Accumulation Of Red Pigment Research Articles

Genome-Wide Analysis of the bHLH Gene Family in Loropetalum chinense var. rubrum: Identification, Classification, Evolution, and Diversity of Expression Patterns under Cultivation.

The basic helix-loop-helix (bHLH) transcription factor family is the second-largest transcription factor family in plants. Members of this family are involved in the processes of growth and development, secondary metabolic biosynthesis, signal transduction, and plant resistance. Loropetalum chinense var. rubrum is a critical woody plant with higher ornamental and economic values, which has been used as ornamental architecture and traditional Chinese herbal medicine plants. However, the bHLH transcription factors in Loropetalum chinense var. rubrum (L. chinense var. rubrum) have not yet been systematically demonstrated, and their role in the biosynthesis of anthocyanin is still unclear. Here, we identified 165 potential LcbHLHs genes by using two methods, and they were unequally distributed on chromosomes 1 to 12 of the genome of L. chinense var. rubrum. Based on an evolutionary comparison with proteins from Arabidopsis and Oryza sativa, these bHLH proteins were categorized into 21 subfamilies. Most LcbHLHs in a particular subfamily had similar gene structures and conserved motifs. The Gene Ontology annotation and Cis-elements predicted that LcbHLHs had many molecular functions and were involved in processes of plant growth, including the biosynthesis of flavonoids and anthocyanins. Transcriptomic analysis revealed different expression patterns among different tissues and cultivars of L. chinense var. rubrum. Many LcbHLHs were expressed in the leaves, and only a few genes were highly expressed in the flowers. Six LcbHLHs candidate genes were identified by bioinformatics analysis and expression analysis. Further Real-time quantitative PCR analysis and protein interaction network analysis showed that LcbHLH156, which is one of the candidate proteins belonging to the IIIf subfamily, could interact with proteins related to anthocyanin synthesis. Therefore, LcbHLH156 was transiently expressed in L. chinense var. rubrum to verify its function in regulating anthocyanin synthesis. Compared with the control group, red pigment accumulation appeared at the wound after injection, and the total anthocyanin content increased at the wound of leaves. These results lay a foundation for the research of the regulation mechanism of leaf colors in L. chinense var. rubrum and also provide a basis for the function of the LcbHLH family.

Chromosome-level genome assembly of Salvia miltiorrhiza with orange roots uncovers the role of Sm2OGD3 in catalyzing 15,16-dehydrogenation of tanshinones.

Salvia miltiorrhiza is well known for its clinical practice in treating heart and cardiovascular diseases. Its roots, used for traditional Chinese medicine materials, are usually brick-red due to accumulation of red pigments, such as tanshinone IIA and tanshinone I. Here we report a S. miltiorrhiza line (shh) with orange roots. Compared with the red roots of normal S. miltiorrhiza plants, the contents of tanshinones with a single bond at C-15,16 were increased, whereas those with a double bond at C-15,16 were significantly decreased in shh. We assembled a high-quality chromosome-level genome of shh. Phylogenomic analysis showed that the relationship between two S. miltiorrhiza lines with red roots was closer than the relationship with shh. It indicates that shh could not be the mutant of an extant S. miltiorrhiza line with red roots. Comparative genomic and transcriptomic analyses showed that a 1.0kb DNA fragment was deleted in shh Sm2OGD3m. Complementation assay showed that overexpression of intact Sm2OGD3 in shh hairy roots recovered furan D-ring tanshinone accumulation. Consistently, in vitro protein assay showed that Sm2OGD3 catalyzed the conversion of cyptotanshinone, 15,16-dihydrotanshinone I and 1,2,15,16-tetrahydrotanshinone I into tanshinone IIA, tanshinone I and 1,2-dihydrotanshinone I, respectively. Thus, Sm2OGD3 functions as tanshinone 15,16-dehydrogenase and is a key enzyme in tanshinone biosynthesis. The results provide novel insights into the metabolic network of medicinally important tanshinone compounds.

Vineyard light manipulation and silicon enhance ethylene-induced anthocyanin accumulation in red table grapes.

