Activation Of Anthocyanin Biosynthesis Research Articles

Regulation of Anthocyanin Accumulation in Tomato Solanum lycopersicum L. by Exogenous Synthetic dsRNA Targeting Different Regions of SlTRY Gene.

RNA interference (RNAi) is a regulatory and protective mechanism that plays a crucial role in the growth, development, and control of plant responses to pathogens and abiotic stresses. In spray-induced gene silencing (SIGS), exogenous double-stranded RNAs (dsRNA) are used to efficiently regulate target genes via plant surface treatment. In this study, we aimed to evaluate the effect of specific exogenous dsRNAs on silencing different regions (promoter, protein-coding and intron) of the target SlTRY tomato gene, encoding an R3-type MYB repressor of anthocyanin biosynthesis. We also assessed the impact of targeting different SlTRY regions on the expression of genes involved in anthocyanin and flavonoid biosynthesis. This study demonstrated the critical importance of selecting the appropriate gene target region for dsRNA action. The highest inhibition of the SlTRY gene expression and activation of anthocyanin biosynthesis was achieved by dsRNA complementary to the protein-coding region of SlTRY gene, compared with dsRNAs targeting the SlTRY promoter or intron regions. Silencing the SlTRY gene increased the content of anthocyanins and boosted levels of other substances in the phenylpropanoid pathway, such as caffeoyl putrescine, chlorogenic acid, ferulic acid glucoside, feruloyl quinic acid, and rutin. This study is the first to examine the effects of four different dsRNAs targeting various regions of the SlTRY gene, an important negative regulator of anthocyanin biosynthesis.

Multiomics analyses of the effects of LED white light on the ripening of apricot fruits

IntroductionApricot (Prunus armeniaca L.) fruits are highly perishable and prone to quality deterioration during storage and transportation. ObjectivesTo investigate the effects of LED white light treatment on postharvest ripening of fruits using metabolomics, transcriptomics, and ATAC-Seq analysis. MethodsFruits were exposed to 5 μmol m−2 s−1 LED white light for 12 h followed by 12 h of darkness at 20 °C daily for 12 days. The effects of the treatments on the physiological and nutritional quality of the fruits were evaluated. These data were combined with transcriptomic, metabolomic, and ATAC-Seq data from fruits taken on 8 d of treatment to provide insight into the potential mechanism by which LED treatment delays ripening. ResultsLED treatment activated pathways involved in ascorbate and aldarate metabolism and flavonoid and phenylpropanoid biosynthesis. Specifically, LED treatment increased the expression of UDP-sugar pyrophosphorylase (USP), L-ascorbate peroxidase (AO), dihydroflavonol 4-reductase (DFR), chalcone synthase (CHS), and caffeoyl-CoA O-methyltransferase (CCOAOMT1), leading to the accumulation of caffeoyl quinic acid, epigallocatechin, and dihydroquercetin and the activation of anthocyanin biosynthesis. LED treatment also affected the expression of genes associated with plant hormone signal transduction, fruit texture and color transformation, and antioxidant activity. The notable genes affected by LED treatment included 1-aminocyclopropane-1-carboxylate synthase (ACS), 1-aminocyclopropane-1-carboxylate oxidase (ACO), hexokinase (HK), lipoxygenase (LOX), malate dehydrogenase (MDH), endoglucanase (CEL), various transcription factors (TCP, MYB, EFR), and peroxidase (POD). ATAC-Seq analysis further revealed that LED treatment primarily regulated phenylpropanoid biosynthesis. ConclusionThe results obtained in this study provide insights into the effects of LED light exposure on apricot fruits ripening. LEDs offer a promising approach for extending the shelf life of other fruits and vegetables.

A transient expression tool box for anthocyanin biosynthesis in Nicotiana benthamiana.

