Anthocyanin Gene Expression Research Articles

Transcriptomic analysis reveals biosynthesis genes and transcription factors related to leaf anthocyanin biosynthesis in Aglaonema commutatum

BackgroundAglaonema commutatum ‘Red Valentine’, as a foliage ornamental plant, is widely used for interior and exterior decoration because of its easy cultivation and management. However, reduced proportion of red foliage during large-scale production of A. commutatum seedlings is a frequent occurrence, which has considerable implications on the plant’s ornamental and market value. However, the molecular mechanisms underlying this phenomenon remain unclear.ResultsTo explore the molecular basis of the variation in leaf color of A. commutatum Red Valentine, we performed transcriptome sequencing with the Illumina platform using two different varieties of A. commutatum, namely Red Valentine and a green mutant, at three different stages of leaf development. We annotated 63,621 unigenes and 14,186 differentially expressed genes by pairwise comparison. Furthermore, we identified 26 anthocyanin biosynthesis structural genes. The transcript per million (TPM) values were significantly higher for Red Valentine than for the green mutant in all three developmental stages, consistent with the high anthocyanin content of Red Valentine leaves. We detected positive transcription factors that may be involved in the regulation of anthocyanin biosynthesis using BLAST and through correlation analysis. Downregulation of these transcription factors may downregulate the expression of anthocyanin genes. We obtained full-length cDNA of the anthocyanin biosynthesis and regulatory genes and constructed phylogenetic trees to ensure accuracy of the analysis.ConclusionsOur study provides insights into the molecular mechanisms underlying leaf variation in A. commutatum Red Valentine and may be used to facilitate the breeding of ornamental cultivars with high anthocyanin levels.

An approach to determining anthocyanin synthesis enzyme gene expression in an evolutionary context: an example from Erica plukenetii.

Floral colour in angiosperms can be controlled by variations in the expression of the genes of the anthocyanin pathway. Floral colour shifts influence pollinator specificity. Multiple shifts in floral colour occurred in the diversification of the genus Erica (Ericaceae), from plesiomorphic pink to, for example, red or white flowers. Variation in anthocyanin gene expression and its effects on floral colour in the red-, pink- and white-flowered Erica plukenetii species complex was investigated. Next generation sequencing, reverse transcriptase PCR and real-time reverse transcriptase quantitative PCR were used to quantify anthocyanin gene expression. Non-homologous mutations causing loss of expression of single genes were found, indicating that the cause was likely to be mutations in transcription factor binding sites upstream of the 5'-untranslated region of the genes, and this was confirmed by sequencing. Independent evolution and subsequent loss of expression of anthocyanin genes may have influenced diversification in the E. plukenetii species complex. The approach developed here should find more general application in studies on the role of floral colour shifts in diversification.

Comparative transcriptome analysis reveals a role for anthocyanin biosynthesis genes in the formation of purple peel in Minhou wild banana (Musa itinerans Cheesman)

ABSTRACTTo investigate the purple peel formation mechanism of the Minhou wild banana (Musa itinerans), epidermal cells of the purple and green parts of peel were subjected to RNA sequencing. In total, 3,640 differently expressed unigenes (DEGs) were identified. A KEGG enrichment analysis showed that these DEGs were mainly involved in flavonoid biosynthesis, phenylpropanoid biosynthesis, plant hormone signal transduction, starch and sucrose metabolism, and phenylalanine metabolism pathways, which suggested that the differential accumulation of secondary metabolites leads to the colour difference. Anthocyanin biosynthesis-related and transcription-factor genes with homologues that were crucial for anthocyanin biosynthesis in other plant species were identified as DEGs. We hypothesised that anthocyanin gene expression and subsequently anthocyanin metabolism contributed to the colour difference. The anthocyanin content in the purple peel was significantly higher than in the green peel, which verified the hypothesis. Quantitative real-time PCR on 11 DEGs was largely consistent with the RNA sequencing, indicating that the transcriptome data was reliable. Thus, the differential expression of anthocyanin biosynthesis-related genes correlates with the formation of the purple peel of Minhou wild banana. The generated comprehensive gene expression data provide insights into the molecular mechanisms underlying anthocyanin biosynthesis in banana peel.