Red color resulted from anthocyanin pigment, is an essential trait for premium table grape production. Anthocyanin biosynthesis occurs through the flavonoid pathway which includes several enzymatic reactions coded by different genes. The expression of these genes is regulated by different cultural practices, cultivars, environmental conditions, and plant hormones. Recently, we reported that the anthocyanin pathway is regulated by several factors such as light and antioxidant activity. Despite the advances in cultural practices, it is still challenging to produce table grapes with high coloration, especially under the current and expected global climate change in warmer areas such as California. In the current study, we deployed two approaches to improve the accumulation of red pigment in table grapes. The first approach involves improving the expression of critical genes involved in the anthocyanin pathway through hormonal treatments and light manipulation using a reflective ground cover (RGC). The second approach was to reduce the negative effect of heat stress through stimulation of the antioxidant pathway to help remove free radicals. Treatments included ethephon (ET) at 600 mg/L, silicon (Si) at 175 mg/L, and a commercial light-reflective white ground cover (RGC) alone and in various combinations. Treatments were conducted either with or without a combination of cluster-zone leaf removal at veraison (LR) on Flame seedless (Vitis vinifera L.). Data collected in 2019 and 2020 showed that the best treatment to improve berry coloration was using ET in combination with Si and RGC, applied at veraison. Adding the LR to this combination did not improve berry color any further, but rather caused a reduction in color development. RGC without conducting LR at veraison significantly increased the quantity of reflected blue and red lights as well as the red (R) to far-red (FR) ratio (R: FR) around clusters. Results were in accordance with the increase in gene expression of flavonoid-3-O-glucosyltransferase (UFGT), a key gene in the anthocyanin biosynthesis pathway, as well as Peroxidase dismutase (POD). Manipulating the light spectrum and application of silicon in combination with the ethephon treatment could be used in table grape vineyards to improve the ethylene-induced anthocyanin accumulation and coloration.

Ripening of Pomegranate Skin as Revealed by Developmental Transcriptomics.

The appearance of pomegranate (Punica granatum L.) fruit is highly important for its marketing. The primary concerns are obtaining sufficient red pigment accumulation and minimal cracking of the fruit skin (the outer red layer of the peel). We analyzed the skin transcriptome of pomegranate cv. Wonderful at distinct time points of fruit development to characterize the processes that occur in the skin during fruit ripening and which may reflect on processes in the whole fruit, such as the non-climacteric nature of pomegranate. The data suggested a ripening mechanism in pomegranate skin that differs from that in strawberry—the model plant for non-climacteric fruit where abscisic acid is the growth regulator that drives ripening—involving ethylene, polyamine, and jasmonic acid pathways. The biosynthetic pathways of important metabolites in pomegranate—hydrolyzable tannins and anthocyanins—were co-upregulated at the ripening stage, in line with the visual enhancement of red coloration. Interestingly, cuticle- and cell-wall-related genes that showed differential expression between the developmental stages were mainly upregulated in the skin of early fruit, with lower expression at mid-growth and ripening stages. Nevertheless, lignification may be involved in skin hardening in the mature fruit.

Integrated Metabolome and Transcriptome Analyses Reveal Dissimilarities in the Anthocyanin Synthesis Pathway Between Different Developmental Leaf Color Transitions in Hopea hainanensis (Dipterocarpaceae).

Changes in plant leaf color during development are directly related to the accumulation or degradation of certain phytochemicals such as anthocyanins. Since some anthocyanins can be beneficial to human health and provide insights into the biology of leaves, the underlying processes and timing by which plants produce these molecules has been the focus of numerous studies. The tree species Hopea hainanensis generally produces green leaves at all growth stages; however, a few explored individuals have been identified possessing red leaves on the top of the seedlings at a young stage. While the phenomenon of leaf color varying with age has been studied in several species, the underlying mechanisms are largely unknown in H. hainanensis. Using a metabolomics approach, the young red leaves in H. hainanensis were found to contain higher levels of anthocyanins and flavonoids than the young green-leaved individuals. Among anthocyanins, pelargonidin and cyanidin were the most likely candidates contributing to the red color of the young leaves. Transcriptome results indicated the genes related to the production of these anthocyanins were significantly upregulated, leading to greater accumulation of red pigments. Specifically, the expression of several MYB and bHLH genes in young red leaf lines was significantly higher than that in the young green leaf lines, especially HhMYB66, HhMYB91, HhMYB6, and HhbHLH70. As such these four transcription factors are probably the main regulatory genes resulting in young red leaves in H. hainanensis. From these results, comparative analyses with other species can be made to better understand the evolution of pigment biosynthesis and how anthocyanins function in plant metabolism and evolution/adaptation.

