Genes In Anthocyanin Biosynthetic Pathway Research Articles

Comparative transcriptome analysis reveals transcriptional regulation of anthocyanin biosynthesis in purple radish (Raphanus sativus L.)

Radish exhibits significant variation in color, particularly in sprouts, leaves, petals, fleshy roots, and other tissues, displaying a range of hues such as green, white, red, purple, and black. Although extensive research has been conducted on the color variation of radish, the underlying mechanism behind the variation in radish flower color remains unclear. To date, there is a lack of comprehensive research investigating the variation mechanism of radish sprouts, leaves, fleshy roots, and flower organs. This study aims to address this gap by utilizing transcriptome sequencing to acquire transcriptome data for white and purple radish flowers. Additionally, the published transcriptome data of sprouts, leaves, and fleshy roots were incorporated to conduct a systematic analysis of the regulatory mechanisms underlying anthocyanin biosynthesis in these four radish tissues. The comparative transcriptome analysis revealed differential expression of the anthocyanin biosynthetic pathway genes DFR, UGT78D2, TT12 and CPC in the four radish tissues. Additionally, the WGCNA results identified RsDFR.9c and RsUGT78D2.2c as hub genes responsible for regulating anthocyanin biosynthesis. By integrating the findings from the comparative transcriptome analysis, WGCNA, and anthocyanin biosynthetic pathway-related gene expression patterns, it is hypothesized that genes RsDFR.9c and RsUGT78D2.2c may serve as pivotal regulators of anthocyanins in the four radish tissues. Furthermore, the tissue-specific expression of the four copies of RsPAP1 is deemed crucial in governing anthocyanin synthesis and accumulation. Our results provide new insights into the molecular mechanism of anthocyanin biosynthesis and accumulation in different tissues of radish.

Transcriptomic and metabolic analysis unveils the mechanism behind leaf color development in Disanthus cercidifolius var. longipes.

Introduction: Leaf coloration in Disanthus cercidifolius var. longipes results from the interplay of various pigments undergoing complex catalytic reactions. Methods: We aimed to elucidate the mechanisms of pigment biosynthesis affecting leaf color transition in D. cercidifolius var. longipes by analyzing variations in pigment accumulation and levels of gene expression. Results: We identified 468, 577, and 215 differential metabolites in green leaves (GL), gradual-color-changing leaves (GCCL), and red leaves (RL), respectively, with 94 metabolites shared across all comparisons. Metabolite accumulation patterns were similar among GL, GCCL, and RL, with flavonoids being the main differential metabolites. Delphinidin, malvidin, and petunidin derivatives were mostly accumulated in GCCL, whereas cyanidin, pelargonidin, and peonidin derivatives accumulated in RL. Transcriptome sequencing was used to identify differentially expressed genes. The expression of anthocyanin biosynthetic pathway genes was associated with anthocyanin accumulation patterns. Discussion: Our findings reveal that the content of delphinidin, malvidin, petunidin, and carotenoids collectively determines the gradual transition of leaf color from green in spring and summer to green, purple, and orange-yellow in early autumn, whereas the content of cyanidin, peonidin, pelargonidin, and carotenoids together causes the autumnal transition to red or orange-red colors as leaves of D. cercidifolius var. longipes age.

CstMYB1R1, a REVEILLE-8-like transcription factor, regulates diurnal clock-specific anthocyanin biosynthesis and response to abiotic stress in Crocus sativus L.

