Anthocyanin Synthase Research Articles

Molecular characterization and expression analyses of an anthocyanin synthase gene from Magnolia sprengeri Pamp.

Anthocyanin synthase (ANS), which catalyzes the conversion of colorless leucoanthocyanins into colored anthocyanins, is a key enzyme in the anthocyanin biosynthetic pathway. It plays important roles in plant development and defense. An ANS gene designated as MsANS was cloned from Magnolia sprengeri using rapid amplification of complementary DNA (cDNA) ends technology. The full-length MsANS is 1171-bp long and contains a 1080-bp open reading frame encoding a 360 amino acid polypeptide. In a sequence alignment analysis, the deduced MsANS protein showed high identity to ANS proteins from other plants: Prunus salicina var. cordata (74 % identity), Ampelopsis grossedentata (74 % identity), Pyrus communis (73 % identity), and Prunus avium (73 % identity). A structural analysis showed that MsANS belongs to 2-oxoglutarate (2OG)- and ferrous iron-dependent oxygenase family because it contains three binding sites for 2OG. Real-time quantitative polymerase chain reaction analyses showed that the transcript level of MsANS was 26-fold higher in red petals than in white petals. The accumulation of anthocyanins in petals of white, pink, and red M. sprengeri flowers was analyzed by HPLC. The main anthocyanin was cyanidin-3-o-glucoside chloride, and the red petals contained the highest concentration of this pigment.

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.

Overexpression of CsDFR and CsANR enhanced flavonoids accumulation and antioxidant potential of roots in tobacco

Flavonoids are widespread throughout the plant kingdom and present in different parts of plants. Tea (Camellia sinensis) is well known for very high content of flavonoids especially flavan-3-ols antioxidants and is an aluminium (Al) accumulator plant. Dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (ANR) are known to be regulatory enzymes of flavonoid biosynthetic pathway. In this study, cDNA encoding DFR (CsDFR) and ANR (CsANR) from tea were overexpressed individually in tobacco to check their influence on accumulation of flavonoid contents and antioxidant potential in roots of transgenic tobacco. Root morphological features, such as total volume and the number of lateral roots were improved in CsDFR and CsANR overexpressing tobacco plants relative to control tobacco plants. Both types of transgenic showed higher content of flavonoids and proanthocyanidins and lower content of anthocyanins in the roots compared to roots of control tobacco. Among flavan-3-ols, only epigallocatechin was observed in the roots and its content was higher in CsDFR and CsANR overexpressing tobacco as compared to control tobacco. Expression of genes encoding various other enzymes of flavonoid pathway like Phenylalanine ammonia-lyase, Chalcone isomerase, Flavanol synthase and Anthocyanin synthase was increased in roots of CsDFR and CsANR overexpressing tobacco plants as compared to control tobacco. The antioxidant potential of root portion of CsDFR and CsANR transgenic tobacco plants was found to be increased as indicated by enhanced total free radical scavenging activity and tolerance against Al toxicity. Taken together, these changes in roots of CsDFR and CsANR transgenic tobacco provided tolerance to aluminum toxicity.

Identification and expression analysis of genes involved in anthocyanin and proanthocyanidin biosynthesis in the fruit of blackberry

Anthocyanin and proanthocyanin are two major secondary metabolites involved in fruit maturation. In this work, the variations of anthocyanin and proanthocyanidin content were determined during blackberry fruit development. Genes associated with biosynthesis of both two compounds were isolated. Expression patterns of these genes were also investigated by quantitative PCR throughout fruit maturation. Anthocyanins, which remained at a low concentration in the early developing stage, increased dramatically as the fruit matured. On the contrary, proanthocyanidins exhibited a continuously decreasing pattern. Transcript levels of genes specifically controlling either of these two compounds: anthocyanin synthase (RuANS) for anthocyanin, leucoanthocyanidin reductase (RuLAR) and anthocyanidin reductase (RuANR) for proanthocyanidin, were generally coordinated with the products’ changing patterns. The expression of the RuMYB10, encoding a transcription factor, was also concomitant with the synthesis of anthocyanins, indicating its specificity regulation role in anthocyanin branch. The other two genes, chalcone synthase (RuCHS) and dihydroflavonol 4-reductase (RuDFR), showed cross regulation for not only the synthesis of anthocyanin and proanthocyanidin, but also other flavonoid compounds. According to the temporal expression patterns, the isolated RuGT gene might encode a glucosyltransferase participating in modifying metabolites besides anthocyanins. Our discoveries of the temporal gene expression and compounds accumulation will shed new light into genetic and physiological metabolisms in blackberry fruit development, thus assisting in future attempts to alter anthocyanin or PA metabolites in fruits.