Study on changes in pigment composition during the blooming period of safflower based on plant metabolomics and semi-quantitative analysis.

Red and yellow pigments are the major ingredients of safflower, often used to color food and cosmetics. Carthamin was the main component of red pigment and hydroxysafflor yellow A and anhydrosafflower yellow B were representative components of yellow pigment. Plant metabolomics and semi-quantitative analysis were used to analyze the changes of pigment composition during the blooming period, especially these characteristic components. Carthamin, hydroxysafflor yellow A, anhydrosafflower yellow B, and other components were screened out as differential metabolites based on plant metabolomics. Then semi-quantitative analysis was used to quantify these three representative components of pigments. Experimental results showed that the content of pigments has dynamic changes along with flowering, in the early blooming period, yellow pigment accumulated much and red pigment was low in content. In the middle period, the accumulation rate of the yellow pigment slowed down and content was stabilized. In the next step, the content of yellow pigments gradually decreased, and the content of red pigments gradually increased. Later, the level of yellow pigment decreased significantly, and the accumulation rate of red pigment increased significantly. Last, the appearance color of safflower was red, with yellow parts barely visible, and accumulation of red pigment was the highest and of the yellow pigment was the lowest in content.

The potential pigmentation-related genes in spider mites revealed by comparative transcriptomes of the red form of Tetranychus urticae.

Colouration in spider mites is due to the presence of carotenoids with diverse colours, including yellows, oranges and reds. Tetranychus urticae has two main colour forms, red and green. Although a ketolase has been implicated in determining the colour, the underlying genetic basis of body colour divergence between the two forms has remained unclear. Based on a combination of comparative transcriptomes and RNA interference, we found that a gene encoding a cytochrome P450 enzyme of the CYP4 clan (CYP389B1) had remarkably high expression in adult females of the red T. urticae, as well as in hybrids obtained by crossing the red and green forms. Down-regulation of this gene by RNA interference resulted in decreased accumulation of red pigment. Up-regulation of the expressions of a scavenger receptor gene (SCARB1) and a mitochondrial glycine transporter (SLC25A38) also strongly contributed to red colour development in adult females. Suppressing the mRNA levels of these genes also resulted in reduced accumulation of red pigment in the three other spider mites with red body colour. Our results provide evidence that the body colour divergence between the two forms is caused by different expressions of pigmentation-related genes, and point to a possible role of a novel cytochrome P450 gene (CYP389B1) in regulating red-orange body colour. These findings expand the number of candidate cytochrome P450 genes involved in endogenous pigmentation and will help to understand their roles in determining colour patterns in mites and other species.

Identification and differential expression analysis of anthocyanin biosynthetic genes in root-skin color variants of radish (Raphanus sativus L.).

Taproot skin color is a major trait for assessing the commercial and nutritional quality of radish, and red-skinned radish is confirmed to improve consumer's interest and health. However, little is known about the molecular mechanisms responsible for controlling the formation of red-skinned radish. This study aimed to identify the differentially expressed anthocyanin biosynthetic genes between red- and white-skinned radishes and understand the molecular regulatory mechanism underlying red-skinned radish formation. Based on the published complete genome sequence of radish, the digital gene expression profiles of Yangzhouyuanbai (YB, white-skinned) and Sading (SD, red-skinned) were analyzed using Illumina sequencing. A total of 3666 DEGs were identified in SD compared with YB. Interestingly, 46 genes encoded enzymes related to anthocyanin biosynthesis and 241 genes encoded transcription factors were identified. KEGG pathway analysis showed that the formation of red-skinned radish was mainly controlled by pelargonidin-derived anthocyanin biosynthetic pathway genes. This process included the upregulation of PAL, C4H, 4CL, CHS, CHI, F3H, DFR, LDOX, and UGT enzymes in SD. CHS genes were specifically expressed in SD, and it might be the key point for red pigment accumulation in red-skinned radish. Furthermore, MYB1/2/75, bHLH (TT8), and WD 40 showed higher expression in SD than in YB. Meanwhile, the corresponding low-abundance anthocyanin biosynthesis enzymes and upregulation of MYB4 might be the factors influencing the formation of white-skinned radish. These findings provide new insights into the molecular mechanisms and regulatory network of anthocyanin biosynthesis in red-skinned radish.

