Search

Background: Sugarcane (Saccharum officinarum) is one of the most valuable feedstocks for sugar production. In addition to the production of industrial raw materials such as alcohol, papermaking, the fiber of livestock feed, respectively, sugarcane can produce bioactive compounds such as anthocyanins. Elucidation of the anthocyanin biosynthesis pathway is critical for the molecular breeding of sugarcane varieties with favorable traits. We aimed to identify candidate genes involved in anthocyanin biosynthesis by transcriptomic and metabolomic analyses., Results: Three varieties of sugarcane displaying different colors were used in this study: FN15 (greed rind), ROC22 (red rind), and Badila (purple rind). Sample materials were subjected to metabolomic analysis using UPLC-Q-TOF/MS and RNA-seq analysis. The metabolomic profiling results showed Cyanidin, Cyanidin (6'-malonylglucoside), Cyanidin O-glucoside, and Peonidin O-glucoside were the main components responsible for the rind color. Then, through RNA-seq analysis, we identified a total of 3137, 3302, 3014 differentially expressed genes (DEGs) between the rind and pith tissues for the corresponding varieties Badila rind, ROC22, and FN15. We then compared the expression levels of genes among the rind tissues from the three varieties. We identified 2901, 2821, and 3071 DEGs between Badila rind vs. ROC22 rind, Badila rind vs. FN15 rind, ROC22 rind vs. FN15 rind, respectively. We identified two enriched pathways, including phenylpropanoid biosynthesis and flavonoid biosynthesis. Sequencing similarity search identified a total of 50 unigenes belonging to 15 enzyme families as putative genes involved in anthocyanin biosynthesis in sugarcane rind. Seven of them were identified as candidate genes related to anthocyanin biosynthesis in the rind of sugarcane through co-localization analysis with the anthocyanin content in sugarcane. In total, 25 unigenes were selected and subjected to RT-qPCR analysis, and qRT-PCR results were consistent with those obtained with the RNA-Seq experiments., Conclusions: We proposed a pathway for anthocyanin biosynthesis in sugarcane rind. This is the first report on the biosynthesis of anthocyanin in sugarcane using the combined transcriptomic and metabolomic methods. The results obtained from this study will lay the foundation for breeding purple pith sugarcane varieties with high anthocyanin contents.

In an elaborate form of inter-species exploitation, many insects hijack plant development to induce novel plant organs called galls that provide the insect with a source of nutrition and a temporary home. Galls result from dramatic reprogramming of plant cell biology driven by insect molecules, but the roles of specific insect molecules in gall development have not yet been determined. Here, we study the aphid Hormaphis cornu, which makes distinctive "cone" galls on leaves of witch hazel Hamamelis virginiana. We found that derived genetic variants in the aphid gene determinant of gall color (dgc) are associated with strong downregulation of dgc transcription in aphid salivary glands, upregulation in galls of seven genes involved in anthocyanin synthesis, and deposition of two red anthocyanins in galls. We hypothesize that aphids inject DGC protein into galls and that this results in differential expression of a small number of plant genes. dgc is a member of a large, diverse family of novel predicted secreted proteins characterized by a pair of widely spaced cysteine-tyrosine-cysteine (CYC) residues, which we named BICYCLE proteins. bicycle genes are most strongly expressed in the salivary glands specifically of galling aphid generations, suggesting that they may regulate many aspects of gall development. bicycle genes have experienced unusually frequent diversifying selection, consistent with their potential role controlling gall development in a molecular arms race between aphids and their host plants., Competing Interests: Declaration of interests HHMI has filed a provisional patent, number 63/092,942, for the inventors A.K. and D.L.S., covering unique aphid polypeptides for use in modifying plants., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

A lot of researches have been focused on the evolution and function of MYB transcription factors (TFs). For revealing the formation of petunia flower color diversity, MYB gene family in petunia was identified and analyzed. In this study, a total of 155 MYB genes, including 40 1R-MYBs, 106 R2R3-MYBs, 7 R1R2R3-MYBs and 2 4R-MYBs, have been identified in the Petunia axillaris genome. Most R2R3 genes contain three exons and two introns, whereas the number of PaMYB introns varies from 0 to 12. The R2R3-MYB members could be divided into 28 subgroups. Analysis of gene structure and protein motifs revealed that members within the same subgroup presented similar exon/intron and motif organization, further supporting the results of phylogenetic analysis. Genes in subgroup 10, 11 and 21 were mainly expressed in petal, not in vegetative tissues. Genes in subgroup 9, 19, 25 and 27 expressed in all tissues, but the expression patterns of each gene were different. According to the promoter analysis, five R2R3-MYB and two MYB-related genes contained MBSI cis-element, which was involved in flavonoid biosynthetic regulation. PaMYB100/DPL has been reported to positively regulate to pigmentation. However, although PaMYB82 , PaMYB68 and Pa1RMYB36 contained MBSI cis-element, their function in flavonoid biosynthesis has not been revealed. Consistent with existing knowledge, PaMYBs in subgroup 11 had similar function to AtMYBs in subgroup 6, genes in which played an important role in anthocyanin biosynthesis. In addition, PaMYB1 and PaMYB40 belonged to P9 (S7) and were potentially involved in regulation of flavonoid synthesis in petunia vegetative organs. This work provides a comprehensive understanding of the MYB gene family in petunia and lays a significant foundation for future studies on the function and evolution of MYB genes in petunia.

Red-fleshed apple fruits are popular because of their high flavonoid content. Although MdMYB10 and its homologs have been identified as crucial regulators of the fruit coloring process, other transcription factors (TFs) contributing to the differences in flesh coloration have not been fully characterized. In this study, we investigated the regulatory effects of MdWRKY41 on anthocyanin and proanthocyanidin (PA) synthesis in red-fleshed apples. The overexpression of MdWRKY41 in red-fleshed apple calli inhibited anthocyanin and PA accumulation by downregulating the expression of a MYB TF gene (MdMYB12) and specific structural genes (MdLAR, MdUFGT, and MdANR). Furthermore, MdWRKY41 was shown to interact with MdMYB16 to form a complex that can further suppress MdANR and MdUFGT expression. Interestingly, MdWRKY41 was targeted by the photoresponse factor MdHY5 and inhibited its transcription. Overall, our findings provide insights into a novel MdHY5-MdWRKY41-MdMYB regulatory module influencing anthocyanin and PA synthesis in red-fleshed apple fruits., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

With diverse genetic backgrounds, soybean landraces are valuable resource for breeding programs. Herein, we apply multi-omic approaches to extensively characterize the molecular basis of drought tolerance in the soybean landrace LX. Initial screens established that LX performed better with PEG6000 treatment than control cultivars. LX germinated better than William 82 under drought conditions and accumulated more anthocyanin and flavonoids. Untargeted mass spectrometry in combination with transcriptomic analyses revealed the chemical diversity and genetic basis underlying the overall performance of LX landrace. Under control and drought conditions, significant differences in the expression of a suite of secondary metabolism genes, particularly those involved in the general phenylpropanoid pathway and flavonoid but not lignin biosynthesis, were seen in LX and William 82. The expression of these genes correlated with the corresponding metabolites in LX plants. Further correlation analysis between metabolites and transcripts identified pathway structural genes and transcription factors likely are responsible for the LX agronomic traits. The activities of some key biosynthetic genes or regulators were confirmed through heterologous expression in transgenic Arabidopsis and hairy root transformation in soybean. We propose a regulatory mechanism based on flavonoid secondary metabolism and adaptive traits of this landrace which could be of relevance to cultivated soybean., (© 2021 John Wiley & Sons Ltd.)