CstMYB1R1 acts as a positive regulator of Crocus anthocyanin biosynthesis and abiotic stress tolerance which was experimentally demonstrated through molecular analysis and over-expression studies in Crocus and Nicotiana. Regulatory mechanics of flavonoid/anthocyanin biosynthesis in Crocus floral tissues along the diurnal clock has not been studied to date. MYB proteins represent the most dominant, functionally diverse and versatile type of plant transcription factors which regulate key metabolic and physiological processes in planta. Transcriptome analysis revealed that MYB family is the most dominant transcription factor family in C. sativus. Considering this, a MYB-related REVEILLE-8 type transcription factor, CstMYB1R1, was explored for its possible role in regulating Crocus flavonoid and anthocyanin biosynthetic pathway. CstMYB1R1 was highly expressed in Crocus floral tissues, particularly tepals and its expression was shown to peak at dawn and dusk time points. Anthocyanin accumulation also peaked at dawn and dusk and was minimum at night. Moreover, the diurnal expression pattern of CstMYB1R1 was shown to highly correlate with Crocus ANS/LDOX gene expression among the late anthocyanin pathway genes. CstMYB1R1 was shown to be nuclear localized and transcriptionally active. CstMYB1R1 over-expression in Crocus tepals enhanced anthocyanin levels and upregulated transcripts of Crocus flavonoid and anthocyanin biosynthetic pathway genes. Yeast one hybrid (Y1H) and GUS reporter assay confirmed that CstMYB1R1 interacts with the promoter of Crocus LDOX gene to directly regulate its transcription. In addition, the expression of CstMYB1R1 in Nicotiana plants significantly enhanced flavonoid and anthocyanin levels and improved their abiotic stress tolerance. The present study, thus, confirmed positive role of CstMYB1R1 in regulating Crocus anthocyanin biosynthetic pathway in a diurnal clock-specific fashion together with its involvement in the regulation of abiotic stress response.

The transcription factors TLR1 and TLR2 negatively regulate trichome density and artemisinin levels in Artemisia annua.

The important antimalarial drug artemisinin is biosynthesized and stored in Artemisia annua glandular trichomes and the artemisinin content correlates with trichome density; however, the factors affecting trichome development are largely unknown. Here, we demonstrate that the A. annua R2R3 MYB transcription factor TrichomeLess Regulator 1 (TLR1) negatively regulates trichome development. In A. annua, TLR1 overexpression lines had 44.7%-64.0% lower trichome density and 11.5%-49.4% lower artemisinin contents and TLR1-RNAi lines had 33%-93.3% higher trichome density and 32.2%-84.0% higher artemisinin contents compared with non-transgenic controls. TLR1 also negatively regulates the expression of anthocyanin biosynthetic pathway genes in A. annua. When heterologously expressed in Arabidopsis thaliana, TLR1 interacts with GLABROUS3a, positive regulator of trichome development, and represses trichome development. Yeast two-hybrid and pull-down assays indicated that TLR1 interacts with the WUSCHEL homeobox (WOX) protein AaWOX1, which interacts with the LEAFY-like transcription factor TLR2. TLR2 overexpression in Arabidopsis and A. annua showed that TLR2 reduces trichome development by reducing gibberellin levels. Furthermore, artemisinin contents were 19%-43% lower in TLR2-overexpressing A. annua plants compared to controls. These data indicate that TLR1 and TLR2 negatively regulate trichome density by lowering gibberellin levels and may enable approaches to enhance artemisinin yields.

MYB-6 and LDOX-1 regulated accretion of anthocyanin response to cold stress in purple black carrot (Daucus carota L.).

Anthocyanin, an essential ingredient of functional foods, is present in a wide range of plants, including black carrots. The current investigation was carried out to analyse the effect of cold stress on the expression of major anthocyanins and anthocyanin biosynthetic pathway genes, MYB6 and LDOX-1. Five cultivated carrotgenotypes belonging to the eastern group, having anthocyanin pigment, were used in the current study. The qRT-PCR analysis revealed that relative gene expression of transcription factor MYB-6 and LDOX1gene was highly expressed upon cold stress compared to non-stress samples. High-performance liquid chromatography-based quantification of Cyanidin 3-O-glucoside (Kuromanin chloride), Ferulic acid, 3,5-Dimethoxy-4-hydroxycinnamic acid (Sinapic acid), and Rutin revealed a significant increase in these major anthocyanins in response to cold stress when compared to control plants. We conclude that MYB6 and LDOX1 gene expression increases upon cold stress, which induces accumulation of major anthocyanins in purple black carrot and suggestsa possible cross-link between cold stress and anthocyanin biosynthesis in purple black carrot.

Genome-wide analysis and transcriptional reprogrammings of MYB superfamily revealed positive insights into abiotic stress responses and anthocyanin accumulation in Carthamus tinctorius L.

