Transcriptome Analysis of Differential Genes Related to Anthocyanin Synthesis in Red and Green Walnut
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ABSTRACT: We report the application of high-throughput analysis of genes related to anthocyanin synthesis in red and green walnuts based on transcriptome sequencing technology. By obtaining a sequence of more than 3 billion bases from mRNA, we found genes that are differentially expressed with anthocyanin synthesis in 4 leaf growth stages and 3 peel growth stages of red and green walnuts.
Project description:Purpose:The red coloration of apple (Malus × domestica Borkh.) is due to the accumulation of anthocyanins in the fruit peel. Light is essential for anthocyanin biosynthesis in apple.Apple peel can quickly turn red under light conditions after unbagging. Therefore, the implementation of transcriptome sequencing to find genes that promote anthocyanin accumulation in response to light signals is necessary to clarify the mechanism of light-induced anthocyanin accumulation in apple peel.
Project description:Tomato MicroTom plants of the genotypes Aft/Aft x atv/atv (Aft/atv) and Aft/Aft x atv/atv x hp2/hp2 (Aft/atv/hp2) were used. Fruits showing purple anthocyanin pigmentation in the skin were collected at at the mature green (MG) and mature red (MR) stages to compare the transcriptomes of exocarp and mesocarp between the two developmental stages and the two genotypes with the objective to highlight the mechanisms through which light induces anthocyanin synthesis in tomato skin and impede it in mesocarp and other internal parts of the fruits.
Project description:Anthocyanins are high value plant antioxidants which are not present in the fruits of cultivated tomato. However, both the dominant gene Anthocyanin fruit (Aft) and the recessive gene atroviolacea (atv), introgressed into domesticated tomato from two different wild Solanum species, stimulate a limited anthocyanin pigmentation. Surprisingly, double mutant Aft/Aft atv/atv tomatoes are characterised by the presence of anthocyanins in the fruit peel, resulting in intensely purple pigmented fruit. We carried out a transcript profiling analysis using GeneChip® Tomato Genome Arrays, in order to identify differentially expressed genes when comparing wild type, Aft/Aft, atv/atv, and Aft/Aft atv/atv fruits. The expression pattern of several genes involved in the anthocyanin pathway was analyzed in detail. Among the fruit peel-associated differentially expressed transcripts, genes involved in phenylpropanoid pathway, cell wall composition, biotic and abiotic stress responses, sugar and hormone metabolism were overrepresented in Aft/Aft atv/atv. Transcriptomic analysis thus revealed that the activation of anthocyanin synthesis in tomato fruit was accompanied by a complex remodulation of gene expression, likely affecting important agronomic and merceological traits. Wild type (Cv. Ailsa Craig, accession number LA2838A), Aft/Aft (accession number LA1996), atv/atv (accession number LA0797) and double mutant (Aft/Aft atv/atv) were grown during the winter season in a controlled heated greenhouse. Fruits were collected at mature green, turning red and red stages of development. The transcriptional profile in Aft/Aft, atv/atv, and Aft/Aft atv/atv fruits when compared to the wild type was analyzed using the GeneChip® Tomato Genome Array.