Transient expression in Nicotiana benthamiana offers a robust platform for the rapid production of complex secondary metabolites. It has proven highly effective in helping identify genes associated with pathways responsible for synthesizing various valuable natural compounds. While this approach has seen considerable success, it has yet to be applied to uncovering genes involved in anthocyanin biosynthetic pathways. This is because only a single anthocyanin, delphinidin 3-O-rutinoside, can be produced in N. benthamiana by activation of anthocyanin biosynthesis using transcription factors. The production of other anthocyanins would necessitate the suppression of certain endogenous flavonoid biosynthesis genes while transiently expressing others. In this work, we present a series of tools for the reconstitution of anthocyanin biosynthetic pathways in N. benthamiana leaves. These tools include constructs for the expression or silencing of anthocyanin biosynthetic genes and a mutant N. benthamiana line generated using CRISPR. By infiltration of defined sets of constructs, the basic anthocyanins pelargonidin 3-O-glucoside, cyanidin 3-O-glucoside and delphinidin 3-O-glucoside could be obtained in high amounts in a few days. Additionally, co-infiltration of supplementary pathway genes enabled the synthesis of more complex anthocyanins. These tools should be useful to identify genes involved in the biosynthesis of complex anthocyanins. They also make it possible to produce novel anthocyanins not found in nature. As an example, we reconstituted the pathway for biosynthesis of Arabidopsis anthocyanin A5, a cyanidin derivative and achieved the biosynthesis of the pelargonidin and delphinidin variants of A5, pelargonidin A5 and delphinidin A5.

Activation of anthocyanin biosynthesis in high light - what is the initial signal?

Due to their sessile nature, plants cannot escape adverse environmental conditions and evolved mechanisms to cope with sudden environmental changes. The reaction to variations in abiotic factors, also summarized as acclimation response, affects all layers of cellular functions and involves rapid modification of enzymatic activities, the metabolome, proteome and transcriptome on different timescales. One trait of plants acclimating to high light (HL) is the rapid transcriptional activation of the flavonoid biosynthesis (FB) pathway resulting in the accumulation of photoprotective and antioxidative flavonoids, such as flavonols and anthocyanins, in the leaf tissue. Although enormous progress has been made in identifying enzymes and transcriptional regulators of FB by forward and reverse genetic approaches in the past, the signals and signalling pathways permitting the conditional activation of FB in HL are still debated. With this Tansley Insight, we summarize the current knowledge on the proposed signals and downstream factors involved in regulating FB and will discuss their contribution to, particularly, HL-induced accumulation of anthocyanins.

Identification of CaAN3 as a fruit-specific regulator of anthocyanin biosynthesis in pepper (Capsicum annuum).

The novel gene CaAN3 encodes an R2R3 MYB transcription factor that regulates fruit-specific anthocyanin accumulation. The key regulatory gene CaAN2 encodes an R2R3 MYB transcription factor that regulates anthocyanin biosynthesis in various tissues in pepper (Capsicum annuum). However, CaAN2 is not expressed in certain pepper accessions showing fruit-specific anthocyanin accumulation. In this study, we identified the novel locus CaAN3 as a regulator of fruit-specific anthocyanin biosynthesis, using an F2 population derived from a hybrid cultivar with purple immature fruits and segregating for CaAN3. We extracted total RNA, assembled two RNA pools according to fruit color, and carried out bulked segregant RNA sequencing. We aligned the raw reads to the pepper reference genome Dempsey and identified 6,672 significant single nucleotide polymorphisms (SNPs) by calculating the Δ(SNP-index) between the two pools. We then conducted molecular mapping to delimit the target region of CaAN3 to the interval 184.6-186.4 Mbp on chromosome 10. We focused on Dem.v1.00043895, encoding an R2R3 MYB transcription factor, as the strongest candidate gene. Sequence analysis revealed four insertion/deletion polymorphisms in the promoter region of the green CaAN3 allele. We employed virus-induced gene silencing and transient overexpression assays to characterize the function of the candidate gene. When Dem.v1.00043895 was silenced in pepper, anthocyanin accumulation decreased in the pericarp, while the transient overexpression of Dem.v1.00043895 in Nicotiana benthamiana leaves resulted in the accumulation of anthocyanins around the infiltration sites. These results showed that Dem.v1.00043895 is CaAN3, an activator of anthocyanin biosynthesis in pepper fruits.

A long noncoding RNA functions in high-light-induced anthocyanin accumulation in apple by activating ethylene synthesis.