The MYB transcription factors comprise one of the largest superfamilies in plants that have been implicated in the regulation of plant-specific metabolites and responses to biotic and abiotic stresses. Here, we present the first comprehensive genome-wide analysis and functional characterization of the CtMYB family in Carthamus tinctorius. A total of 272 CtMYBs were identified and classified into 12 subgroups using comparative phylogenetic analysis with Arabidopsis and rice orthologs. The overview of conserved motifs, gene structures, and cis elements as well as the expression pattern of CtMYB genes indicated the diverse roles of these transcription factors during plant growth, regulation of secondary metabolites, and various abiotic stress responses. The subcellular localization and transactivation analysis of four CtMYB proteins indicated predominant localization in the nuclei with enhanced transcriptional activation in yeast. The expression of CtMYB63 induced with various abiotic stress conditions showed upregulation in its transcription level. In addition, the expression analysis of the core structural genes of anthocyanin biosynthetic pathway under drought and cold stress in CtMYB63 overexpressed transgenic lines also supports the notion of CtMYB63 transcriptional reprogramming in response to abiotic stress by upregulating the anthocyanin biosynthesis. Together, our findings revealed the underlying regulatory mechanism of CtMYB TF network involving enhanced cold and drought stress tolerance through activating the rapid biosynthesis of anthocyanin in C. tinctorius. This study also presents useful insights towards the establishment of new strategies for crop improvements.

VvMYBAs induce expression of a series of anthocyanin biosynthetic pathway genes in red grapes (Vitis vinifera L.)

The role of MYBA transcription factors in anthocyanin biosynthesis has been established in many plants including grapevine (Vitis vinifera L.); however, not all of the target genes of MYBAs in grapevine have been elucidated. The aim of this study was therefore to identify targets of VvMYBAs by using berry skin from a red grape (Pinot Noir) and its white mutant (Pinot Blanc), and from white grapes (Italia and Muscat of Alexandria) and their red mutants (Benitaka and Flame Muscat). First, microarray and reverse-transcription real-time PCR data revealed that, in addition to the anthocyanin specific biosynthetic genes, i.e. UDP-glucose flavonoid:3-O-glucosyltransferase (UFGT) and O-methyltransferase (OMT), a gene involved in anthocyanin transfer into vacuoles, i.e. glutathione S-transferase (GST), as well as flavonoid-3’,5’-hydroxylase (F3’,5’H) were highly upregulated in red grapes. From collective findings, it is likely that these genes are probably induced only by VvMYBAs. By contrast, genes involved in upstream flavonoid biosynthesis, such as phenylalanine ammonia-lyase (PAL), chalcone synthase 3 (CHS3), flavanone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), and leucoanthocyanidin dioxygenase (LDOX), were expressed in white grapes and moderately upregulated in red grapes. These genes seem to be induced by both VvMYBAs and other transcription factors. The electrophoretic mobility shift assays results revealed that VvMYBA1 and/or VvMYBA2 proteins interact with the promoter region of UFGT, CHS3, and LDOX. Collectively, our findings suggest that VvMYBAs regulate the expression of all genes involved in anthocyanin biosynthesis, and that VvMYBA protein directly interacts with not only UFGT, but also CHS3 and LDOX.

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.

StMYB44 negatively regulates anthocyanin biosynthesis at high temperatures in tuber flesh of potato.

High temperatures are known to reduce anthocyanin accumulation in a number of diverse plant species. In potato (Solanum tuberosum L.), high temperature significantly reduces tuber anthocyanin pigment content. However, the mechanism of anthocyanin biosynthesis in potato tuber under heat stress remains unknown. Here we show that high temperature causes reduction of anthocyanin biosynthesis in both potato tuber skin and flesh, with white areas forming between the vasculature and periderm. Heat stress reduced the expression of the R2R3 MYB transcription factors (TFs) StAN1 and StbHLH1, members of the transcriptional complex responsible for coordinated regulation of the skin and flesh pigmentation, as well as anthocyanin biosynthetic pathway genes in white regions. However, the core phenylpropanoid pathway, lignin, and chlorogenic acid (CGA) pathway genes were up-regulated in white areas, suggesting that suppression of the anthocyanin branch may result in re-routing phenylpropanoid flux into the CGA or lignin biosynthesis branches. Two R2R3 MYB TFs, StMYB44-1 and StMYB44-2, were highly expressed in white regions under high temperature. In transient assays, StMYB44 represses anthocyanin accumulation in leaves of Nicotiana tabacum and N. benthamiana by directly suppressing the activity of the dihydroflavonol reductase (DFR) promoter. StMYB44-1 showed stronger repressive capacity than StMYB44-2, with both predicted proteins containing the repression-associated EAR motif with some variation. StMYB44-1 conferred repression without a requirement for a basic helix-loop-helix (bHLH) partner, suggesting a different repression mechanism from that of reported anthocyanin repressors. We propose that temperature-induced reduction of anthocyanin accumulation in potato flesh is caused by down-regulation of the activating anthocyanin regulatory complex, by enhancing the expression of flesh-specific StMYB44 and alteration of phenylpropanoid flux.