KEY MESSAGE: we identified a functional chromogen gene C from wild rice, providing a new insight of anthocyanin biosynthesis pathway in indica and japonica. Accumulation of anthocyanin is a desirable trait to be selected in rice domestication, but the molecular mechanism of anthocyanin biosynthesis in rice remains largely unknown. In this study, a novel allele of chromogen gene C, OrC1, from Oryza rufipongon was cloned and identified as a determinant regulator of anthocyanin biosynthesis. Although OrC1 functions in purple apiculus, leaf sheath and stigma in indica background, it only promotes purple apiculus in japonica. Transcriptome analysis revealed that OrC1 regulates flavonoid biosynthesis pathway and activates a few bHLH and WD40 genes of ternary MYB-bHLH-WD40 complex in indica. Differentially expressed genes and metabolites were found in the indica and japonica backgrounds, indicating that OrC1 activated the anthocyanin biosynthetic genes OsCHI, OsF3H and OsANS and produced six metabolites independently. Artificial selection and domestication of C1 gene in rice occurred on the coding region in the two subspecies independently. Our results reveal the regulatory system and domestication of C1, provide new insights into MYB transcript factor involved in anthocyanin biosynthesis, and show the potential of engineering anthocyanin biosynthesis in rice.

Anthocyanin biosynthesis is one of the most studied pathways in plants due to the important ecological role played by these compounds and the potential health benefits of anthocyanin consumption. Given the interest in identifying new genetic factors underlying anthocyanin content we studied a diverse collection of diploid potatoes by combining a genome-wide association study and pathway-based analyses. By using an expanded SNP dataset, we identified candidate genes that had not been associated with anthocyanin variation in potatoes, namely a Myb transcription factor, a Leucoanthocyanidin dioxygenase gene and a vacuolar membrane protein. Importantly, a genomic region in chromosome 10 harbored the SNPs with strongest associations with anthocyanin content in GWAS. Some of these SNPs were associated with multiple anthocyanin compounds and therefore could underline the existence of pleiotropic genes or anthocyanin biosynthetic clusters. We identified multiple anthocyanin homologs in this genomic region, including four transcription factors and five enzymes that could be governing anthocyanin variation. For instance, a SNP linked to the phenylalanine ammonia-lyase gene, encoding the first enzyme in the phenylpropanoid biosynthetic pathway, was associated with all of the five anthocyanins measured. Finally, we combined a pathway analysis and GWAS of other agronomic traits to identify pathways related to anthocyanin biosynthesis in potatoes. We found that methionine metabolism and the production of sugars and hydroxycinnamic acids are genetically correlated to anthocyanin biosynthesis. The results contribute to the understanding of anthocyanins regulation in potatoes and can be used in future breeding programs focused on nutraceutical food., Competing Interests: MAPG is a paid employee of Medcolcanna S.A.S, but began their employment after concluding the research included in this manuscript, so Medcolcanna S.A.S. did not provide any support for this study. This affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Anthocyanins are pigments present in blood oranges which can be enriched by post-harvest cold storage. Additionally, citrus fruits contain appreciable levels of other flavonoids, whose content increases under post-harvest heat treatments. Here, we investigated the effects of curing (37 °C for 3 days) and storage at low-temperature (9 °C) during 15, 30 and 45 days on accumulation of anthocyanins and other flavonoids in Moro and Sanguinelli Polidori blood oranges (Citrus sinensis L. Osbeck). Cured fruits reached up to 191.4 ± 1.4 mg/L of anthocyanins in their juice after cold storage and a 3-fold enrichment of other flavonoids such as flavones and flavanones, compared to 85.7 ± 3.3 mg/L anthocyanins from fruits with cold storage alone. Concomitantly, qPCR analysis showed that curing enhanced upregulation of the main structural and transcription factor genes regulating the flavonoid pathway. GC-MS analysis showed that no unpleasant compounds were generated in the cured plus cold-stored juice volatilome., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Anthocyanins are important flavonoid pigments in plants. Malonyl CoA is an important intermediate in anthocyanin synthesis, and citrate, formed by citrate synthase (CS) catalysing oxaloacetate, is the precursor for the formation of malonyl-CoA. CS is composed of two isoforms, mitochondrial citrate synthase (mCS), a key enzyme of the tricarboxylic acid (TCA) cycle, and citrate synthase (CSY) localizated in microbodies in plants. However, no CS isoform involvement in anthocyanin synthesis has been reported. In this study, we identified the entire CS family in petunia (Petunia hybrida): PhmCS, PhCSY1 and PhCSY2. We obtained petunia plants silenced for the three genes. PhmCS silencing resulted in abnormal development of leaves and flowers. The contents of citrate and anthocyanins were significantly reduced in flowers in PhmCS-silenced plants. However, silencing of PhCSY1 and/or PhCSY2 did not cause a visible phenotype change in petunia. These results showed that PhmCS is involved in anthocyanin synthesis and the development of leaves and flowers, and that the citrate involved in anthocyanin synthesis mainly derived from mitochondria rather than microbodies in petunia., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Anthocyanins are flavonoid pigments providing plants a range of colors from red, pink, orange to blue. Anthocyanins are synthesized in the cytosol but accumulate predominantly in the vacuoles through vacuolar sequestration involving glutathione S-transferases (GSTs) and multidrug and toxic compound extrusion (MATE) and the ATP binding cassette (ABC) transporters. However, little is known about anthocyanin-related GSTs in Upland cotton (Gossypium hirsutum L.). In this study, we performed genome-wide identification of GST genes in Upland cotton and identified GST genes functioning in accumulation of anthocyanins. We demonstrated that GhGSTF12 was able to complement the defective leaf color phenotypes of the Arabidopsis tt19 mutant caused by mutation in a GSTF gene. Virus-induced silencing of GhGSTF12 in the red leaf cultivar turned its red color to green and transient overexpression of GhGSTF12 accelerated anthocyanin accumulation in the red leaf cultivar but not in the green leaf cultivar. Collectively, GhGSTF12 may be involved in transport of anthocyanins from cytosol to vacuoles in cotton. These results also demonstrated a conserved function of plant GSTF genes in anthocyanin accumulation and provide a candidate gene for manipulating pigmentation in cotton tissues., (Copyright © 2021 Elsevier B.V. All rights reserved.)

As one of the largest transcription factor family, basic helix-loop-helix (bHLH) transcription factor family plays an important role in plant metabolism, physiology and growth. Berry color is one of the important factors that determine grape quality. However, the bHLH transcription factor family's function in anthocyanin synthesis of grape berry has not been studied systematically. We identified 115 bHLH transcription factors in grape genome and phylogenetic analysis indicated that bHLH family could be classified into 25 subfamilies. First, we screened six candidate genes by bioinformatics analysis and expression analysis. We found one of the candidate genes VdbHLH037 belonged to III (f) subfamily and interacted with genes related to anthocyanin synthesis through phylogenetic analysis and interaction network prediction. Therefore, we speculated that VdbHLH037 participated in the anthocyanin synthesis process. To confirm this, we transiently expressed VdbHLH037 in grape and Arabidopsis transformation. Compared with the control, transgenic materials can accumulate more anthocyanins. These results provide a good base to study the function of the VdbHLH family in anthocyanin synthesis of grape berry.

Anthocyanins are natural water-soluble pigments that are important in plants because they endow a variety of colors to vegetative tissues and reproductive plant organs, mainly ranging from red to purple and blue. The colors regulated by anthocyanins give plants different visual effects through different biosynthetic pathways that provide pigmentation for flowers, fruits and seeds to attract pollinators and seed dispersers. The biosynthesis of anthocyanins is genetically determined by structural and regulatory genes. MYB ( v-myb avian myeloblastosis viral oncogene homolog) proteins are important transcriptional regulators that play important roles in the regulation of plant secondary metabolism. MYB transcription factors (TFs) occupy a dominant position in the regulatory network of anthocyanin biosynthesis. The TF conserved binding motifs can be combined with other TFs to regulate the enrichment and sedimentation of anthocyanins. In this study, the regulation of anthocyanin biosynthetic mechanisms of MYB-TFs are discussed. The role of the environment in the control of the anthocyanin biosynthesis network is summarized, the complex formation of anthocyanins and the mechanism of environment-induced anthocyanin synthesis are analyzed. Some prospects for MYB-TF to modulate the comprehensive regulation of anthocyanins are put forward, to provide a more relevant basis for further research in this field, and to guide the directed genetic modification of anthocyanins for the improvement of crops for food quality, nutrition and human health.