Anthocyanin production in apple (Malus domestica) fruit and their consequent coloration can be induced by high-light treatment. The hormone ethylene is also essential for this coloration, but the regulatory relationships that link ethylene and light with anthocyanin-associated coloration are not well defined. In this study, we observed that high-light treatment of apple fruit increased anthocyanin accumulation more than moderate-light treatment did and was the main contributor of induced ethylene production and activation of anthocyanin biosynthesis. A transcriptome study of light-treated apple fruit suggested that a long noncoding RNA (lncRNA), MdLNC610, the corresponding gene of which is physically located downstream from the 1-aminocyclopropane-1-carboxylate oxygenase (ACO) ethylene biosynthesis gene MdACO1, likely affects anthocyanin biosynthesis under high-light treatment. Expression and promoter β-glucuronidase reporter analyses further showed that MdLNC610 upregulates expression of MdACO1 and so likely participates in high-light-induced ethylene biosynthesis. Overexpression of MdACO1 and MdLNC610 in apple fruit and calli indicated that a major increase in MdLNC610 expression activates MdACO1 expression, thereby causing an increase in ethylene production and anthocyanin levels. These results suggest that MdLNC610 participates in the regulation of high-light-induced anthocyanin production by functioning as a positive regulator to promote MdACO1 expression and ethylene biosynthesis. Our study provides insights into the relationship between mRNA and lncRNA networks in the ethylene biosynthetic pathway and anthocyanin accumulation in apple fruit.

A Transcriptional Network Promotes Anthocyanin Biosynthesis in Tomato Flesh

Dietary anthocyanins are important health-promoting antioxidants that make a major contribution to the quality of fruits. It is intriguing that most tomato cultivars do not produce anthocyanins in fruit. However, the purple tomato variety Indigo Rose, which has the dominant Aft locus combined with the recessive atv locus from wild tomato species, exhibits light-dependent anthocyanin accumulation in the fruit skin. Here, we report that Aft encodes a functional anthocyanin activator named SlAN2-like, while atv encodes a nonfunctional version of the anthocyanin repressor SlMYBATV. The expression of SlAN2-like is responsive to light, and the functional SlAN2-like can activate the expression of both anthocyanin biosynthetic genes and their regulatory genes, suggesting that SlAN2-like acts as a master regulator in the activation of anthocyanin biosynthesis. We further showed that cultivated tomatoes contain nonfunctional alleles of SlAN2-like and therefore fail to produce anthocyanins. Consistently, expression of a functional SlAN2-like gene driven by the fruit-specific promoter in a tomato cultivar led to the activation of the entire anthocyanin biosynthesis pathway and high-level accumulation of anthocyanins in both the peel and flesh. Taken together, our study exemplifies that efficient engineering of complex metabolic pathways could be achieved through tissue-specific expression of master transcriptional regulators.

Ectopic Expression of Multiple Chrysanthemum (Chrysanthemum × morifolium) R2R3-MYB Transcription Factor Genes Regulates Anthocyanin Accumulation in Tobacco.

The generation of chrysanthemum (Chrysanthemum × morifolium) flower color is mainly attributed to the accumulation of anthocyanins. In the anthocyanin biosynthetic pathway in chrysanthemum, although all of the structural genes have been cloned, the regulatory function of R2R3-MYB transcription factor (TF) genes, which play a crucial role in determining anthocyanin accumulation in many ornamental crops, still remains unclear. In our previous study, four light-induced R2R3-MYB TF genes in chrysanthemum were identified using transcriptomic sequencing. In the present study, we further investigated the regulatory functions of these genes via phylogenetic and alignment analyses of amino acid sequences, which were subsequently verified by phenotypic, pigmental, and structural gene expression analyses in transgenic tobacco lines. As revealed by phylogenetic and alignment analyses, CmMYB4 and CmMYB5 were phenylpropanoid and flavonoid repressor R2R3-MYB genes, respectively, while CmMYB6 was an activator of anthocyanin biosynthesis, and CmMYB7 was involved in regulating flavonol biosynthesis. Compared with wild-type plants, the relative anthocyanin contents in the 35S:CmMYB4 and 35S:CmMYB5 tobacco lines significantly decreased (p < 0.05), while for 35S:CmMYB6 and 35S:CmMYB7, the opposite result was obtained. Both in the 35S:CmMYB4 and 35S:CmMYB5 lines, the relative expression of several anthocyanin biosynthetic genes in tobacco was significantly downregulated (p < 0.05); on the contrary, several genes were upregulated in the 35S:CmMYB6 and 35S:CmMYB7 lines. These results indicate that CmMYB4 and CmMYB5 negatively regulate anthocyanin biosynthesis in chrysanthemum, while CmMYB6 and CmMYB7 play a positive role, which will aid in understanding the complex mechanism regulating floral pigmentation in chrysanthemum and the functional divergence of the R2R3-MYB gene family in higher plants.