The Effect of Maturity Status on Biochemical Composition, Antioxidant Activity, and Anthocyanin Biosynthesis Gene Expression in a Pomegranate (Punica granatum L.) Cultivar with Red Flowers, Yellow Peel, and Pinkish Arils

Pomegranate (Punica granatum L.) has widely been used as a fruit and in folk medicine since ancient civilizations in the world. It is now known that bioactive compounds present in pomegranates attribute to its therapeutic potential. Harvesting at the correct maturity stage is one of the key factors deciding the quality of harvest for consumption in fresh or value-added forms. Identification of the correct maturity stage (harvesting index) is particularly difficult for cultivars having yellowish peel and pinkish arils (sarcotesta). We studied the changes in total phenolic content (TPC), antioxidant activity (AOX), punicalagin α and β contents, color indices, total soluble solids (TSS), and expression of anthocyanin biosynthetic pathway genes from flowering to harvesting in the pomegranate cultivar Nimali, having red flowers, yellow peels, and pinkish arils at maturity over two growing seasons. Interestingly, there was no seasonal variation observed in any of the parameters over two cultivation seasons. Although the β punicalagin content did not change, the TPC, AOX, punicalagin α contents in both peel and arils gradually decreased from flowering to maturity. Though the TPC of both peels and arils, AOX and total punicalagin content of peel did not change significantly 140 days after flower initiation, the TPC and the total punicalagin of arils reached a stable level at 160 days. The TSS in both peels and arils increased significantly with the maturity having the highest values at 180 days. The peel color changed from green to yellow with maturity with a significant increase in l* and b* values and significant decrease in a* value. Nevertheless, the aril color changed from pale white to pink with the maturity with significant reduction of l* and b* values and significant increase of a* value. Changes in pomegranate dihydroflavonol 4-reductase (DFR), flavanone 3-hydroxylase (F3H), and leucoanthocyanidin dioxygenase (anthocyanidin synthase-ANS) gene expression in Nimali arils correlated with its color changes during maturity. These findings support to identify the harvesting index of Nimali ensuring the maximum nutritional and health benefits of pomegranate flower, arils, and peels for different downstream uses.

Transcriptomic profiling of two Pak Choi varieties with contrasting anthocyanin contents provides an insight into structural and regulatory genes in anthocyanin biosynthetic pathway

BackgroundThe accumulation of anthocyanin in horticultural crops not only improves their stress tolerances but also their nutritional values. Many key regulatory and structural genes in anthocyanin biosynthesis have been identified in model plants, but limited information is available for non-model plant species featured with colored leaves. In this study, two Pak Choi varieties with green or purple leaves were selected to analyze the anthocyanin biosynthesis through RNA-Seq.ResultsA total of 2475 unigenes were differentially expressed between these tested varieties, including 1303 down-regulated and 1172 up-regulated genes in the purple-leafed one. The reliability of the RNA-Seq was further confirmed by using real-time quantitative PCR. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the differentially expressed genes revealed ‘flavonoid biosynthesis’ was the only enriched pathway in the purple-leafed variety: In the pathway of phenylpropanoid metabolism, Bra017210, Bra039777, and Bra021637 were expressed at higher levels in the purple-leafed variety; among the early anthocyanin biosynthetic genes, Bra037747 transcripts were only detected in the purple-leafed variety but not in the green-leafed one; among the late anthocyanin biosynthetic genes, Bra027457, Bra013652, Bra019350, Bra003021, Bra035004, and Bra038445 were all up-regulated in purple-leafed variety; and genes encoding anthocyanin-related transcription factors, such as Bra016164, and genes encoding anthocyanin transportation, such as GST F12, were also identified as up-regulated ones in the purple-leafed variety.ConclusionsThe current result provided a valuable insight into the anthocyanin accumulation in the purple-leafed variety of Pak Choi and a bioinformatic resource for further functional identification of key allelic genes determining the difference of anthocyanin content between Pak Choi varieties.