Project description:Gene-to-gene coexpression analysis is a powerful approach to infer function of uncharacterized genes. To perform non-targeted coexpression analysis of tomato genes, we collected a developmental gene expression dataset using various tissues of tomato plant. Expression data are collected from 24 different tissue types including root, hypocotyl, cotyledon, leaf at different stages, and fruit tissues at 4 different ripening stages from 4 different Solanum lycopersicum cultivars. Fruits were separated to the flesh and the peel. These two tissue types indeed showed remarkably different gene expression profiles. We also collected data from 4 different ripening stages (mature green, yellow, orange, and red) to detail the changes during ripening. By using this gene expression dataset, we calculated pair-wise Pearsonâs correlation coefficients, and performed network-based coexpression analysis. The analysis generated a number of coexpression modules, some of which showed an enrichment of genes associated with specific functional categories. This result will be useful in inferring functions of uncharacterized tomato genes, and in prioritizing genes for further experimental analysis. We used Affymetrix GeneChip Tomato genome Arrays to detail the global gene expression change using 24 different tomato tissue types (67 hybridizations). We collected gene expression data from 24 different tomato tissue types using 67 hybridizations. Root, hypocotyl, cotyledon, and leaf were sampled from 3-week-old or 5-weekâold plant of Solanum lycopersicum cultivar Micro-Tom. Fruit tissues were sampled from S. lycopersicum cultivars Micro-Tom, Anthocyanin fruit (Aft, LA1996), Line27859, and Momotaro 8 (Takii, Japan). From Micro-Tom fruit, the peel and the flesh were separately sampled from 4 different ripening stages: mature green (MG, approximately 30 day after anthesis), yellow (Y, approximately 35 days after anthesis), orange (O, approximately 38-40 days after anthesis), and red (R, approximately 45-48 days after anthesis). From fruits of Aft and Line27859, the peel and the flesh were sampled at mature green (MG, approximately 40 days after anthesis) and red (R, approximately 50-55 days after anthesis) stages. From Momotaro 8, the peel and the flesh were sampled at red (R, 50- approximately 50-55 days after anthesis) stages. For each tissue type, 2-4 biological replicates were made in RNA preparation.
Project description:Peel color is a key factor that affects the fruit’s aesthetic and economic values. In Red Sugar pineapple, the peels’ red color reduces during maturation. Limited knowledge is available on the regulation of pineapple peel discoloration, which makes it important to study the molecular mechanisms associated with this important trait. Here, we report that a decrease in anthocyanin biosynthesis is predominantly associated with the pineapple peel color change during maturation. Particularly the exclusive accumulation of cyanidin in 60 days after flowering (DAF) as compared to 120 DAF gives the fruit peel its distinct reddish color. Our findings suggest that the changes in the expression of key structural genes (early and late biosynthetic genes) of the anthocyanin (cyanidin) biosynthesis pathway are responsible for peel discoloration. Based on a gene co-expression analysis and a transient expression, we identified two transcription factors i.e., AcHOX21 and AcMYB12, and showed that their downregulation leads to the reduced anthocyanin accumulation with fruit maturation.
Project description:Environmental factors play an important role in anthocyanin biosynthesis, and potassium, an essential nutrient for blueberry growth, can act as an enzyme activator. However, few reports exist on the transcriptional and anthocyanin metabolic changes in blueberries regulated by potassium. In this study, blueberries treated with potassium at different stages were compared for changes in enzyme activity, transcription, and metabolism related to anthocyanin synthesis. The results showed that potassium treatment significantly enhanced the activities of key enzymes F3H, F3'5'H, and UFGT in the anthocyanin synthesis pathway of blueberry fruit. Metabolomic results indicated that the contents of malvidin, petunidin, and delphinidin were higher with potassium fertilization, and potassium treatment promoted the early color change of blueberry fruit. The transcriptome analysis identified 102 glucose metabolism-related genes and 12 differential potassium transport genes potentially involved in potassium-regulated anthocyanin synthesis and accumulation. It was found that thirteen genes relate to anthocyanin synthesis. UFGT, F3H, CHI, HCT, C12RT1, DFR, and F3'5'H were all closely associated with potassium-controlled flavonoid and anthocyanin metabolite synthesis. It provides valuable insights into the molecular mechanisms that regulate the synthesis of anthocyanins in blueberries.