The N-terminal of Myc-like basic helix-loop-helix transcription factors (bHLH TFs) contains an interaction domain, namely the MYB-interacting region (MIR), which interacts with the R2R3-MYB proteins to regulate genes involved in the anthocyanin biosynthetic pathway. However, the functions of MIR-domain bHLHs in this pathway are not fully understood. In this study, PbbHLH2 containing the MIR-domain was identified and its function investigated. The overexpression of PbbHLH2 in "Zaosu" pear peel increased the anthocyanin content and the expression levels of late biosynthetic genes. Bimolecular fluorescence complementation showed that PbbHLH2 interacted with R2R3-MYB TFs PbMYB9, 10, and 10b in onion epidermal cells and confirmed that MIR-domain plays important roles in the interaction between the MIR-domain bHLH and R2R3-MYB TFs. Moreover, PbbHLH2 bound and activated the dihydroflavonol reductase promoter in yeast one-hybrid (Y1H) and dual-luciferase assays. Taken together these results suggested that the MIR domain of PbbHLH2 regulated anthocyanin biosynthesis in pear fruit peel.

Black rice is considered to be functional food containing anthocyanins as bioactive compounds. This study examined the genomic and proteomic patterns in local black rice from Java Island, Indonesia, with attention to the mechanism of anthocyanin synthesis. Three kinds of black rice from Java Island, including black rice from East Java (BREJ), black rice from Central Java (BRCJ), and black rice from West Java (BRWJ), were studied in comparison to white rice (WREJ) and red rice (RREJ). Genomic profiling was done by simple sequence repeat (SSR) analysis, and sequencing of red coleoptile (Rc) and glycosyltransferase (GT) genes, followed by in silico analysis. Total anthocyanin was investigated by ultra-high performance liquid chromatography- diode array detector (UHPLC-DAD). The proteomic profiles were determined by liquid-chromatography and mass spectrometry of tryptic peptides. The SSR profiles showed a specific band in each black rice variant. The Rc gene exon-2 sequences were similar in the three black rice cultivars. The GT gene sequence was identified as a new variant that correlates with the purple stem, leaf, bran, and whole grain morphology seen exclusively in the BRWJ cultivar. The anthocyanin composition in Java black rice is diverse. The highest cyanidin level was seen in BRWJ and the highest level of peonidin-3-O-glucoside in BREJ. Proteomic profiling of the black rice cultivars demonstrated that the expression of proteins that might be related to the levels of anthocyanin synthesis varied. These studies conclude that the genomic, proteomic and anthocyanins composition of Java black rice cultivars may be used the improvement of their functional nutrition values.

Plants exposed to environmental contaminants often synthesize anthocyanins (ATHs) as an approach to safeguard themselves from adverse impact. However, the overload of ATHs in plant cells can threaten their growth and development through proteins oxidization and intercalating with DNAs inside cells. In the present study, a microcosm hydroponic experiment was conducted using rice seedlings to investigate the molecular signaling pathways involved in regulating and controlling ATHs synthesis and transport exposed to thiocyanate (SCN - ). Our results indicated that SCN - exposure significantly (p < 0.05) increased the expression of ATHs synthesis related genes (i.e., PAL, CHS, ANS, UFGT genes) in rice tissues, altered the activities of these ATHs synthesis related enzymes, and consequently elevated the ATHs content. However, SCN - exposure significantly decreased the expression of ATHs transport related genes (i.e., GST, ABC, MATE genes) in rice seedlings, suggesting that SCN - exposure have restrained ATHs transport from cytosol to vacuole in cells, eventually posing a significant adverse effect on cells survival. Our findings highlight on one of the plant aspects in managing the toxicity triggered by secondary metabolites under stress conditions., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Abscisic acid (ABA) induces anthocyanin biosynthesis in many plant species. However, the molecular mechanism of ABA-regulated anthocyanin biosynthesis remains unclear. As a crucial regulator of ABA signaling, ABSCISIC ACID-INSENSITIVE5 (ABI5) is involved in many aspects of plant growth and development, yet its regulation of anthocyanin biosynthesis has not been elucidated. In this study, we found that MdABI5, the apple homolog of Arabidopsis ABI5, positively regulated ABA-induced anthocyanin biosynthesis. A series of biochemical tests showed that MdABI5 specifically interacts with basic helix-loop-helix 3 (MdbHLH3), a positive regulator of anthocyanin biosynthesis. MdABI5 enhanced the binding of MdbHLH3 to its target genes dihydroflavonol 4-reductase (MdDFR) and UDP flavonoid glucosyl transferase (MdUF3GT). In addition, MdABI5 directly bound to the promoter of MdbHLH3 to activate its expression. Moreover, MdABI5 enhanced ABA-promoted interaction between MdMYB1 and MdbHLH3. Finally, antisense suppression of MdbHLH3 significantly reduced anthocyanin biosynthesis promoted by MdABI5, indicating that MdABI5-promoted anthocyanin biosynthesis was dependent on MdbHLH3. Taken together, our data suggest that MdABI5 plays a positive role in ABA-induced anthocyanin biosynthesis by modulating the MdbHLH3-MdMYB1 complex. Our work broadens the regulatory network of ABA-mediated anthocyanin biosynthesis, providing new insights to further study the transcriptional regulatory mechanisms behind this process., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Carrot (Daucus carota L.) is one of the most cultivated vegetable in the world and of great importance in the human diet. Its storage organs can accumulate large quantities of anthocyanins, metabolites that confer the purple pigmentation to carrot tissues and whose biosynthesis is well characterized. Long non-coding RNAs (lncRNAs) play critical roles in regulating gene expression of various biological processes in plants. In this study, we used a high throughput stranded RNA-seq to identify and analyze the expression profiles of lncRNAs in phloem and xylem root samples using two genotypes with a strong difference in anthocyanin production. We discovered and annotated 8484 new genes, including 2095 new protein-coding and 6373 non-coding transcripts. Moreover, we identified 639 differentially expressed lncRNAs between the phenotypically contrasted genotypes, including certain only detected in a particular tissue. We then established correlations between lncRNAs and anthocyanin biosynthesis genes in order to identify a molecular framework for the differential expression of the pathway between genotypes. A specific natural antisense transcript linked to the DcMYB7 key anthocyanin biosynthetic transcription factor suggested how the regulation of this pathway may have evolved between genotypes.

B-BOX proteins are zinc finger transcription factors that play important roles in plant growth, development, and abiotic stress responses. In this study, we identified 15 PavBBX genes in the genome database of sweet cherry. We systematically analyzed the gene structures, clustering characteristics, and expression patterns of these genes during fruit development and in response to light and various hormones. The PavBBX genes were divided into five subgroups. The promoter regions of the PavBBX genes contain cis -acting elements related to plant development, hormones, and stress. qRT-PCR revealed five upregulated and eight downregulated PavBBX genes during fruit development. In addition, PavBBX6 , PavBBX9 , and PavBBX11 were upregulated in response to light induction. We also found that ABA, BR, and GA 3 contents significantly increased in response to light induction. Furthermore, the expression of several PavBBX genes was highly correlated with the expression of anthocyanin biosynthesis genes, light-responsive genes, and genes that function in multiple hormone signaling pathways. Some PavBBX genes were strongly induced by ABA, GA, and BR treatment. Notably, PavBBX6 and PavBBX9 responded to all three hormones. Taken together, BBX proteins likely play major roles in regulating anthocyanin biosynthesis in sweet cherry fruit by integrating light, ABA, GA, and BR signaling pathways.