Diversifying selection of the anthocyanin biosynthetic downstream gene UFGT accelerates floral diversity of island Scutellaria species.

BackgroundAdaptive divergence, which usually explains rapid diversification within island species, might involve the positive selection of genes. Anthocyanin biosynthetic pathway (ABP) genes are important for floral diversity, and are related to stress resistance and pollination, which could be responsible for species diversification. Previous studies have shown that upstream genes of ABP are subject to selective constraints and have a slow evolutionary rate, while the constraints on downstream genes are lower.ResultsIn this study, we confirmed these earlier observations of heterogeneous evolutionary rate in upstream gene CHS and the downstream gene UFGT, both of which were expressed in Scutellaria sp. inflorescence buds. We found a higher evolutionary rate and positive selection for UFGT. The codons under positive selection corresponded to the diversified regions, and the presence or absence of an α-helix might produce conformation changes in the Rossmann-like fold structure. The significantly high evolutionary rates for UFGT genes in Taiwanese lineages suggested rapid accumulation of amino acid mutations in island species. The results showed positive selection in closely related species and explained the high diversification of floral patterns in these recently diverged species. In contrast, non-synonymous mutation rate decreases in long-term divergent species could reduce mutational load and prevent the accumulation of deleterious mutations.ConclusionsTogether with the positive selection of transcription factors for ABP genes described in a previous study, these results confirmed that positive selection takes place at a translational level and suggested that the high divergence of ABP genes is related to the floral diversity in island Scutellaria species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0759-0) contains supplementary material, which is available to authorized users.

Multiple post-domestication origins of kabuli chickpea through allelic variation in a diversification-associated transcription factor.

Chickpea (Cicer arietinum) is among the founder crops domesticated in the Fertile Crescent. One of two major forms of chickpea, the so-called kabuli type, has white flowers and light-colored seed coats, properties not known to exist in the wild progenitor. The origin of the kabuli form has been enigmatic. We genotyped a collection of wild and cultivated chickpea genotypes with 538 single nucleotide polymorphisms (SNPs) and examined patterns of molecular diversity relative to geographical sources and market types. In addition, we examined sequence and expression variation in candidate anthocyanin biosynthetic pathway genes. A reduction in genetic diversity and extensive genetic admixture distinguish cultivated chickpea from its wild progenitor species. Among germplasm, the kabuli form is polyphyletic. We identified a basic helix-loop-helix (bHLH) transcription factor at chickpea's B locus that conditions flower and seed colors, orthologous to Mendel's A gene of garden pea, whose loss of function is associated invariantly with the kabuli type of chickpea. From the polyphyletic distribution of the kabuli form in germplasm, an absence of nested variation within the bHLH gene and invariant association of loss of function of bHLH among the kabuli type, we conclude that the kabuli form arose multiple times during the phase of phenotypic diversification after initial domestication of cultivated chickpea.

Molecular characterization of genes encoding leucoanthocyanidin reductase involved in proanthocyanidin biosynthesis in apple.

Proanthocyanidins (PAs) are the major component of phenolics in apple, but mechanisms involved in PA biosynthesis remain unclear. Here, the relationship between the PA biosynthesis and the expression of genes encoding leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) was investigated in fruit skin of one apple cultivar and three crabapples. Transcript levels of LAR1 and ANR2 genes were significantly correlated with the contents of catechin and epicatechin, respectively, which suggests their active roles in PA synthesis. Surprisingly, transcript levels for both LAR1 and LAR2 genes were almost undetectable in two crabapples that accumulated both flavan-3-ols and PAs. This contradicts the previous finding that LAR1 gene is a strong candidate regulating the accumulation of metabolites such as epicatechin and PAs in apple. Ectopic expression of apple MdLAR1 gene in tobacco suppresses expression of the late genes in anthocyanin biosynthetic pathway, resulting in loss of anthocyanin in flowers. Interestingly, a decrease in PA biosynthesis was also observed in flowers of transgenic tobacco plants overexpressing the MdLAR1 gene, which could be attributed to decreased expression of both the NtANR1 and NtANR2 genes. Our study not only confirms the in vivo function of apple LAR1 gene, but it is also helpful for understanding the mechanism of PA biosynthesis.