A β-carotene-binding protein carrying a red pigment regulates body-color transition between green and black in locusts

Changes of body color have important effects for animals in adapting to variable environments. The migratory locust exhibits body color polyphenism between solitary and gregarious individuals, with the former displaying a uniform green coloration and the latter having a prominent pattern of black dorsal and brown ventral surface. However, the molecular mechanism underlying the density-dependent body color changes of conspecific locusts remain largely unknown. Here, we found that upregulation of β-carotene-binding protein promotes the accumulation of red pigment, which added to the green color palette present in solitary locusts changes it from green to black, and that downregulation of this protein led to the reverse, changing the color of gregarious locusts from black to green. Our results provide insight that color changes of locusts are dependent on variation in the red β-carotene pigment binding to βCBP. This finding of animal coloration corresponds with trichromatic theory of color vision.

Rapid colour changes in Euglena sanguinea (Euglenophyceae) caused by internal lipid globule migration

ABSTRACTThe accumulation of red pigments, frequently carotenoids, under chronic stress is a response observed in diverse kinds of eukaryotic photoautotrophs. It is thought that red pigments protect the chlorophyll located underneath by a light-shielding mechanism. However, the synthesis or degradation of carotenoids is a slow process and this response is usually only observed when the stress is maintained over long periods of time. In contrast, rapid colour changes have been reported in the euglenophyte Euglena sanguinea. Here we study the ecophysiological process behind this phenomenon through chlorophyll fluorescence, and pigment, colour and ultrastructural analyses. Reddening in E. sanguinea was due to the presence of a large amount of free and esterified astaxanthin (representing 80% of the carotenoid pool). The process was highly dynamic, shifting from green to red in 8 min (and vice-versa in 20 min). This change was not due to de novo carotenogenesis, but to the relocation of cytoplasmic lipid globules where astaxanthin accumulates. Thus, red globules were observed to migrate from the centre of the cell to peripheral locations when exposed to high light. Globule migration seems to be so efficient that other classical photoprotective mechanisms are not operative in this species. Despite the presence and operation of the diadino-diatoxanthin cycle, non-photochemical quenching was almost undetectable. Since E. sanguinea forms extensive floating colonies, reddening can be observed at a much greater scale than at the cellular level and the mechanism described here is one of the fastest and most dramatic colour changes attributable to photosynthetic organisms at cell and landscape level. In conclusion E. sanguinea shows an extremely dynamic and efficient photoprotective mechanism, based more on organelle migration than on carotenoid biosynthesis, which prevents excess light absorption by chlorophylls reducing the need for other protective processes related to energy dissipation.

Comprehensive analysis of wintersweet flower reveals key structural genes involved in flavonoid biosynthetic pathway

Wintersweet (Chimonanthus praecox (L.)), with an over-one-thousand-years long history in cultivation, is still a popular ornamental woody plant in China. The tepals of wintersweet flower are waxy in nature and the overall color of the flower is yellow, while the inner tepals range from yellow to red, which makes it an ideal plant to study floral color formation in ornamental shrubs. In our current work, HPLC analysis revealed that the principal pigments in tepals were the metabolite of flavonoids. All the tepals were containing quercetin, kaempferol 3‑O‑rutinoside and rutin while cyanidin‑3‑O‑glucoside and cyanidin‑3‑O‑rutinoside were only found in the in the red tepals. Moreover, we found the rutin as the principal component of all the pigments revealed. As well as in this study, a reference transcriptome library constructed from two varieties H29 and H64 flower. Further, 30 proteins of flavonoid biosynthesis pathway were identified in H29 flower using proteome analysis. Based on these dataset, the flavonoid biosynthesis pathway was also speculated. After quantitative analysis of gene expression, we found that ANS act as an on-off switch for the accumulation of red pigments and had positive correlations with various steps genes of the flavonoid pathway. This expression profiling demonstrates that no gene products compete for common substrates to redirect the metabolic flux in wintersweet. It is also demonstrated that high expression of F3′H would provide sufficient content of the precursor, dihydroquercetin, for both flavonol and anthocyanin biosynthesis. The results help us to deepen and enrich the gene resource of color formation in wintersweet flower, and provide specific breeding strategies for increasing diversity of flower color.