Project description:Anthocyanins are flavonoid compounds responsible for red/purple colours in the leaves, fruit and flowers of many plant species. They are produced through a multistep pathway which is controlled by MYB transcription factors. VvMYBA1 and VvMYBA2 activate anthocyanin biosynthesis in grapevine (Vitis vinifera) and are non-functional in white grapevine cultivars. In this study, transgenic grapevines with altered VvMYBA gene expression were developed, and transcript analysis was carried out on berries using a microarray technique. The results showed that VvMYBA is a positive regulator of the later stages of anthocyanin synthesis, modification and transport in Shiraz. One up-regulated gene ANTHOCYANIN 3- O-GLUCOSIDE-6”-O-ACYLTRANSFERASE (Vv3AT) encodes a BAHD acyltransferase protein, belonging to a clade separate from most anthocyanin acyltransferases. Functional studies (in planta and in vitro) show that Vv3AT has a broad anthocyanin substrate specificity and can also utilise both aliphatic and aromatic acyl donors, a novel activity for this enzyme family found in nature. In V. vinifera cv. Pinot Noir, a red-berried grapevine mutant lacking acylated anthocyanins, Vv3AT contains a nonsense mutation encoding a truncated protein that lacks two motifs required for BAHD protein activity. Promoter activation assays confirm that Vv3AT transcription is activated by VvMYBA1, which adds to the current understanding of the regulation of the BAHD gene family. The flexibility of Vv3AT to use both classes of acyl donors will be useful in the engineering of anthocyanins in planta or in vitro.
Project description:MicroRNA172 (miR172) plays a role in regulating a diverse range of plant developmental processes. However, its role in regulating anthocyanin biosynthesis is unclear. In this study, we show that transgenic apple plants over-expressing miR172 show a reduction in red colouration and anthocyanin accumulation in multiple tissue types. This reduction was consistent with decreased expression of APETALA2 homolog MdAP2_1a (a miR172 target gene), MdMYB10, and targets of MdMYB10, as demonstrated by both RNA-seq and RT-PCR analyses. During the onset of anthocyanin biosynthesis in apple fruit skin, miR172 transcript abundance was negatively correlated and MdAP2_1a mRNA concentration was positively correlated with fruit skin red-colouration. The positive role of MdAP2_1a in regulating anthocyanin biosynthesis was supported by the enhanced petal anthocyanin accumulation in transgenic tobacco plants overexpressing MdAP2_1a, and by the reduction in anthocyanin accumulation in apple and cherry fruits transfected with an MdAP2_1a virus-induced-gene-silencing construct. We demonstrated that MdAP2_1a could bind directly to the promoter and protein sequences of MdMYB10 in yeast and tobacco, and enhance MdMYB10 promotor activity. In Arabidopsis, over-expression of miR172 reduced anthocyanin concentration and RNA transcript abundance of anthocyanin genes in plantlets cultured on medium containing 7% sucrose. The anthocyanin content and RNA abundance of anthocyanin genes could be partially restored by using a synonymous mutant of MdAP2_1a, which had lost the miR172 target sequences at mRNA level, but not restored by using a WT MdAP2_1a. These results indicate that miR172 inhibits anthocyanin biosynthesis through suppressing the expression of an AP2 transcription factor that positively regulates MdMYB10.
Project description:Anthocyanins are colorful plant pigments with antioxidant properties, and a diet rich in these flavonoids bears health benefits. Therefore, a strong anthocyanin accumulation in edible plant parts is of significant interest, and in Malus domestica, the domesticated apple, certain red-fleshed apple varieties exhibit this trait. Enhanced anthocyanin accumulation in the flesh of apple fruits is attributed to the hyperactivation of the MYB transcription factor MdMYB10, which act as a key regulators of anthocyanin biosynthesis by inducing the expression of multiple biosynthetic genes. While several studies have explored the underlying genetic mutations and resulting transcriptome changes, there is a lack of research on proteome alterations that cause the red-fleshed apple phenotype. To address this gap, a mass spectrometry-based proteomics approach was employed. Comparative proteomics identified differentially abundant proteins in young and mature fruits of the red-fleshed ‘Bay13645’ variety compared to the white-fleshed ‘Royal Gala’. Whereas several MYB transcription factors were enriched during early fruit development, they were no longer among the hyper-abundant proteins in ripe fruits of the red-fleshed genotype. In contrast, anthocyanin biosynthetic enzymes were enriched more strongly in ripe fruits of the red-fleshed cultivar, indicating developmental stage-specific differences in the control of the pigmentation process. The proteomic approach also identified novel regulatory factors and enzymes that may contribute to the red-fleshed apple phenotype, including a BAHD acyltransferase, Mal d proteins, and transcription factors of diverse families, and their potential relevance for the exhibition of this trait is discussed.