Validation of a Carnation-Specific Gene, ANS, Used as an Endogenous Reference Gene in Qualitative and Real-Time Quantitative PCR for Carnations

The validation of the anthocyanin synthase (ANS) gene as a carnation endogenous reference gene applicable both in classical and real-time PCR methods is a prerequisite for the development of PCR assays for genetically modified (GM) carnation detection. This is important due to the fact that GM carnation lines, developed by Florigene Pty Ltd, have been approved for commercialization. In this study, both methods were tested on 14 different carnation cultivars, and identical amplification products were obtained with all of them. No amplification products were observed with samples from 14 other plant species, which demonstrated that the system was specific to carnation. The results of Southern blot analysis confirmed that the ANS gene had a low copy number in the 10 tested carnation varieties. In qualitative and real-time PCR assays, the LOD values of 0.05 and 0.005 ng carnation DNA, respectively, were validated. Moreover, the real-time PCR system was validated with high PCR efficiency and linearity. Thus, the ANS gene had species specificity, low heterogeneity, and low copy number among the tested cultivars. These results provide evidence that the gene can be used as an endogenous reference gene of carnation, as well as in qualitative and quantitative PCR systems.

Functional divergence within class B MADS-box genes TfGLO and TfDEF in Torenia fournieri Lind

Homeotic class B genes GLOBOSA (GLO)/PISTILLATA (PI) and DEFICIENS (DEF)/APETALA3 (AP3) are involved in the development of petals and stamens in Arabidopsis. However, functions of these genes in the development of floral organs in torenia are less well known. Here, we demonstrate the unique floral phenotypes of transgenic torenia formed due to the modification of class B genes, TfGLO and TfDEF. TfGLO-overexpressing plants showed purple-stained sepals that accumulated anthocyanins in a manner similar to that of petals. TfGLO-suppressed plants showed serrated petals and TfDEF-suppressed plants showed partially decolorized petals. In TfGLO-overexpressing plants, cell shapes on the surfaces of sepals were altered to petal-like cell shapes. Furthermore, TfGLO- and TfDEF-suppressed plants partially had sepal-like cells on the surfaces of their petals. We isolated putative class B gene-regulated genes and examined their expression in transgenic plants. Three xyloglucan endo-1,4-beta-d-glucanase genes were up-regulated in TfGLO- and TfDEF-overexpressing plants and down-regulated in TfGLO- and TfDEF-suppressed plants. In addition, 10 anthocyanin biosynthesis-related genes, including anthocyanin synthase and chalcone isomerase, were up-regulated in TfGLO-overexpressing plants and down-regulated in TfGLO-suppressed plants. The expression patterns of these 10 genes in TfDEF transgenic plants were diverse and classified into several groups. HPLC analysis indicated that sepals of TfGLO-overexpressing plants accumulate the same type of anthocyanins and flavones as wild-type plants. The difference in phenotypes and expression patterns of the 10 anthocyanin biosynthesis-related genes between TfGLO and TfDEF transgenic plants indicated that TfGLO and TfDEF have partial functional divergence, while they basically work synergistically in torenia.Electronic supplementary materialThe online version of this article (doi:10.1007/s00438-010-0574-z) contains supplementary material, which is available to authorized users.

Epistatic Interactions Influencing Anthocyanin Gene Expression in Capsicum annuum

Anthocyanin pigmentation in leaves, flowers, and fruit imparts violet to black color and enhances both ornamental and culinary appeal. Shades of violet to black pigmentation in Capsicum annuum L. are attributed to anthocyanin accumulation. Anthocyanin production is markedly influenced by numerous environmental factors, including temperature and light stress. The objective of this study was to determine the genetic basis for differences in C. annuum anthocyanin content in response to varying environments. Growth experiments conducted under controlled environment conditions demonstrated that anthocyanin concentration was significantly higher in mature leaves in comparison with immature leaves under high light (435 μmol·s−1·m−2) conditions. High (30 °C day/25 °C night) versus low (20 °C day/15 °C night) temperature had no significant effect on anthocyanin concentration regardless of leaf maturity stage. Foliar anthocyanin concentration in plants grown under short days (10 h) with low light intensity (215 μmol·s−1·m−2) was significantly less than under long days (16 h) with low light. Under high light intensity, daylength had no effect on anthocyanin content. Three structural genes [chalcone synthase (Chs), dihydroflavonol reductase (Dfr), anthocyanin synthase (Ans)] and three regulatory genes (Myc, MybA , Wd40) were selected for comparison under inductive and noninductive environmental conditions for anthocyanin accumulation. Expression of Chs, Dfr, and Ans was significantly higher in mature leaves in comparison with younger leaves. Consistent with anthocyanin concentration, temperature had no effect on structural gene expression, whereas light positively influenced expression. Under low light conditions, temperature had no effect on Myc, MybA , and Wd40 expression; whereas under high light conditions, temperature only had an effect on MybA expression. The study of anthocyanin leaf pigmentation in C. annuum under inductive and noninductive environments provides a new approach for elucidating the molecular genetic basis of epistatic gene interactions and the resulting phenotypic plasticity.