Evaluating the involvement and interaction of abscisic acid and miRNA156 in the induction of anthocyanin biosynthesis in drought-stressed plants

ABA is involved in anthocyanin synthesis through the regulation of microRNA156, augmenting the level of expression of anthocyanin synthesis-related genes and, therefore, increasing anthocyanin level. Drought stress is the main cause of agricultural crop loss in the world. However, plants have developed mechanisms that allow them to tolerate drought stress conditions. At cellular level, drought stress induces changes in metabolite accumulation, including increases in anthocyanin levels due to upregulation of the anthocyanin biosynthetic pathway. Recent studies suggest that the higher anthocyanin content observed under drought stress conditions could be a consequence of a rise in the abscisic acid (ABA) concentration. This plant hormone crosses the plasma membrane by specific transporters, and it is recognized at the cytosolic level by receptors known as pyrabactin resistance (PYR)/regulatory component of ABA receptors (PYR/RCARs) that regulate downstream components. In this review, we discuss the hypothesis regarding the involvement of ABA in the regulation of microRNA156 (miRNA156), which is upregulated as part of dehydration stress responsiveness in different species. The miRNA156 upregulation produces a greater level of anthocyanin gene expression, forming the multienzyme complex that will synthesize an increased level of anthocyanins at the cytosolic face of the rough endoplasmic reticulum (RER). After synthesis, anthocyanins are transported from the RER to the vacuole by two possible models of transport: (1) membrane vesicle-mediated transport, or (2) membrane transporter-mediated transport. Thus, the aim was to analyze the recent findings on synthesis, transport and the possible mechanism by which ABA could increase anthocyanin synthesis under drought stress conditions potentially throughout microRNA156 (miRNA156).

Naturally Occurring Anthocyanin, Structure, Functions and Biosynthetic Pathway in Fruit Plants

Anthocyanins are naturally occurring compounds, member of the flavonoid groups of photochemical, involved in defense against the damaging effects of UV irradiation in plants and protect from many oxidants. The anthocyanins, group of pigments are relatively small and diverse flavonoid family in nature, and responsible for the attractive colors, red and purple to blue in many plants. Presence of pigments in flowers and fruits seems to provide attraction for pollination and aiding seed distribution, it also provides antiviral and antimicrobial activities, however their occurrence in the vacuoles remains ambiguous. During the last decades, anthocyanin gene expression and many structural genes encoding enzymes has been extensively studied in fruits, flowers and leaves in many plants. In addition, the genetic regulating mechanism, their biosynthesis and other factors involved are well described. The biosynthesis pathway of anthocyanin is a complex with diverse branches responsible to produce variety of metabolites. In general Anthocyanins, production through the flavonoid path, are a class of vital phenolic compounds. Over six thousand diverse anthocyanins have been reported from various species. So far, the potential health benefits of anthocyanins have been reported in the contexts of their antioxidant properties. Anthocyanins are also extensively studied for their several positive effects on body. Based on these facts, the present review briefly summarizes recent advances, highlighting the importance of biosynthetic pathway of anthocyanins, thus will serve to encourage advance investigation in this field.

Time-dependent sequestration of RVE8 by LNK proteins shapes the diurnal oscillation of anthocyanin biosynthesis