Effect of low temperature-storage on the proteome of ‘Moro’ blood orange flesh

Anthocyanins are a subclass of flavonoid pigments with important therapeutic properties beneficial to human health. Blood oranges are rich in these pigments, which present synergistic effects (i.e., anti-inflammatory and anti-obesity) with other orange juice phytonutrients. Anthocyanin biosynthesis is cold induced in blood citrus cultivars, requiring a broad day/night thermal range to get purple pigmentation in fruit. This cold-dependency limits geographically a reliable quality for commercial production to only a few regions worldwide. For example, cultivation of blood oranges under tropical/subtropical climates, as those of Brazil, yields fruit with very low level or lack of anthocyanins. A feasible alternative in tropical countries to enhance anthocyanin content is cold postharvest storage, as it has been shown that anthocyanin synthesis is induced when fruits are kept below 10°C for a few weeks after harvesting. Blood oranges kept at 9°C showed higher expression levels of anthocyanin biosynthetic genes, increased anthocyanin content and reached a darker purple coloration than those stored at 4°C. In order to gain insight on the effect of storage temperature on activation of anthocyanin biosynthesis and accumulation as well as on purple orange coloration, we have investigated the proteome of 'Moro' sweet orange [Citrus sinensis (L.) Osbeck] fruit stored either at 4°C (low temperature) or at 9°C (moderate temperature). Results on increased or reduced accumulation of specific proteins in the 'Moro' orange pulp upon storage at different temperatures reinforces that postharvest storage at 9°C could be better to enhance anthocyanin biosynthesis and accumulation on blood orange juices.

Effect of low temperature-storage on the proteome of ‘Moro’ blood orange flesh

Anthocyanins are a subclass of flavonoid pigments with important therapeutic properties beneficial to human health. Blood oranges are rich in these pigments, which present synergistic effects (i.e., anti-inflammatory and anti-obesity) with other orange juice phytonutrients. Anthocyanin biosynthesis is cold induced in blood citrus cultivars, requiring a broad day/night thermal range to get purple pigmentation in fruit. This cold-dependency limits geographically a reliable quality for commercial production to only a few regions worldwide. For example, cultivation of blood oranges under tropical/subtropical climates, as those of Brazil, yields fruit with very low level or lack of anthocyanins. A feasible alternative in tropical countries to enhance anthocyanin content is cold postharvest storage, as it has been shown that anthocyanin synthesis is induced when fruits are kept below 10°C for a few weeks after harvesting. Blood oranges kept at 9°C showed higher expression levels of anthocyanin biosynthetic genes, increased anthocyanin content and reached a darker purple coloration than those stored at 4°C. In order to gain insight on the effect of storage temperature on activation of anthocyanin biosynthesis and accumulation as well as on purple orange coloration, we have investigated the proteome of 'Moro' sweet orange [Citrus sinensis (L.) Osbeck] fruit stored either at 4°C (low temperature) or at 9°C (moderate temperature). Results on increased or reduced accumulation of specific proteins in the 'Moro' orange pulp upon storage at different temperatures reinforces that postharvest storage at 9°C could be better to enhance anthocyanin biosynthesis and accumulation on blood orange juices.