Anthocyanin accumulation is a striking symptom of plant environmental response and plays an important role in plant adaptation to adverse stimuli. PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) is a member of the PIFs family that directly interacts with light-activated phytochromes, and it can not only regulate various light responses but also optimize growth as a key integrator of multiple signaling pathways. However, the mechanism by which PIF4 participates in the regulation of anthocyanin accumulation remains to be elucidated. In this study, we found that anthocyanin accumulation was effectively induced by white light in Arabidopsis Col-0, but such an effect was impaired in the overexpression line PIF4OX. Consistently, the transcript level of PAP1 that encodes a key transcript factor involved in regulating anthocyanin biosynthesis was significantly decreased in PIF4OX compared with Col-0. Moreover, the expression of PAP1 was markedly lower in pap1-D/PIF4OX than pap1-D, as a result, the phenotype that highly accumulates anthocyanins in leaves of pap1-D caused by PAP1 overexpressing was almost eliminated in pap1-D/PIF4OX. Analyses through chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA) revealed that PIF4 could directly bind to the G-box motif present in the promoter of PAP1. Furthermore, transient transcriptional expression analysis showed that PIF4 could weaken the transcriptional activity of the PAP1 promoter, and the G-box motif is necessary for the effect of PIF4. Subsequently, when the seedlings shifted from darkness to light and grew under constant red light and short-day photoperiod, it was found that the PAP1 transcription level and anthocyanin content in pif4-2/pap1-D were significantly higher than pap1-D, implying that PIF4 mutation can strengthen PAP1's effect on anthocyanin biosynthesis under these conditions. Taken together, the results indicate that PIF4 negatively regulates anthocyanin accumulation in Arabidopsis through transcriptional suppression of PAP1 by directly binding to the G-box motif of the promoter., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Plants experiencing abiotic stress react by generating reactive oxygen species (ROS), compounds that, if allowed to accumulate to excess, repress plant growth and development. Anthocyanins induced by abiotic stress are strong antioxidants that neutralize ROS, whereas their over-accumulation retards plant growth. Although the mechanism of anthocyanin synthesis has been revealed, how plants balance anthocyanin synthesis under abiotic stress to maintain ROS homeostasis is unknown. Here, ROS-related proteins, SIMILAR TO RCD-ONEs (SROs), were analysed in Zea mays (maize), and all six SRO1 genes were inducible by a variety of abiotic stress agents. The constitutive expression of one of these genes, ZmSRO1e, in maize as well as in Arabidopsis thaliana increased the sensitivity of the plant to abiotic stress, but repressed anthocyanin biosynthesis and ROS scavenging activity. Loss-of-function mutation of ZmSRO1e enhanced ROS tolerance and anthocyanin accumulation. We showed that ZmSRO1e competed with ZmR1 (a core basic helix-loop-helix subunit of the MYB-bHLH-WD40 transcriptional activation complex) for binding with ZmPL1 (a core MYB subunit of the complex). Thus, during the constitutive expression of ZmSRO1e, the formation of the complex was compromised, leading to the repression of genes, such as ZmA4 (encoding dihydroflavonol reductase), associated with anthocyanin synthesis. Overall, the results have revealed a mechanism that allows the products of maize SRO1e to participate in the abiotic stress response., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Anthocyanidins are important pigments that cause plant tissues to develop colors. They have attracted much attention due to their crucial regulatory roles in plant growth as well as their health benefits. In order to reveal the molecular mechanism of anthocyanidin synthesis and regulation in purple corn (Zea mays L.) in this study, purple corn 963 was used to compare differences in gene expression during three stages of grain development by transcriptome analysis. A total of 17,168 differentially expressed genes (DEGs) (7564 up-regulated and 9604 down-regulated DGEs) were identified. The DEGs were significantly enriched in "Phenylpropanoid biosynthesis", "Biosynthesis of secondary metabolites", and "Plant hormone signal transduction". In addition, 72 % of the structural genes that regulate anthocyanidin synthesis were up-regulated, and the transcription factors related to the accumulation of anthocyanidins were enriched during grain development. Moreover, the differential expression of phytohormone genes might also be an important factor in anthocyanidin accumulation. Transcriptomic analysis presents a molecular basis for the study of grain color changes in the three stages of grain development, and provides information for further research on the mechanism of anthocyanidin synthesis., (Copyright © 2020. Published by Elsevier GmbH.)

Anthocyanins, a major class of compounds derived from the flavonoid pathway, are important pigments of apple fruit. They can also prevent certain diseases and are beneficial to human health. Fruit pigmentation is a key quality trait that influences consumer preference; therefore, it is of great importance to investigate its regulatory mechanism. Here, we identified a MYB transcription factor (TF), MdMYB114, whose transcript level increased in the skin of the deep red apple fruit. It was determined to belong to the R2R3-MYB TF family and was localized in the nucleus. MdMYB114 overexpression led to anthocyanin accumulation in apple calli. MdMYB114 was not able to form an MBW complex but could enhance anthocyanin biosynthesis and transport by directly binding to the promoters of MdANS, MdUFGT, and MdGST to promote their expression. In addition, multiple assays revealed that MdbZIP4-like, a basic leucine-zipper TF, could directly bind to the MdMYB114 promoter to enhance its expression. Taken collectively, our results provide evidence that MdMYB114 is a positive regulator of anthocyanin biosynthesis and transport and it functions downstream of MdbZIP4-like in apple fruit., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Background: Chrysanthemum morifolium is one of the most economically important and popular floricultural crops in the family Asteraceae. Chrysanthemum flowers vary considerably in terms of colors and shapes. However, the molecular mechanism controlling the development of chrysanthemum floral colors and shapes remains an enigma. We analyzed a cut-flower chrysanthemum variety that produces normal capitula composed of ray florets with normally developed pistils and purple corollas and mutant capitula comprising ray florets with green corollas and vegetative buds instead of pistils., Results: We conducted a whole-transcriptome analysis of the differentially expressed genes (DEGs) in the mutant and normal capitula using third-generation and second-generation sequencing techniques. We identified the DEGs between the mutant and normal capitula to reveal important regulators underlying the differential development. Many transcription factors and genes related to the photoperiod and GA pathways, floral organ identity, and the anthocyanin biosynthesis pathway were differentially expressed between the normal and mutant capitula. A qualitative analysis of the pigments in the florets of normal and mutant capitula indicated anthocyanins were synthesized and accumulated in the florets of normal capitula, but not in the florets of mutant capitula. These results provide clues regarding the molecular basis of the replacement of Chrysanthemum morifolium ray florets with normally developed pistils and purple corollas with mutant ray florets with green corollas and vegetative buds. Additionally, the study findings will help to elucidate the molecular mechanisms underlying floral organ development and contribute to the development of techniques for studying the regulation of flower shape and color, which may enhance chrysanthemum breeding., Conclusions: The whole-transcriptome analysis of DEGs in mutant and normal C. morifolium capitula described herein indicates the anthocyanin deficiency of the mutant capitula may be related to the mutation that replaces ray floret pistils with vegetative buds. Moreover, pistils may be required for the anthocyanin biosynthesis in the corollas of chrysanthemum ray florets.

Background: In plants, CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is a key negative regulator in photoperiod response. However, the biological function of COP1-interacting protein 1 (CIP1) and the regulatory mechanism of the CIP1-COP1 interaction are not fully understood., Results: Here, we identified the apple MdCIP1 gene based on the Arabidopsis AtCIP1 gene. Expression pattern analysis showed that MdCIP1 was constitutively expressed in various tissues of apple, and responded to stress and hormone signals at the transcriptional level. Ectopic expression of MdCIP1 complemented the phenotypes of the Arabidopsis cip1 mutant, and MdCIP1 inhibited anthocyanin biosynthesis in apple calli. In addition, the biochemical assay demonstrated that MdCIP1 could interact with MdCOP1 protein by their coiled-coil domain, and MdCIP1-OX/cop1-4 had a similar phenotype in photomorphogenesis with the cop1-4 mutant, suggesting that COP1 is epistatic to CIP1. Furthermore, the transient transformation assay indicated that MdCIP1 repressed anthocyanin biosynthesis in an MdCOP1-mediated pathway., Conclusion: Take together, this study finds that MdCIP1 acts as a repressor in regulating hypocotyl elongation and anthocyanin biosynthesis through MdCOP1 in apple.

