinidin), violdelphin (7-O-(6-O-(4-O-(6-O-(4-hydroxybenzoyl)-beta-D-glucopyranosyl)-oxybenz oyl)-beta- D-glucopyranosyl)-3-O-(6-O-alpha-L-rhamnopyranosyl-beta-D-glucopyranosyl ) delphinidin), an unknown anthocyanin and campanin (7-O-(6-O-(4-O-(6-O-(4-O-(6-O-p-hydroxybenzoyl-beta-D-glucopyranosyl) oxybenzoyl)-beta-D-glucopyranosyl)oxybenzoyl)-beta-D-glucopy ranosyl)-3-O-(6-O- (alpha-L-rhamnopyranosyl)-beta-D-glucopyranosyl)delphinidin). The structure of the unknown anthocyanin has been determined as 7-O-(6-O-(4-O-(6-O-(4-O-(beta-D-glucopyranosyl)oxybenzoyl)-beta-D- glucopyranosyl)oxybenzoyl)-beta-D-glucopyranosyl)-3-O-(6-O-(alpha- L- rhamnopyranosyl)-beta-D-glucopyranosyl)-delphinidin, i.e. monodeacylcampanin. The genetic evidence is consistent with a biosynthetic pathway in which bisdeacylplatyconin, violdelphin and monodeacylcampanin are intermediates in the biosynthesis of campanin.

In earlier studies [G. Hrazdina, G. J. Wagner, and H. W. Siegelman (1978) Phytochemistry 17, 53-56; G. J. Wagner and G. Hrazdina (1984) Plant Physiol. 74, 901-906], evidence was obtained suggesting that the endoplasmic reticulum was a site for phenylpropanoid and flavonoid metabolism in petal tissue, and that (a) multienzyme complex(es) might be involved in this metabolism. Now, the possible role of membrane-bound multienzyme complexes in phenylpropanoid and flavonoid metabolism in three tissues has been investigated by (1) correlating enzyme induction kinetics and rates, (2) examining the molecular weight of putative complexes, (3) channeling of substrates, (4) determining the susceptibility of bound activities to trypsin digestion, and (5) investigating the structurally linked latency of bound activities. Results suggest that at least a part--and possibly the entire pathway--from phenylalanine to flavonoids is membrane (endoplasmic reticulum) associated, and that this metabolism is facilitated by a multienzyme complex. Phenylalanine ammonia lyase, the first enzyme of the biosynthetic sequence, and a flavonoid glucosyltransferase, the last, appear to be located in the lumen of the membranes. Cinnamate 4-hydroxylase is membrane embedded, while other enzyme activities appear to be weakly associated with the cytoplasmic face of endoplasmic reticulum membranes.