How to make red yeast: An undergraduate laboratory using CRISPR/Cas9 technology to target ADE2 in budding yeast

The ability of CRISPR/Cas9 to edit genomic sequence in a multitude of organisms has revolutionized modern science and is an ideal topic to engage undergraduate students. The ease and speed of this technology combined with the short generation time, well‐characterized genome, established protocols, and relatively limited financial demands of Saccharomyces cerevisiae (bakers yeast) make for a practical undergraduate laboratory. Students, working in pairs were assigned the task of replacing the chromosomal copy of ADE2 with HIS3. Over the course of several weeks students cloned the ADE2 gRNA into an expression vector, PCR amplified a HIS3 construct for homologous recombination, and transformed these components into yeast. ADE2 encodes for Phosphoribosylaminoimidazole carboxylase, an enzyme that catalyzes a reaction in the de novo synthesis of purine nucleotides. Disruption of this gene results in the accumulation of red pigment in the yeast cells. Students were thus able to screen for disruption of the ADE2 gene and subsequent replacement with HIS3 with a specific growth phenotype: red yeast capable of growing on media lacking histidine. The methods used in this lab included: basic cell culture, primer design, PCR, plasmid isolation, transformation, restriction digestion, ligation and DNA sequencing. To reinforce scientific writing and analysis skills the culmination of this project was a manuscript describing their results and a final class lab meeting where each group presented their data and conclusions. Informal assessments indicated that participating in this lab increased student's interest in attending graduate schools and pursing careers in biomedical research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Iron deficiency stimulates anthocyanin accumulation in grapevine apical leaves

Iron chlorosis is a diffuse disorder affecting Mediterranean vineyards. Beside the commonly described symptom of chlorophyll decrease, an apex reddening was recently observed. Secondary metabolites, such as anthocyanins, are often synthetized to cope with stresses in plants. The present work aimed to evaluate grapevine responses to iron deficiency, in terms of anthocyanin metabolism (reflectance spectrum, total anthocyanin content, HPLC profile and gene expression) in apical leaves of Cabernet sauvignon and Sangiovese grown in hydroponic conditions. Iron supply interruption produced after one month an increasing of anthocyanin content associated to a more stable profile in both cultivars. In Cabernet sauvignon, the higher red pigment accumulation was associated to a lower intensity of chlorotic symptoms, while in Sangiovese, despite the activation of the metabolism, the lower anthocyanin accumulation was associated to a stronger decrease in chlorophyll concentration. Gene expression data showed a significant increase of anthocyanin biosynthesis. The effects on the expression of structural and transcription factor genes of phenylpropanoid pathway were cultivar dependent. F3H, F3′H, F3′5′H and LDOX genes, in Cabernet sauvignon, and AOMT1 and AOMT genes, in Sangiovese, were positively affected by the treatment in response to iron deficiency. All data support the hypothesis of an anthocyanin biosynthesis stimulation rather than a decreased degradation of them due to iron chlorosis.

Chlorophyll catabolites in conditioned media of green microalga Desmodesmus subspicatus.

Although the appearance of coloured chlorophyll degradation products of higher plants is well known, knowledge about such compounds produced and released particularly by planktonic algae is still limited. Colourless conditioned media (CM) obtained from autotrophic cultures of unicellular green alga Desmosdemus subspicatus turn red after acidification. The accumulation of red pigments in the medium and the growth rate of algae were inversely correlated. The red, crude solution isolated from CM by dialysis and ion exchange chromatography, and next purified by means of high-performance liquid chromatography, appeared to be a mixture of three compounds with characteristic UV/VIS absorption maxima near 330 and 505 nm. Electrospray ionization (ESI) mass spectrometry analysis revealed that the molecular mass of the most polar and most abundant compound was 637 Da and molecular masses of two other ones were 641 and 607 Da. Addition of 15 N isotope to the culture medium and subsequent mass spectrometry measurements revealed the occurrence of four nitrogen atoms per each molecule. The data suggest that red pigments isolated from algal-conditioned media are chlorophyll degradation compounds, the production of which depends on light intensity, and are released mainly during the stationary phase of growth.

Yeast red pigment modifies Amyloid beta growth in Alzheimer disease models in both Saccharomyces cerevisiae and Drosophila melanogaster

The effect of yeast red pigment on amyloid-β (Aβ) aggregation and fibril growth was studied in yeasts, fruit flies and in vitro. Yeast strains accumulating red pigment (red strains) contained less amyloid and had better survival rates compared to isogenic strains without red pigment accumulation (white strains). Confocal and fluorescent microscopy was used to visualise fluorescent Aβ-GFP aggregates. Yeast cells containing less red pigment had more Aβ-GFP aggregates despite the lower level of overall GFP fluorescence. Western blot analysis with anti-GFP, anti-Aβ and A11 antibodies also revealed that red cells contained a considerably lower amount of Aβ GFP aggregates as compared to white cells. Similar results were obtained with exogenous red pigment that was able to penetrate yeast cells. In vitro experiments with thioflavine and TEM showed that red pigment effectively decreased Aβ fibril growth. Transgenic flies expressing Aβ were cultivated on medium containing red and white isogenic yeast strains. Flies cultivated on red strains had a significant decrease in Aβ accumulation levels and brain neurodegeneration. They also demonstrated better memory and learning indexes and higher locomotor ability.