Key Message: Overexpression and virus-induced gene silencing verified BoDFR1 conferred the anthocyanin accumulation in pink-leaved ornamental kale. Leaf color is an essential trait in the important horticultural biennial plant ornamental kale (Brassica oleracea var. acephala). The identity of the gene conferring this striking trait and its mode of inheritance are topics of debate. Based on an analysis of F 1 , F 2 , BC 1 P 1 , and BC 1 P 2 ornamental kale populations derived from a cross between a pink-leaved P28 and white-leaved D10 line, we determined that the pink leaf trait is controlled by a semi-dominant gene. We cloned two genes potentially involved in anthocyanin biosynthesis in ornamental kale: Bo9g058630 and Bo6g100940. Based on their variation in sequence, we speculated that Bo9g058630, encoding the kale dihydroflavonol-4 reductase (BoDFR1) enzyme, plays a critical role in the development of the pink leaf trait. Indeed, an InDel marker specific for BoDFR1 completely co-segregated with the pink leaf trait in our F 2 population. We then generated the 35Spro: DFR-GUS overexpression vector, which we transformed into D10. Overexpression of BoDFR1 indeed restored some anthocyanin accumulation in this white-leaved parental line. In addition, we targeted BoDFR1 in P28 using virus-induced gene silencing. Again, silencing of BoDFR1 resulted in a substantial decrease in anthocyanin accumulation. This work lays the foundation for further exploration of the mechanism underlying anthocyanin accumulation in pink-leaved ornamental kale.

Fruit development and ripening are essential components of human and animal diets. Fruit ripening is also a vital plant trait for plant shelf life at the commercial level. In the present study, two apple cultivars, Hanfu wild (HC) and Hanfu mutant (HM), were employed for RNA-Sequencing (RNA-Seq) to explore the genes involved in fruit ripening. We retrieved 2642 genes, differentially expressed in HC and HM apple cultivars. Gene ontology (GO) analysis revealed the 569 categories, significantly enriched in biological process, cellular component, and molecular function. KEGG analysis exhibited the plant hormone transduction and flavonoid-anthocyanin biosynthesis pathways, might be involved in the fruit ripening and anthocyanin biosynthesis mechanism. A cluster of 13 and 26 DEGs was retrieved, representing the plant hormones and transcription factors, respectively, that may be important for early ripening in HM genotype. This transcriptome study would be useful for researchers to functionally characterize the DEGs responsible for early ripening., (Copyright © 2020. Published by Elsevier Inc.)

Lotus is an important aquatic ornamental plant, whose flower color is one of the key horticultural traits that determines its ornamental value. Previous studies revealed that anthocyanins largely determined the red color of lotus flower, which are also the main component that has beneficial effects on human health. However, the regulation mechanism of flower pigmentation in lotus flower remains unclear. In the present study, in order to further understand the regulatory mechanism underlying the anthocyanin biosynthesis, a bHLH gene NnTT8 was characterized to be phylogenetically close to AtTT8 and the bHLH proteins from other plant species that have been indicated to be involved in the positive regulation of anthocyanin biosynthesis. Complementation analysis in Arabidopsis tt8 mutant showed that NnTT8 could function similarly to AtTT8 in regulating anthocyanin and proanthocyanin biosynthesis. An MYB transcription factor capable of interacting with NnTT8 was also characterized from lotus. The identification of a bHLH transcription factor playing regulatory roles in anthocyanin biosynthesis is crucial, as it might help to obtain more in-depth insight into the coloration of lotus and help in breeding high anthocyanin content lotus variety that can be explored for lotus flower beverages., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Sweetpotato [Ipomoea batatas (L.) Lam] is an economically important crop for fresh and processed consumption and is widely cultivated worldwide, especially in China. Various sweetpotato cultivars with different storage root colors are presently available. The purple-fleshed sweetpotato obtains its color from anthocyanin accumulation in the storage roots, which is beneficial for both plant and human health. To date, the molecular mechanism of this anthocyanin accumulation has not been studied in detail. In our study, three cDNA libraries generated from 'Xuzi8' with dark-purple flesh, 'Xuzi6' with light-purple flesh, and 'Xu28' with white flesh were sequenced utilizing an Illumina HiSeq™ 2500 platform. Corresponding totals of 28,093,466, 29,239,729 and 27,217,440 raw reads were obtained from the three libraries and assembled into 137,625 unigenes with an average length of 481 bp. Moreover, 79,203 unigenes (57.55%) were found to be annotated in several public databases, and 1285 unigenes were differentially expressed among the Xu28 vs Xuzi8, Xu28 vs Xuzi6, and Xuzi6 vs Xuzi8 libraries. After functional category enrichment analysis of differential expression genes (DEGs), 25 genes were selected as the candidate genes related to anthocyanin accumulation. Furthermore, the expression patterns of some selected DEGs were verified by quantitative real-time PCR (qRT-PCR), and the correlation between expression levels of relevant genes involved in anthocyanin biosynthesis and anthocyanin content was determined. Taken together, the results compose a transcriptomic analysis to investigate the differences in purple flesh formation in the storage roots among different sweetpotato varieties, with the notable outcome that several key genes can now be closely linked to anthocyanin biosynthesis., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Anthocyanins are a group of secondary metabolites that protect plants from biotic and abiotic stresses. The research on anthocyanins has been well-received due to their colorfulness and human health benefits. In this study, we used the photosensitive eggplant cultivar 'Lanshan Hexian' as the research material and reported the functional characterization of SmMYB86, a negative regulator involved in the anthocyanin biosynthesis in eggplant. Our results suggested that SmMYB86 was a nuclear protein that was particularly expressed in leaves, stems, and peels. Overexpression of SmMYB86 in eggplant indicated that the accumulation of anthocyanins was reduced. Silencing of SmMYB86 in eggplant fruit peel significantly increased the anthocyanin content and expression levels of SmCHS, SmF3H, and SmANS. Yeast one-hybrid and dual-luciferase assays showed that SmMYB86 could directly bind to the promoters of SmCHS, SmF3H, and SmANS and suppress their activities. SmTTG1 binded to the promoter of SmCHS and promoted its activating. SmMYB86 interacted with SmTTG1 and inhibited its promotive role in SmCHS expression. This study provides some insights into the regulatory roles of SmMYB86 on key structural genes in the anthocyanin synthesis pathway in eggplant., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ethylene induces anthocyanin biosynthesis in most fruits, including apple (Malus domestica), strawberry (Fragaria × ananassa), and plum (Prunus spp.). However, ethylene inhibits anthocyanin biosynthesis in pear (Pyrus spp.), but the underlying molecular mechanism has not been characterized. In this study, ethylene induced the expression of PpERF105, which encodes a transcription factor. PpERF105 functioned as a transcriptional activator, but it inhibited anthocyanin biosynthesis in pear. A transcriptome analysis revealed that PpERF105 activated the expression of PpMYB140, which encodes an R2R3-MYB transcriptional repressor. Moreover, PpMYB140 directly inhibited the expression of anthocyanin-related structural genes. It also competed with PpMYB114 for the binding to bHLH3, ultimately resulting in the formation of the MYB140-bHLH-WDR complex rather than the conventional MBW complex, thereby further inhibiting anthocyanin biosynthesis. Furthermore, PpMYB140 prevented the overaccumulation of anthocyanins in the absence of ethylene. Collectively, our study data indicate that ethylene-induced PpERF105 inhibits anthocyanin biosynthesis by upregulating PpMYB140 expression. Our findings may be useful for elucidating the molecular basis of the ethylene-mediated inhibition of anthocyanin biosynthesis in fruit., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