Circadian clocks sustain 24-h rhythms in physiology and metabolism that are synchronized with the day/night cycle. In plants, the regulatory network responsible for the generation of rhythms has been broadly investigated over the past years. However, little is known about the intersecting pathways that link the environmental signals with rhythms in cellular metabolism. Here, we examine the role of the circadian components REVEILLE8/LHY-CCA1-LIKE5 (RVE8/LCL5) and NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED genes (LNK) shaping the diurnal oscillation of the anthocyanin metabolic pathway. Around dawn, RVE8 up-regulates anthocyanin gene expression by directly associating to the promoters of a subset of anthocyanin biosynthetic genes. The up-regulation is overcome at midday by the repressing activity of LNK proteins, as inferred by the increased anthocyanin gene expression in lnk1/lnk2 double mutant plants. Chromatin immunoprecipitation assays using LNK and RVE8 misexpressing plants show that RVE8 binding to target promoters is precluded in LNK overexpressing plants and conversely, binding is enhanced in the absence of functional LNKs, which provides a mechanism by which LNKs antagonize RVE8 function in the regulation of anthocyanin accumulation. Based on their previously described transcriptional coactivating function, our study defines a switch in the regulatory activity of RVE8-LNK interaction, from a synergic coactivating role of evening-expressed clock genes to a repressive antagonistic function modulating anthocyanin biosynthesis around midday.

Genetic basis for a rare floral mutant in an Andean species of Solanaceae.

White forms of typically pigmented flowers are one of the most common polymorphisms in flowering plants. Although the range of genetic changes that give rise to white phenotypes is well known from model systems, few studies have identified causative mutations in natural populations. Here we combine genetic studies, in vitro enzyme assays, and biochemical analyses to identify the mechanism underlying the loss of anthocyanin pigment production in the naturally occurring white-flowered morph of Iochroma calycinum (Solanaceae). Comparison of anthocyanin gene sequences revealed a putative loss-of-function mutation, an 11 amino-acid deletion in dihydroflavonol 4-reductase (DFR), in the white morph. Functional assays of Dfr alleles from blue and white morphs demonstrated that this deletion results in a loss of enzymatic activity, indicating that the deletion could be solely responsible for the lack of pigment production. Consistent with this hypothesis, quantitative PCR showed no significant differences in expression of anthocyanin genes between the morphs. Also, thin layer chromatography confirmed that the white morph continues to accumulate compounds upstream of the DFR enzyme. Collectively, these experiments indicate that the structural mutation at Dfr underlies the rare white flower morph of I. calycinum. This study is one of only a few examples where a flower color polymorphism is due to a loss-of-function mutation in the coding region of an anthocyanin enzyme. The rarity of such mutations in nature suggests that negative consequences prevent fixation across populations.

High AN1 variability and interaction with basic helix-loop-helix co-factors related to anthocyanin biosynthesis in potato leaves.

AN1 is a regulatory gene that promotes anthocyanin biosynthesis in potato tubers and encodes a R2R3 MYB transcription factor. However, no clear evidence implicates AN1 in anthocyanin production in leaves, where these pigments might enhance environmental stress tolerance. In our study we found that AN1 displays intraspecific sequence variability in both coding/non-coding regions and in the promoter, and that its expression is associated with high anthocyanin content in leaves of commercial potatoes. Expression analysis provided evidence that leaf pigmentation is associated to AN1 expression and that StJAF13 acts as putative AN1 co-regulator for anthocyanin gene expression in leaves of the red leaf variety 'Magenta Love,' while a concomitant expression of StbHLH1 may contribute to anthocyanin accumulation in leaves of 'Double Fun.' Yeast two-hybrid experiments confirmed that AN1 interacts with StbHLH1 and StJAF13 and the latter interaction was verified and localized in the cell nucleus by bimolecular fluorescence complementation assays. In addition, transgenic tobacco (Nicotiana tabacum) overexpressing a combination of either AN1 with StJAF13 or AN1 with StbHLH1 showed deeper purple pigmentation with respect to AN1 alone. This further confirmed AN1/StJAF13 and AN1/StbHLH1 interactions. Our findings demonstrate that the classical loci identified for potato leaf anthocyanin accumulation correspond to AN1 and may represent an important step to expand our knowledge on the molecular mechanisms underlying anthocyanin biosynthesis in different plant tissues.