Impact of alg3 gene deletion on growth, development, pigment production, protein secretion, and functions of recombinant Trichoderma reesei cellobiohydrolases in Aspergillus niger

Dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl α-1,3-mannosyltransferase (also known as “asparagine-linked glycosylation 3”, or ALG3) is involved in early N-linked glycan synthesis and thus is essential for formation of N-linked protein glycosylation. In this study, we examined the effects of alg3 gene deletion (alg3Δ) on growth, development, pigment production, protein secretion and recombinant Trichoderma reesei cellobiohydrolase (rCel7A) expressed in Aspergillus niger. The alg3Δ delayed spore germination in liquid cultures of complete medium (CM), potato dextrose (PD), minimal medium (MM) and CM with addition of cAMP (CM+cAMP), and resulted in significant reduction of hyphal growth on CM, potato dextrose agar (PDA), and CM+cAMP and spore production on CM. The alg3Δ also led to a significant accumulation of red pigment on both liquid and solid CM cultures. The relative abundances of 54 of the total 215 proteins identified in the secretome were significantly altered as a result of alg3Δ, 63% of which were secreted at higher levels in alg3Δ strain than the parent. The rCel7A expressed in the alg3Δ mutant was smaller in size than that expressed in both wild-type and parental strains, but still larger than T. reesei Cel7A. The circular dichroism (CD)-melt scans indicated that change in glycosylation of rCel7A does not appear to impact the secondary structure or folding. Enzyme assays of Cel7A and rCel7A on nanocrystalline cellulose and bleached kraft pulp demonstrated that the rCel7As have improved activities on hydrolyzing the nanocrystalline cellulose. Overall, the results suggest that alg3 is critical for growth, sporulation, pigment production, and protein secretion in A. niger, and demonstrate the feasibility of this alternative approach to evaluate the roles of N-linked glycosylation in glycoprotein secretion and function.

Isolation and expression analysis of six putative structural genes involved in anthocyanin biosynthesis in Tulipa fosteriana

Getting basic molecular information of pigment formation in tulip flower is a prerequisite for enhancing the range of flower colors. In this study, six cDNA clones encoding putative anthocyanin biosynthesis enzymes were isolated from petals of tulip (Tulipa fosteriana) ‘Shangnong Zaoxia’ by rapid-amplification of cDNA ends (RACE): chalcone synthase (TfCHS1), chalcone isomerase (TfCHI1), flavanone 3-hydroxylase (TfF3H1), flavonoid 3′-hydroxylase (TfF3′H1), dihydroflavonol 4-reductase (TfDFR1) and anthocyanidin synthase (TfANS1). Southern blot analysis showed they all present as multiple copies in the tulip genome. In parallel, the expression profiles of these genes in different tulip tissues as well as in petals during flower development were measured by real-time quantitative PCR. The expression levels of TfCHS1, TfCHI1 and TfF3H1 were higher in stems and leaves than those in floral tissues (stamens, pistils, petals), the expression of TfF3′H1, TfDFR1 and TfANS1 showed highest levels in petals compared with other tissues. During flower development, the transcript levels of TfCHS1 and TfF3H1 were continuously increased, which were in general constant with the changing pattern of anthocyanin accumulation; the mRNA levels of TfDFR1 and TfANS1 and anthocyanin accumulation were both significantly increased; a low level transcript of TfF3′H1 was detected in petals throughout pigmentation process; TfCHI1 expression level firstly dramatically decreased and then gently increased. These results suggest that the red pigment accumulation in petals of T. fosteriana was positively related to the expression levels of TfCHS1, TfF3H1, TfDFR1 and TfANS1.