The purplish-red color of "Tailihong" jujube fruit skins is caused primarily by anthocyanin accumulation, but the mechanisms that underlie anthocyanin biosynthesis in jujube fruit have rarely been studied. We performed metabolomic and transcriptomic analyses of jujube fruit skins at different developmental stages and identified a total of 158 flavonoids, among which cyanidin-3- O -rutinoside and peonidin-3,5- O -diglucoside were the primary anthocyanins. During fruit development and maturation, the anthocyanin content was strongly correlated with the expression of ZjANS and ZjUGT79B1 , suggesting that these are key genes in the anthocyanin biosynthesis process. Transcriptomic analysis indicated that the transcription factors ZjMYB5, ZjTT8, and ZjWDR3 regulated anthocyanin biosynthesis in jujube fruit skins. Subcellular localization experiments confirmed that ZjANS and ZjUGT79B1 were localized to the nucleus and the endoplasmic reticulum. ZjMYB5 and ZjTT8 were found only in the nucleus, whereas strong fluorescence signals from ZjWDR3 were observed in the nucleus and cytoplasm. Prokaryotic expression and in vitro enzyme activity assays showed that the recombinant ZjANS protein catalyzed the formation of cyanidin from (+)-catechin. Secondary glycosylation by ZjUFGT79B1 modified cyanidin-3- O -glucoside to produce cyanidin-3- O -rutinoside, and ZjCCoAOMT readily catalyzed the production of the methylated anthocyanin peonidin-3,5- O -diglucoside from cyanidin 3,5- O -glucoside. Dual-Luciferase and GUS activity assays showed that the ZjANS and ZjUGT79B1 promoters were activated by ZjMYB5, ZjTT8, and ZjWDR3. All data indicated that these three transcription factors played important roles in anthocyanin biosynthesis in the color mutant Ziziphus jujuba cv. Tailihong, contributing to anthocyanin accumulation by enhancing the expression of ZjANS and ZjUGT79B1 .

Anthocyanins and PAs are the two most common flavonoids, which are widely present among diverse species. Great progress has been made in their synthesis and regulation. In this study, we analyzed the metabolic fluxes from their synthetic precursor leucoanthocyanins, which were obtained by overexpression of dihydroflavonol 4-reductase (DFR) in vitro and in vivo . The unstable product leucocyanidin generated in the CsDFRa enzymatic reaction was easily converted into C-type carbocations under weak acidic conditions, which could be further involved in the synthesis of C-type PAs in vitro . Additionally, the metabolites in tobacco overexpressing CsDFRa and Arabidopsis thaliana DFR and anthocyanidin synthase (ANS) mutants were investigated. In CsDFRa transgenic tobacco, the content of anthocyanins in the petals was greatly increased, but no catechin or PA was detected. In A. thaliana , EC-type carbocation was mainly accumulated in the wild type (WT), and the C-type carbocation was only detected in the ans mutant. In tea plant, the accumulation of C-type PAs is strong positively correlated with the expression of CsDFRa . In summary, leucocyanidin is not only involved in the synthesis of downstream anthocyanin and epicatechin but also can be converted into C-type carbocation to participate in the synthesis of C-type PAs. Hence, from leucocyanidin, three metabolic fluxes were formed toward catechin, cyanidin, and C-type carbocation. These results enriched the metabolic fluxes of leucoanthocyanins and further elaborated the roles of DFR in the process of C-type PA formation.

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.)

Light is indispensable for the anthocyanin accumulation of red pear ( Pyrus pyrifolia ). Anthocyanin biosynthesis is catalyzed by a series of enzymes encoded by structural genes, which are regulated by a MYB-basic/helix-loop-helix-WD repeat (MYB-bHLH-WDR [MBW]) complex. The bHLH proteins of subgroup (SG) IIIf are believed to be involved in the regulation of anthocyanin accumulation. In this study, we revealed that pear PpbHLH64, which belongs to SGIIIb, positively regulates anthocyanin biosynthesis and is regulated by light at the transcriptional and posttranslational levels. Specifically, an exposure to light induced PpbHLH64 expression and anthocyanin accumulation in pear fruit and calli. Under light conditions, pear calli overexpressing PpbHLH64 exhibited enhanced red coloration, whereas the anthocyanin accumulation decreased in the PpbHLH64 -RNA interference calli. Additionally, the transient overexpression of PpbHLH64 in pear fruit peel increased anthocyanin accumulation, whereas the virus-induced gene silencing of PpbHLH64 had the opposite effect. Further analyses indicated that PpbHLH64 is a transcriptional activator that directly binds to the promoter of UDP-GLUCOSE:FLAVONOID 3-O-GLYCOSYLTRANFERASE to upregulate expression. Moreover, PpbHLH64 interacted with PpMYB10, but not with PpMYB114, to form an MBW complex that significantly induces the accumulation of anthocyanins. Furthermore, PpbHLH64 was targeted by CONSTITUTIVE PHOTOMORPHOGENIC1 in darkness for subsequent degradation by the 26S proteasome. A genetic analysis indicated that PpbHLH64 functions downstream of B-BOX18, a component of the light signal transduction pathway. However, we were unable to detect the direct interaction between PpbHLH64 and PpBBX18. The characterization of PpbHLH64 in this study highlights the importance of SGIIIb bHLH proteins for light-induced anthocyanin accumulation., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

During the course of evolution of land plants, different classes of flavonoids, including flavonols and anthocyanins, sequentially emerged, facilitating adaptation to the harsh terrestrial environment. Flavanone 3β-hydroxylase (F3H), an enzyme functioning in flavonol and anthocyanin biosynthesis and a member of the 2-oxoglutarate-dependent dioxygenase (2-ODD) family, catalyzes the hydroxylation of ( 2S )-flavanones to dihydroflavonols, but its origin and evolution remain elusive. Here, we demonstrate that functional flavone synthase Is (FNS Is) are widely distributed in the primitive land plants liverworts and evolutionarily connected to seed plant F3Hs. We identified and characterized a set of 2-ODD enzymes from several liverwort species and plants in various evolutionary clades of the plant kingdom. The bifunctional enzyme FNS I/F2H emerged in liverworts, and FNS I/F3H evolved in Physcomitrium ( Physcomitrella ) patens and Selaginella moellendorffii , suggesting that they represent the functional transition forms between canonical FNS Is and F3Hs. The functional transition from FNS Is to F3Hs provides a molecular basis for the chemical evolution of flavones to flavonols and anthocyanins, which contributes to the acquisition of a broader spectrum of flavonoids in seed plants and facilitates their adaptation to the terrestrial ecosystem., (© 2020 American Society of Plant Biologists. 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.)

We examined the effects of high temperature (HT) at veraison (the onset of ripening) on coloration and anthocyanin biosynthesis in berry skins of 'Kyoho' grapevines (Vitis labruscana L.). The vines were subjected to control, HT (6 °C higher than the control for 10 days), and intermittent HT (IHT; 6 °C higher than the control for 4 days followed by control temperature for 3 days and then 6 °C higher than the control for another 3 days) conditions from 50 to 60 days after full bloom (DAFB) in temperature-controlled rooms. Under control conditions, berry skins were tinted purple from 55 DAFB and turned to reddish-purple thereafter until 80 DAFB, concurrently with the anthocyanin accumulation. The HT and IHT treatments greatly inhibited the coloration and anthocyanin accumulation, with greater inhibition by the HT treatment. The HT and IHT treatments significantly inhibited the expressions of early (EBGs) and late anthocyanin biosynthetic genes (LBGs), and the transcription factor gene VlMYBA2. Abscisic acid (ABA) contents in the control berry skins increased from 50 DAFB, peaked at 55 DAFB, and decreased thereafter. The HT and IHT treatments reduced the increase in ABA contents, with no significant difference between HT- and IHT-treated vines. Gibberellin (GA) contents decreased during veraison in the berry skins of control and IHT-treated vines, but remained unchanged in those of HT-treated vines. These results suggest that the coloration and anthocyanin biosynthesis in berry skins are associated with changes in the ABA/GA ratio., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Anthocyanins are polyphenol compounds that render various hues of pink, red, purple, and blue in flowers, vegetables, and fruits. Anthocyanins also play significant roles in plant propagation, ecophysiology, and plant defense mechanisms. Structurally, anthocyanins are anthocyanidins modified by sugars and acyl acids. Anthocyanin colors are susceptible to pH, light, temperatures, and metal ions. The stability of anthocyanins is controlled by various factors, including inter and intramolecular complexations. Chromatographic and spectrometric methods have been extensively used for the extraction, isolation, and identification of anthocyanins. Anthocyanins play a major role in the pharmaceutical; nutraceutical; and food coloring, flavoring, and preserving industries. Research in these areas has not satisfied the urge for natural and sustainable colors and supplemental products. The lability of anthocyanins under various formulated conditions is the primary reason for this delay. New gene editing technologies to modify anthocyanin structures in vivo and the structural modification of anthocyanin via semi-synthetic methods offer new opportunities in this area. This review focusses on the biogenetics of anthocyanins; their colors, structural modifications, and stability; their various applications in human health and welfare; and advances in the field.