Brassica yellows virus\u2019 movement protein upregulates anthocyanin accumulation, leading to the development of purple leaf symptoms on Arabidopsis thaliana

Poleroviruses are widely distributed and often of great economic importance because they cause a variety of symptoms, such as the rolling of young leaves, leaf color changes, and plant decline, in infected plants. However, the molecular mechanism behind these viral-induced symptoms is still unknown. Here, we verified the pathogenicity of the polerovirus Brassica yellows virus (BrYV) by transforming its full-length amplicon into Arabidopsis thaliana, which resulted in many abnormal phenotypes. To better understand the interactions between BrYV and its host, global transcriptome profiles of the transgenic plants were compared with that of non-transgenic Arabidopsis plants. An association between the BrYV- induced purple leaf symptoms and the activation of anthocyanin biosynthesis was noted. Using the transgenic approach, we found that movement protein of BrYV was responsible for the induction of these coloration symptoms. Collectively, our findings demonstrate the BrYV’ pathogenicity and show that the BrYV-induced purple leaf symptom resulted from its movement protein stimulating anthocyanin accumulation.

Activation of anthocyanin biosynthesis by expression of the radish R2R3-MYB transcription factor gene RsMYB1

RsMYB1, a MYB TF of red radish origin, was characterized as a positive regulator to transcriptionally activate the anthocyanin biosynthetic machinery by itself in Arabidopsis and tobacco plants. Anthocyanins, providing the bright red-orange to blue-violet colors, are flavonoid-derived pigments with strong antioxidant activity that have benefits for human health. We isolated RsMYB1, which encodes an R2R3-MYB transcription factor (TF), from red radish plants (Raphanus sativus L.) that accumulate high levels of anthocyanins. RsMYB1 shows higher expression in red radish than in common white radish, in both leaves and roots, at different growth stages. Consistent with RsMYB1 function as an anthocyanin-promoting TF, red radishes showed higher expression of all six anthocyanin biosynthetic and two anthocyanin regulatory genes. Transient expression of RsMYB1 in tobacco showed that RsMYB1 is a positive regulator of anthocyanin production with better efficiency than the basic helix-loop-helix (bHLH) TF gene B-Peru. Also, the synergistic effect of RsMYB1 with B-Peru was larger than the effect of the MYB TF gene mPAP1D with B-peru. Arabidopsis plants stably expressing RsMYB1 produced red pigmentation throughout the plant, accompanied by up-regulation of the six structural and two regulatory genes for anthocyanin production. This broad transcriptional activation of anthocyanin biosynthetic machinery in Arabidopsis included up-regulation of TRANSPARENT TESTA8, which encodes a bHLH TF. These results suggest that overexpression of RsMYB1 promotes anthocyanin production by triggering the expression of endogenous bHLH genes as potential binding partners for RsMYB1. In addition, RsMYB1-overexpressing Arabidopsis plants had a higher antioxidant capacity than did non-transgenic control plants. Taken together, RsMYB1 is an actively positive regulator for anthocyanins biosynthesis in radish plants and it might be one of the best targets for anthocyanin production by single gene manipulation being applicable in diverse plant species.

Stability and fitness impact of the visually discernible Rosea1 marker in the Tobacco etch virus genome.

Antirrhinum majus Rosea1 (Ros1) is an MYB-related transcription factor that induces anthocyanin biosynthesis in plant tissues, and has been shown to be suitable for visual tracking of virus infection in plants. However, activation of anthocyanin biosynthesis has far reaching effects on plant physiology and could consequently have negative effects on viral replication. Therefore, viruses carrying the Ros1 marker might have a low fitness and consequently rapidly lose the marker. To compare the stability of the Ros1 marker, we generated Tobacco etch virus (TEV) based constructs containing either Ros1 or the enhanced green fluorescent protein (eGFP) between the NIb and CP cistrons (TEV-Ros1 and TEV-eGFP, respectively). We measured the within-host competitive fitness of both viruses by direct competitions with a common competitor during infection of Nicotiana tabacum. The fitness of TEV-Ros1 was significantly lower than that of TEV-eGFP, and both recombinant viruses had a significantly lower fitness than the wild-type virus. Nevertheless, after seven weeks of infection in N. tabacum, similar levels of marker gene instability where found for both viruses. Despite lower fitness of the marked virus, Ros1 is therefore a viable alternative marker for tracking viral infection in plants.