Isolation and analysis of the promoter of an anthocyanin synthase gene from purple-fleshed sweet potato tubers

Purple-fleshed sweet potato (Ipomoea batatas (L.) Lam) accumulates a large amount of anthocyanins in its tubers. Activation of anthocyanin gene expression requires transcription factors such as MYB domain, basic helix-loop-helix domain, or WD40-repeat domain-containing proteins. However, the mechanisms controlling pigmentation in underground organs remain unresolved. We used a principal component analysis to identify the most important gene in anthocyanin biosynthesis in pigmented sweet potato tubers, because this gene was the most likely to be regulated by IbMYB1. Anthocyanidin synthase was identified as the most important gene. Functional analysis of its promoter identified four MYB DNA-binding sites. In gel mobility shift experiments with recombinant IbMYB1, the IbMYB1 protein bound specifically to TAACCG box and TATCC box motifs in vitro. We conducted transient expression experiments in which various promoter fragments were used to drive expression of the LUC reporter gene. The reporter gene was strongly expressed under the control of the full-length promoter, but weakly expressed under the control of promoter fragments that lacked the MYB DNA-binding domains. This provided direct evidence that IbMYB1 activates the expression of this structural anthocyanin gene. Together, these results show that IbMYB1 is important in controlling the expression of genes in the anthocyanin biosynthetic pathway in cells.

Identification and expression analysis of chalcone synthase and dihydroflavonol 4-reductase in Clivia miniata

Clivia miniata is a popular breeding variety. The production of anthocyanin has been studied in Clivia species and the presence of key genes in anthocyanin production, chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR) confirmed. However, it is currently unknown to what extent these genes are expressed in different flower tissue during flower development. Thus the aim of this study was to determine the expression of CHS and DFR in C. miniata var. miniata, an orange flowered variety, and C. miniata var. citrina, a yellow flowered variety, in tepal, carpel and stamen at flower developmental stage two to six. As expected, the anthocyanin content in orange flowers was higher than that of yellow flowers. The expression of CHS and DFR correlated to anthocyanin content. Anthocyanin gene expression and production was found primarily in the tepal. There was a high correlation between CHS and DFR expression suggesting that these genes are subject to coordinate regulation in C. miniata.

Expression and mapping of anthocyanin biosynthesis genes in carrot

Anthocyanin gene expression has been extensively studied in leaves, fruits and flowers of numerous plants. Little, however, is known about anthocyanin accumulation in roots of carrots or other species. We quantified expression of six anthocyanin biosynthetic genes [phenylalanine ammonia-lyase (PAL3), chalcone synthase (CHS1), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR1), leucoanthocyanidin dioxygenase (LDOX2), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT)] in three carrot inbreds with contrasting root color: solid purple (phloem and xylem); purple outer phloem/orange xylem; and orange phloem and xylem. Transcripts for five of these genes (CHS1, DFR1, F3H, LDOX2, PAL3) accumulated at high levels in solid purple carrots, less in purple-orange carrot, and low or no transcript in orange carrots. Gene expression coincided with anthocyanin accumulation. In contrast, UFGT expression was comparable in purple and orange carrots and relatively unchanged during root development. In addition, five anthocyanin biosynthesis genes [FLS1 (flavonol synthase), F3H, LDOX2, PAL3, and UFGT] and three anthocyanin transcription factors (DcEFR1, DcMYB3 and DcMYB5) were mapped in a population segregating for the P 1 locus that conditions purple root color. P 1 mapped to chromosome 3 and of the eight anthocyanin biosynthesis genes, only F3H and FLS1 were linked to P 1. The gene expression and mapping data suggest a coordinated regulatory control of anthocyanin expression in carrot root and establish a framework for studying the anthocyanin pathway in carrots, and they also suggest that none of the genes evaluated is a candidate for P 1.