The MYB transcription factors are involved in the regulation of plant secondary metabolism, cell development and morphogenesis, and stress response. Here, a full-length, 816-bp NtMYB4a cDNA, which encodes a protein comprising 271 amino acids, was isolated from tobacco leaves. Phylogenetic analysis revealed that NtMYB4a is most similar to Nicotiana. attenuata MYB4, followed by Eriobotrya japonica MYB4, and NtMYB4a clustered with transcriptional activators rather than repressors. Subcellular localization assays showed that NtMYB4 localized in the nucleus, membrane, and cytoplasm. Expression analyses revealed differential expression of NtMYB4a among different tissues and organs and between different developmental stages, with most expression occurring in the stems and leaves during the full-bloom stage. Moreover, NtMYB4a expression was induced by cold, NaCl, PEG, abscisic acid, methyl jasmonate, and dark stressors, and the expression patterns and maximum expression levels varied with the type of stress. Overexpression of NtMYB4a upregulated NtPAL, Nt4CL, NtCHS, NtCHI, NtF3H, NtDFR, NtANS, and NtUFGT, which resulted in increased anthocyanin content in the tobacco corolla and darker colors. However, CRISPR/Cas9-mediated knockout of NtMYB4a downregulated NtPAL, NtC4H, Nt4CL, NtCHS, NtCHI, NtF3H, NtANS, and NtUFGT, which resulted in reduced anthocyanin content, and lighter corolla colors. These results indicated that NtMYB4a positively regulates anthocyanin biosynthesis and is involved in abiotic stress responses in tobacco plants., 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 B.V. All rights reserved.)

Plant secondary metabolism is complex in its diverse chemical composition and dynamic regulation of biosynthesis. How the functional diversification of enzymes contributes to the diversity is largely unknown. In the flavonoids pathway, dihydroflavonol 4-reductase (DFR) is a key enzyme mediating dihydroflavanol into anthocyanins biosynthesis. Here, the DFR homolog was identified from Camellia nitidissima Chi. ( CnDFR ) which is a unique species of the genus Camellia with golden yellow petals. Sequence analysis showed that CnDFR possessed not only conserved catalytic domains, but also some amino acids peculiar to Camellia species. Gene expression analysis revealed that CnDFR was expressed in all tissues and the expression of CnDFR was positively correlated with polyphenols but negatively with yellow coloration. The subcellular localization of CnDFR by the tobacco infiltration assay showed a likely dual localization in the nucleus and cell membrane. Furthermore, overexpression transgenic lines were generated in tobacco to understand the molecular function of CnDFR . The analyses of metabolites suggested that ectopic expression of CnDFR enhanced the biosynthesis of polyphenols, while no accumulation of anthocyanins was detected. These results indicate a functional diversification of the reductase activities in Camellia plants and provide molecular insights into the regulation of floral color.

Anthocyanins are a group of important secondary metabolites, functioning as colorant in plant organs as well as protective agents against several stresses. Sub-red plant (Rs) cottons, accumulating moderate level of anthocyanins in shoots, had increased photosynthesis efficiency compared to green- (GL) and red-plant (R1) cottons. The present work aimed to clarify the molecular base of anthocyanin regulation in Rs cotton. It was found that GhPAP1A was significantly up-regulated in Rs plants compared to GL cottons, but its expression level is lower than that of GhPAP1D in R1 plants. Virus induced gene silencing of GhPAP1s inhibited the red pigmentation in Rs plants. Comparative cloning revealed a 50-bp tandem repeat in the promoter of GhPAP1A in Rs cotton, which showed stronger activity to drive the expression of downstream genes in petals. Considered that the coding sequence of GhPAP1As from Rs and GL cottons had similar functions to promote anthocyanin biosynthesis in transgenic tobaccos, we attributed moderate anthocyanin accumulation in Rs cotton to increased transcription of GhPAP1A, resulted from varied promoter structure. Our works suggested GhPAP1s as useful tool to manipulate anthocyanin level and several breeding targets, including herbivore- and pathogen- resistance, high photosynthesis efficiency and colored fibers.

Purple leaves are rich in health-protecting anthocyanins and food colorants in Brassica juncea. But the causal gene, which is related to leaf color formation, have not been reported in B. juncea. Anthocyanins mainly accumulated throughout the adaxial and abaxial epidermal leaf cells of purple leaves. A genetic analysis indicated that an incompletely dominant gene controls the purple leaf trait in B. juncea. Furthermore, the BjPur gene, which increased anthocyanin accumulation in purple-leaf mustard, was cloned. Blast and phylogenetic analyses revealed that BjPur encodes a new R2R3-MYB transcription factor. Sequence analysis of two alleles revealed a DNA sequence insertion in the first intron of BjPur in green leaves parent line (LY) when compared with the BjPur gene in the purple-leaf parent line (ZY). And this insertion greatly reduced the transcription of BjPur in green leaves. In purple-leaf plants, the transcript level of BjPur was significantly higher in leaves than in roots, stems, siliques, and flower buds. Additionally, molecular markers linked to leaf color were developed to distinguish different genotypes of B. juncea. These results will be helpful for the genetic improvement of the purple leaf color in B. juncea.