Anthocyanin content in rice is related to expression levels of anthocyanin biosynthetic genes

The composition of anthocyanins was analyzed in two rice cultivais, ‘llpum’ showed no detectable levels, while ‘Heugjinju’ contained three types of anthocyanins: cyanidin, cyanidin 3-O-glucoside, and peonidin 3-O-glucoside. We also assessed the expression of anthocyanin biosynthetic genes — for phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), flavanone 3β-hydroxylase (F3H), dihydroflavonol reductase (DFR), and anthocyanin synthase (ANS)-in different tissues of those cultivars. All five genes were expressed more in the leaves and seeds of ‘Heugjinju’ than in ‘Ilpum’, with the greatest amount of transcript being detected in ‘Heugjinju’ seeds. Two genes,DFR andANS, had relatively high expression levels and were specific for anthocyanin biosynthesis. Furthermore, expression of CHS,F3H, DFR, andANS was enhanced during seed maturation and was correlated with ambient temperature during seedling growth.

Identification of the fourth allele of the ANS (anthocyanidin synthase) gene and its effect on red color intensity in onions (Allium cepa)

Bulb color in onions (Allium cepa) is an important trait, and homogenous red coloration is desirable in red onion cultivars. The gene encoding anthocyanin synthase (ANS) is required for anthocyanin biosynthesis in onions. We have previously described three different alleles of the ANS gene. Here we report identification of the fourth allele of ANS, ANS-h1, found in a dark red doubled haploid line. ANS-h1 is similar to a non-functional allele found in Brazilian yellow cultivars except that it has several point mutations and indels throughout the promoter and coding regions, none of which are predicted to inactivate enzymatic activity. F2 and backcross populations originating from the crosses between wild-type (ANS-L) allele-containing red and pink (ANS-p) allele-containing white or yellow parents show a discrete segregation ratio of 3 red to 1 light pink, indicating that the wild-type allele is completely dominant over the pink allele. In contrast, segregating populations derived from the crosses between ANS-h1 allele-containing red and the same white or yellow parents show a gradient of red intensity from light pink to dark red, suggesting that other genetic factors may affect expression of ANS-h1. A newly developed PCR-based marker and two previously developed markers for allelic selection of the ANS gene were used to examine allele composition in fifty-six breeding lines and commercial cultivars. Most lines are heterogeneous for the ANS gene with two or three alleles detected. The frequency of the pink allele is low in red breeding lines, but it is predominant in white and yellow lines.

Differential Expression of Capsicum Anthocyanin Structural Genes in Response to Temperature Stress

Anthocyanins contribute to color development in economically important vegetables, fruits and floral crops. Their expression is critical to product sensory quality attributes, potential nutritive value, and stress response. Anthocyanins are synthesized in response to numerous environmental factors including temperature and light stress and pathogen attack. We have developed several Capsicum lines, including `02C27', expressing anthocyanin pigmentation differentially in various tissues (leaf, stem, fruit and flower). HPLC analysis demonstrated that the anthocyanins within the fruit, flower and leaves of Capsicum `02C27' were identical and that the major anthocyanidin was a delphinidin glycoside. Line `02C27' exhibits anthocyanin foliar pigmentation that is accumulated differentially in response to temperature stress. Under unfavorable low temperature (20 °C day/18 °C night), mature Capsicum leaves contained 4.6 times less anthocyanin per gram fresh weight than under high (30 °C day/28 °C; day/night) temperatures. Besides containing less anthocyanin in mature leaves, young immature leaves did not develop color as quickly under the lower temperature. Utilizing cloned and sequenced gene fragments of pepper chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), we evaluated the role of transcription in regulation of flavonol biosynthesis. Analysis of anthocyanin composition and gene expression data indicated that the block in anthocyanin formation in less pigmented leaves occurred at anthocyanin synthase. In contrast to wild tupe plants, this mutant also exhibited reduced flowering and failed to set fruit under high temperature, long day conditions.