Computational identification of seed-specific transcription factors involved in anthocyanin production in black rice

To better understand the functional characterization of anthocyanin gene expression in black rice, we performed a detailed computational examination. The experiment was performed using the newly designed 135K Oryza sativa microarray to assess gene expression at three time points after the heading stage in Ds insertion lines. Transcription factors related to anthocyanin pigmentation biosynthesis were clustered. A total of 8,517 genes exhibited greater than 2.5-fold up- or downregulation in each comparison between three rice groups and three seed developmental stages. The resultant 82 transcription factor genes found to be associated with anthocyanin were classified into 12 groups. In addition, 12 unknown and hypothetical genes were identified from comparisons between the white cultivar and two black Ds insertion mutants tested. We identified 12 unknown and hypothetical genes involved in anthocyanin biosynthesis. These genes likely play either a regulatory role in the anthocyanin production process or are related to anthocyanin metabolism during flavonoid biosynthesis. While these genes require further characterization and validation, the results here underline the potential use of the newly designed microarray.

Environmental regulation of floral anthocyanin synthesis in Ipomoea purpurea

Responses of metabolites to environmental fluctuations may play large roles in biological adaptation, yet how these responses initiate in the natural environment and the molecular mechanisms remain unclear. Synthesis of floral anthocyanins, as typical examples of secondary metabolites, is known to respond to the physical environment and therefore an ideal system for understanding the process of the environmental regulation. Here, by simultaneous monitoring of six natural environmental variables and anthocyanin content of daily opening flowers throughout a natural flowering season ( approximately 50 days) of Ipomoea purpurea, we have identified significant and positive correlations of temperature (3-days ago) and ultraviolet (UV) light intensity (5-days ago) with the floral anthocyanin content. We sequenced all known (seven structural and three regulatory) anthocyanin genes in I. purpurea flowers and examined their transcript quantities in the natural environment across eight floral developmental stages (covering 0-96 h before anthesis). The anthocyanin gene expression patterns corroborated with the inferred effects from the time-series data, and further showed that the positive UV effect became negative on transcript levels about 36 h before anthesis. With falling natural temperature, content of the principal anthocyanin declined, whereas that of an alternative anthocyanin with fewer glucose and caffeic acid moieties increased. Our data suggest that environmental regulation of the anthocyanin pathway may account for more than half of the flux variation in the floral limb, and is influenced mainly by daily average temperature and UV light intensity that modulate anthocyanin transcript levels (most likely via myb1) at floral developmental stages.

Ratio of Myc and Myb Transcription Factors Regulates Anthocyanin Production in Orchid Flowers

Many studies have examined anthocyanin gene expression in colorless tissues by introducing anthocyanin regulatory genes of the MYC/R and MYB/C1 families. Expression of the two regulatory genes under the control of a strong promoter generally results in high anthocyanin accumulation. However, such approaches usually have a negative effect on growth and development of the recovered plants. In this study the author used two promoters of different strengths—a weak (Solanum tuberosum L. polyubiquitin Ubi3) and a strong (double 35S) promoter—and generated two sets of expression constructs with the Zea mays L. anthocyanin regulatory genes MycLc and MybC1 . A transient expression system was developed using biolistic bombardment of white Phalaenopsis amabilis (L.) Blume flowers, which the authors confirmed to be anthocyanin regulatory gene mutants. Transient expression of different combinations of the four constructs would generate three different MycLc -to-MybC1 ratios (>1, 1, <1). The enhanced green florescent protein gene (EGFP) was cotransformed as an internal control with the two anthocyanin regulatory gene constructs. These results demonstrate that the ratio of the two transcription factors had a significant influence on the amount of anthocyanin produced. Anthocyanin accumulation occurred only when MybC1 was under the control of the 35S promoter, regardless of whether MycLC was driven by the 35S or Ubi3 promoter.