Anthocyanins are plant-specific pigments, the biosynthesis of which is stimulated by pathogen infection in several plant species. A. thaliana seedlings injected with airborne fungi can accumulate a high content of anthocyanins. The mechanism involved in fungus-induced anthocyanin accumulation in plants has not been fully described. In this study, the fungus Penicillium corylophilum (P. corylophilum), isolated from an Arabidopsis culture chamber, triggered jasmonic acid (JA), salicylic acid (SA), and anthocyanin accumulation in A. thaliana. Inhibitors of JA and SA biosynthesis suppressed the anthocyanin accumulation induced by P. corylophilum. The anthocyanin content was minimal in both the null mutant of JA-receptor coi1 and the null mutant of SA-receptor npr1 under P. corylophilum stimulation. The results indicate that JA and SA signaling mediated fungus-induced anthocyanin biosynthesis in A. thaliana. P. corylophilum led to different levels of anthocyanin generation in null mutants for MYB75, bHLH, EGL3, and GL3 transcription factors and WD40 protein, demonstrating that multiple MYB-bHLH-WD40 transcription factor complexes participated in fungus-induced anthocyanin accumulation in A. thaliana. The present study will help further elucidate the mechanism of plant resistance to pathogen infection., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The application of one or two short-term treatments with high levels of CO 2 during 3 days at 0 °C maintained the quality of Autumn Royal table grapes (Vitis vinifera) during storage at 0 °C. We have analyzed how the application of a 3-day gaseous treatment, for one or two times at 0 °C, influences on common (VviPAL, VviCHS, VviCHI, VviF3'H, VviF3'5'H, VviF3H and VviLDOX) and branch-specific (VviFLS1, VviLAR1, VviLAR2, VviANR and VviUFGT) flavonoid gene expression in the skin of Autumn Royal table grapes. Likewise, the content of flavonols, flavan-3-ols and anthocyanins were identified with Q-TOF equipment and quantified by HPLC-quadrupole together with the total phenolic content and the antioxidant capacity by the ABTS and FRAP methods. Moreover, we have also used a solid-state voltammetry methodology to compare the effect of the application of one or two gaseous treatments in the skin of table grapes stored at 0 °C. Results revealed that the application of one or two gaseous treatments modulated the expression of flavonoid gene expression and the levels of catechin, in the case of one application, or quercetin-3-glucoside and five anthocyanins in fruit treated twice, maintaining their levels after 28 days of storage at 0 °C similar to those recorded in freshly harvested fruit. Satisfactorily, the electrochemical approach was useful to distinguish between treated and non-treated samples not only in the first stage of storage but also after 16 days., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Purple-fleshed sweetpotato (PFSP) accumulates high amounts of anthocyanins that are beneficial to human health. Although biosynthesis of such secondary metabolites has been well studied in aboveground organs of many plants, the mechanisms underlying anthocyanin accumulation in underground tuberous roots of sweetpotato are less understood. To identify genes and metabolites involved in anthocyanin accumulation in sweetpotato, we performed comparative transcriptomic and metabolomic analysis of (PFSP) and white-fleshed sweetpotato (WFSP). Anthocyanin-targeted metabolome analysis revealed that delphinidin, petunidin, and rosinidin were the key metabolites conferring purple pigmentation in PFSP as they were highly enriched in PFSP but absent in WFSP. Transcriptomic analysis identified 358 genes that were potentially implicated in multiple pathways for the biosynthesis of anthocyanins. Although most of the genes were previously known for their roles in anthocyanin biosynthesis, we identified 26 differentially expressed genes that are involved in Aux/IAA-ARF signaling. Gene-metabolite correlation analysis also revealed novel genes that are potentially involved in the anthocyanin accumulation in sweetpotato. Taken together, this study provides insights into the genes and metabolites underlying anthocyanin enrichment in underground tuberous roots of sweetpotato., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Dihydroflavonol 4-reductase (DFR) catalyzes a committed step in anthocyanin and proanthocyanidin biosynthesis by reducing dihydroflavonols to leucoanthocyanidins. However, the role of this enzyme in determining flower color in the economically important crop chrysanthemum ( Chrysanthemum morifolium Ramat.) is unknown. Here, we isolated cDNAs encoding DFR from two chrysanthemum cultivars, the white-flowered chrysanthemum "OhBlang" (CmDFR-OB) and the red-flowered chrysanthemum "RedMarble" (CmDFR-RM) and identified variations in the C-terminus between the two sequences. An enzyme assay using recombinant proteins revealed that both enzymes catalyzed the reduction of dihydroflavonol substrates, but CmDFR-OB showed significantly reduced DFR activity for dihydrokaempferol (DHK) substrate as compared with CmDFR-RM. Transcript levels of anthocyanin biosynthetic genes were consistent with the anthocyanin contents at different flower developmental stages of both cultivars. The in planta complementation assay, using Arabidopsis thaliana dfr mutant ( tt3-1 ), revealed that CmDFR-RM, but not CmDFR-OB, transgenes restored defective anthocyanin biosynthesis of this mutant at the seedling stage, as well as proanthocyanidin biosynthesis in the seed. The difference in the flower color of two chrysanthemums can be explained by the C-terminal variation of CmDFR combined with the loss of CmF3H expression during flower development.

Azaleas (Ericaceae) comprise one of the most diverse ornamental plants, renowned for their cultural and economic importance. We present a chromosome-scale genome assembly for Rhododendron simsii, the primary ancestor of azalea cultivars. Genome analyses unveil the remnants of an ancient whole-genome duplication preceding the radiation of most Ericaceae, likely contributing to the genomic architecture of flowering time. Small-scale gene duplications contribute to the expansion of gene families involved in azalea pigment biosynthesis. We reconstruct entire metabolic pathways for anthocyanins and carotenoids and their potential regulatory networks by detailed analysis of time-ordered gene co-expression networks. MYB, bHLH, and WD40 transcription factors may collectively regulate anthocyanin accumulation in R. simsii, particularly at the initial stages of flower coloration, and with WRKY transcription factors controlling progressive flower coloring at later stages. This work provides a cornerstone for understanding the underlying genetics governing flower timing and coloration and could accelerate selective breeding in azalea.

The flavonoids, one of the largest classes of plant secondary metabolites, are found in lineages that span the land plant phylogeny and play important roles in stress responses and as pigments. Perhaps the most well-studied flavonoids are the anthocyanins that have human health benefits and help plants attract pollinators, regulate hormone production, and confer resistance to abiotic and biotic stresses. The canonical biochemical pathway responsible for the production of these pigments is well-characterized for flowering plants yet its conservation across deep divergences in land plants remains debated and poorly understood. Many early land plants such as mosses, liverworts, and ferns produce flavonoid pigments, but their biosynthetic origins and homologies to the anthocyanin pathway remain uncertain. We conducted phylogenetic analyses using full genome sequences representing nearly all major green plant lineages to reconstruct the evolutionary history of the anthocyanin biosynthetic pathway then test the hypothesis that genes in this pathway are present in early land plants. We found that the entire pathway was not intact until the most recent common ancestor of seed plants and that orthologs of many downstream enzymes are absent from seedless plants including mosses, liverworts, and ferns. Our results also highlight the utility of phylogenetic inference, as compared to pairwise sequence similarity, in orthology assessment within large gene families that have complex duplication-loss histories. We suggest that the production of red-violet flavonoid pigments widespread in seedless plants, including the 3-deoxyanthocyanins, requires the activity of novel, as-yet discovered enzymes, and represents convergent evolution of red-violet coloration across land plants., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Key Message: FtMYB18 plays a role in the repression of anthocyanins and proanthocyanidins accumulation by strongly down-regulating the CHS and DFR genes in Tartary buckwheat, and the C5 motif plays an important role in this process. Anthocyanins and proanthocyanidins (PAs) are important flavonoids in Tartary buckwheat (Fagopyrum tataricum Gaertn.), which provides various vibrant color and stronge abiotic stress resistance. Their synthesis is generally regulated by MYB transcription factors at transcription level. However, the negative regulations of MYB and their effects on flavonol metabolism are poorly understood. A SG4-like MYB subfamily TF, FtMYB18, containing C5 motif was identified from Tartary buckwheat. The expression of FtMYB18 was not only showed a negative correlation with anthocyanins and PAs content but also strongly respond to MeJA and ABA. As far as the transgenic lines with FtMYB18 overexpression, anthocyanins and PAs accumulations were decreased through down-regulating expression levels of NtCHS and NtDFR in tobacco, AtDFR and AtTT12 in Arabidopsis, FtCHS, FtDFR and FtANS in Tartary buckwheat hairy roots, respectively. However, FtMYB18 showed no effect on the FLS gene expression and the metabolites content in flavonol synthesis branch. The further molecular interaction analysis indicated FtMYB18 could mediate the inhibition of anthocyanins and PAs synthesis by forming MBW transcriptional complex with FtTT8 and FtTTG1, or MYB-JAZ complex with FtJAZ1/-3/-4/-7. Importantly, in FtMYB18 mutant lines with C5 motif deletion (FtMYB18-C), both of anthocyanins and PAs accumulations had recovered to the similar level as that in wild type, which was attributed to the weakened MBW complex activity or the deficient molecular interaction between FtMYB18ΔC5 with FtJAZ3/-4. The results showed that FtMYB18 could suppress anthocyanins and PAs synthesis at transcription level through the specific interaction of C5 motif with other proteins in Tartary buckwheat.