CAROTENOID PIGMENT CONTENT IN NEW CULTIVARS OF PAPRIKA PEPPERS

The fruits of Capsicum owe their intense red color to carotenoid pigments that are synthetized massively during fruit ripening. In the industry, carotenoids from paprika are used as natural colorants. All the carotenoid pigments present in the pepper are isoprenoids containing nine conjugated double bonds in the central polyenic chain, although with different end groups, allowing them to be classified in two isochromic families: red (R) and yellow (Y). Since the commercial value of paprika is determined by the intensity of its red coloration, the selection of cultivars must not only increase the total carotenoid content, but also increase, if possible, the R/Y ratio. Chlorophyll pigments decrease and disappear from the red cultivars during ripening. In contrast in the stay-green cultivars, with the recessive gene cl (chloro-phyll retainer) a chocolate brown color of the ripe fruits results from lack of chloro-phyll degradation and the simultaneous accumulation of red pigments. In the present work, the local cultivar 'Bola' and six cultivars of paprika selected by IMIDA Horticulture Department, the wild-type red cultivars 'Costal', 'Datler' and 'Rodacol' and the stay-green cultivars 'Negral', 'Datler Negro' and 'Nedacol', have been characterized by their carotenoid pigment content, the R/Y ratio and °ASTA value, with the following results: except for the red cultivar 'Costal' with 7917.51 mg/kg carotenoid content, the highest total carotenoid content were the stay-green cultivars 'Datler Negro' with 6358.59 mg/kg, and 'Negral' and 'Nedacol' with 6236.61 and 5984.09 mg/kg respectively.

DYNAMICS OF THE DEGRADATION OF CHLOROPHYLLS IN CULTIVARS OF PAPRIKA PEPPERS

The aim of the present study is to investigate the behavior, in terms of evolution of chlorophylls in fruits, of seven cultivars selected and bred by IMIDA Horticulture Department: the wild-type red cultivars ‘Costal’, ‘Bola’, ‘Datler’ and ‘Rodacol’ and the stay-green cultivars, with the recessive gene cl (chlorophyll retainer) ‘Negral’, ‘Datler Negro’ and ‘Nedacol’. The fruit have been classified in 8 ripening stages: stages 1-4 green to mature green, stage 5 breaker and stage 6 to 8 red succulent to red partially dry (over-ripe). In each of the stages we have studied the following parameters: total chlorophyll content, chlorophyll a, chlorophyll b and the chlorophyll a/b ratio. The results were as follows: the red cultivars degrade the chlorophyll when the process of fruit ripening starts. Thus, in stage 7 (fully ripe fruits) chlorophyll disappears completely. In the stay-green cultivars, however, there is no chlorophyll degradation at the beginning of ripening. A chocolate brown color of the ripe fruit results from lack of chlorophyll degradation and the simultaneous accumulation of red pigments. In some stay-green cultivars, such as ‘Datler Negro’, the chlorophyll values in over-ripe fruit range from 528.26 mg/kg at the beginning of maturation to 222.68 mg/kg in the over-ripe stage. Therefore, the chlorophyll catabolism pathway is not completely inhibited but delayed.

Effect of red pigment on amyloidization of yeast

Amyloid-bound thioflavin T fluorescence was studied in lysates of yeast strains that carry mutations in the ADE1 or ADE2 genes and accumulate red pigment as a result of the polymerization of aminoimidazole ribotide (an intermediate of adenine biosynthesis). The fluorescence is drastically enhanced in cells grown in media with high concentrations of adenine (100 mg/l), which suppresses the accumulation of red pigment. Mutations that block the first stages of purine biosynthesis de novo also impede the accumulation of red pigment and produce the same effect on thioflavin fluorescence. Mutations in ADE1 or ADE2 genes in originally white prototrophic strains considerably suppress fluorescence. The fraction of protein polymers was studied by agarose gel electrophoresis, which permitted us to conclude that reduced fluorescence intensity was associated with decreased amyloid content in cells that accumulate red pigment. Model experiments with insulin fibers demonstrate that red pigment binds fibrils and blocks their interaction with thioflavin T. A comparison of lysate pellet proteins from red and white isogenic strains separated by 2D electrophoresis followed by MALDI analysis allowed us to identify 23 pigment-dependent proteins. These proteins mostly belong to functional classes of chaperones and proteins involved in glucose metabolism, which closely correspond to the prion-dependent proteins that we characterized previously. We suppose that the binding of red pigment with amyloid fibrils prevents the generation of prion aggregates and impedes prion propagation by blocking fibril contact with chaperones.