Plant Genomics

Plant Genomics

Expression of Anthocyanin Genes in Potato Periderm

Anthocyanins are a class of flavonoids that are responsible for pigments in flowers, fruit, and potato periderm. Developing `Norland' potatoes synthesize anthocyanins in periderm tissue when the tubers are mere swollen stolon tips. As the tubers enlarge, anthocyanin accumulation seems to stop, and anthocyanins synthesized early in development seem to be diluted as the tubers enlarge. Expression of dihydroflavonol reductase (DFR) limits anthocyanin synthesis in grape and maize fruit, and in petunia and snapdragon flowers. However, DFR expression in periderm tissue occurred throughout tuber development (Hung et al., 1999). To determine if expression of late anthocyanin pathway genes limit anthocyanin synthesis in developing potato tubers, we performed RNA gel blot analyses. Expression of leucoanthocyanidin dioxygenase and UDP glucose: flavonoid 3-O-glucosyl transferase was observed in swollen stolon tips but not in periderm of later tuber development stages. Surprisingly, expression was also observed in cortex tissue, although that tissue remained white throughout tuber growth.

Transient expression of anthocyanin genes in barley epidermal cells : potential for use in evaluation of disease response genes

A transient assay is described that should allow evaluation of the role of host genes in disease response by enhancing or disrupting expression of those genes in specific cells and looking for effects on disease development. The assay also has the potential for assessing utility of host and non-host genes in enhancing resistance to disease in transgenic plants. Particle bombardment with a helium discharge particle gun was utilized to transiently express genes in epidermal cells of coleoptiles of barley (Hordeum vulgare). An anthocyanin reporter gene construct provided a means of identifying those cells that were transiently expressing introduced DNA. Optimal transient expression rates were achieved two days following bombardment with 1800 psi helium pressure, 1.0 μm diameter gold particles, and coleoptile pre- and post-treatment in 0.30--0.35 m mannitol/sorbitol. Under optimal conditions, at least 35 cells expressed anthocyanin per bombardment. Transiently expressing cells were inoculated with the fungal pathogen, Erysiphe graminis f. sp. hordei, and fungal development observed. Neither the bombardment procedures, the presence of nearby dead cells, nor accumulation of anthocyanin within living cells affected fungal development in living cells. Therefore, incorporation of disease-related genes onto the same plasmid as the reporter genes will allow evaluation of the role of those genes in disease development or suppression. Since particle bombardment is possible with a great range of different plant tissues, the described methodology should exhibit wide applicability for evaluating genes in diverse plant-pathogen interactions, as well as genes involved in many other biological processes

Establishment of embryogenic callus and transient gene expression in selected sugarcane varieties

Twelve commercial sugarcane (Saccharum spp. hybrids) varieties were compared for both their ability to form white, embryogenic callus and their transformation potential. Of the 12 varieties investigated, NCo310, NCo376, N8, N12, N16, N18 and N20 produced large proportions of white, embryogenic (Type 3) callus in relation to total callus volume, especially when subcultured weekly on 1 mg l− 1 2,4-dichlorophenoxyacetic acid (2,4-D) for up to six weeks. Fortnightly subculture on 1 mg l− 1 2,4-D and on variable levels of 2,4-D were less effective in Type 3 callus maintenance. Transformation potential of the white callus from seven varieties was assessed by bombarding callus with a plasmid containing the genes which encode two anthocyanin transcriptional activators (pDP687). Expression of the inserted genes was measured after 48 h. Of all the varieties, NCo310 produced higher proportions of white callus, showed the best transient anthocyanin gene expression, and subsequent plantlet regeneration occurred, indicating that this variety is suitable for further transformation studies.

Multiple genes are transcribed in Hordeum vulgare and Zea mays that carry the DNA binding domain of the myb oncoproteins.

cDNA clones were isolated from tissue specific cDNA libraries of barley and maize using as a probe the cDNA of the maize gene C1, a regulator of anthocyanin gene expression. C1-related homology for all of the four cDNAs characterized by sequence analysis is restricted to the N-terminal 120 amino acids of the putative proteins. This region shows striking homology to the N-proximal domain of the myb oncoproteins from vertebrates and invertebrates. Within the myb proto-oncogene family this part of the respective gene products functions as a DNA binding domain. Acidic domains are present in the C-proximal protein segments. Conservation of these sequences, together with the genetically defined regulator function of the C1 gene product, suggest that myb-related plant genes code for trans-acting factors which regulate gene expression in a given biosynthetic pathway.