Molecular Cloning and Expression Analysis of an ANS Gene Encoding Anthocyanidin Synthase from Purple-Fleshed Sweet Potato [Ipomoea batatas (L.) Lam

Anthocyanidin synthase (ANS), a 2-oxoglutarate (2OG) iron-dependent oxygenase, catalyzes the penultimate step in the biosynthesis of anthocyanin. This reaction is responsible for the formation of the colored anthocyanidins from the colorless leucoanthocyanidins. A full-length cDNA was isolated from purple-fleshed sweet potato (Ipomoea batatas (L.) Lam) cv. Yamakawamurasaki, designated IbANS, containing a 1,086-bp open reading frame encoding a 362-amino-acid polypeptide. Multiple alignments revealed that the deduced IbANS protein had high identity to ANS proteins of other plants such as Ipomoea nil (90.8% identities), Ipomoea purpurea (91.4% identities), and Brassica juncea (72.7% identities). Structural analysis showed that the IbANS protein might belong to the 2OG and Fe(II)-dependent oxygenase, containing three binding sites of 2OG (H236, D238, and H292) and three binding sites of Fe(II) (Y221, R302, and S304). Phylogenetic tree analysis revealed that IbANS shared the close relationships with I. nil and I. purpurea. Southern blotting showed that there were two copies of the IbANS gene in this genome. Real-time quantitative polymerase chain reaction revealed that expression of the IbANS gene was highest in storage roots and lowest in leaves. IbANS was expressed most abundantly during the formation of storage roots. In five cultivars of sweet potato, IbANS expression was strongly associated with anthocyanin accumulation, suggesting that ANS gene expression was associated with activation of anthocyanin biosynthesis.

Activation of anthocyanin biosynthesis in Gerbera hybrida (Asteraceae) suggests conserved protein-protein and protein-promoter interactions between the anciently diverged monocots and eudicots.

We have identified an R2R3-type MYB factor, GMYB10, from Gerbera hybrida (Asteraceae) that shares high sequence homology to and is phylogenetically grouped together with the previously characterized regulators of anthocyanin pigmentation in petunia (Petunia hybrida) and Arabidopsis. GMYB10 is able to induce anthocyanin pigmentation in transgenic tobacco (Nicotiana tabacum), especially in vegetative parts and anthers. In G. hybrida, GMYB10 is involved in activation of anthocyanin biosynthesis in leaves, floral stems, and flowers. In flowers, its expression is restricted to petal epidermal cell layers in correlation with the anthocyanin accumulation pattern. We have shown, using yeast (Saccharomyces cerevisiae) two-hybrid assay, that GMYB10 interacts with the previously isolated bHLH factor GMYC1. Particle bombardment analysis was used to show that GMYB10 is required for activation of a late anthocyanin biosynthetic gene promoter, PGDFR2. cis-Analysis of the target PGDFR2 revealed a sequence element with a key role in activation by GMYB10/GMYC1. This element shares high homology with the anthocyanin regulatory elements characterized in maize (Zea mays) anthocyanin promoters, suggesting that the regulatory mechanisms involved in activation of anthocyanin biosynthesis have been conserved for over 125 million years not only at the level of transcriptional regulators but also at the level of the biosynthetic gene promoters.

Transient expression of maize anthocyanin regulatory genes influences anthocyanin production in white clover and peas

Transient expression of the maize anthocyanin regulatory genes, B-Peru and C1, was examined in maize cell suspensions, and in pea and white clover tissues in order to determine if these maize regulators can function in legume tissues. Optimal ratios and amounts of B-Peru and C1 genes for anthocyanin production were determined in maize cell suspensions. Activation of anthocyanin biosynthesis in legume tissues was observed following expression of the B-Peru and C1 genes, delivered in the pre-determined optimal ratio and amounts by microparticle bombardment. Tissue-specific expression of these genes was observed in the legumes tested and the exact sites of anthocyanin expression in pea depended on the cultivar tested. Maize anthocyanin regulatory genes can activate anthocyanin biosynthesis in legume tissues, but appear to be subject to tissue specific controls. Three of the four varieties of pea tested were able to accumulate anthocyanin when the B-Peru coding region alone was biolistically expressed.