CDNA-AFLP analysis on 2 Osmanthus fragrans cultivars with different flower color and molecular characteristics of OfMYB1 gene

In this study, we carried out cDNA-AFLP analysis on petals of 2 Osmanthus fragrans cultivars, yielding 102 cDNA fragments. OfMYB1 gene was cloned and the molecular characteristics were studied. The Osmanthus fragrans cultivars ‘Dangui’ and ‘Yingui’ differ considerably in floral coloration and volatiles. We carried out cDNA-AFLP analysis on petals of the two cultivars to isolate genes involved in the production of floral pigments and aromatic chemicals. Using 256 primer combinations, we detected 390 differentially expressed fragments, yielding 102 cDNA fragments. Of the differentially expressed transcripts, 45 were upregulated in ‘Yingui’ and 57 were upregulated in ‘Dangui’. The differentially expressed genes were involved in several metabolic and signal transduction pathways. According to the cDNA-AFLP analysis, a 72-bp fragment was upregulated in ‘Dangui’, sequence comparison of the differentially expressed transcript-derived fragment showed this to be a MYB transcription factor. The cDNA of OfMYB1 was 749 bp long and contained a coding sequence of 585 bp encoding a polypeptide of 195 amino acids. A phylogenetic analysis placed the OfMYB1 gene within the subgroup containing “nyWsV/M E/DDlWP/S” motif. Protoplast transformation experiment showed that OfMYB1 protein accumulated in cell nuclei. Yeast one hybrid assay confirmed that OfMYB1 acted as a transcriptional activator to regulate expression of OfPAL gene. The differential expression patterns of OfMYB1 and anthocyanin biosynthetic pathway genes (OfCHS, OfCHI, OfDFR and OfANS) in the 2 O. fragrans cultivars suggested that OfMYB1 may not regulate the expression of these genes.

Transcription factors and anthocyanin genes related to low-temperature tolerance in rd29A:RdreB1BI transgenic strawberry

Dehydration-responsive element-binding (DREB) transcription factors play critical roles in plant stress responses and signal transduction. To further understand how DREB regulates genes expression to promote cold-hardiness, Illumina/Solexa sequencing technology was used to compare the transcriptomes of non-transgenic and rd29A:RdreB1BI transgenic strawberry plants exposed to low temperatures. Approximately 3.5 million sequence tags were obtained from non-transgenic (NT) and transgenic (T) strawberry untreated (C) or low-temperature treated (LT) leaf samples. Over 1000 genes were differentially expressed between the NT-C and T-C plants, and also the NT-C and NT-LT, as well as the T-C and T-LT plants. Analysis of the genes up-regulated following low-temperature treatment revealed that the majority are linked to metabolism, biosynthesis, transcription and signal transduction. Uniquely up-regulated transcription factors as well as anthocyanin biosynthetic pathway genes are discussed. Accumulation of anthocyanin in the stolon and the base of the petiole differed between non-treated NT and T plants, and this correlated with gene expression patterns. The differentially expressed genes that encode transcription factors and anthocyanin enzymes may contribute to the cold hardiness of RdreB1BI transgenic strawberry. The transcriptome data provide a valuable resource for further studies of strawberry growth and development and DREB-mediated gene regulation under low-temperature stress.

COORDINATE INDUCTION OF ANTHOCYANIN BIOSYNTHETIC PATHWAY GENES BY VVMYBAS

Recent studies in grapevine revealed that VvMYBA transcription factors control the expression from UDP-glucose flavonoid: 3-O-glucosyltransferase (UFGT) to the candidate genes putatively involved in the vacuolar anthocyanin transport. Additionally, data on northern blot analysis had revealed that, in addition to UFGT, other anthocyanin biosynthesis pathway genes were also expressed coordinately at higher levels in red skin grapes than in white skin grapes. Hence, molecular targets of VvMYBAs may be a set of anthocyanin biosynthetic pathway genes. Therefore, we compared GeneChip data of the berry skin of ?Pinot Noir? and its white sport ?Pinot Blanc? to reveal the targets of VvMYBAs. Seventy highly hybridized probe sets (=5 folds) of ?Pinot Noir? were mostly related to anthocyanin biosynthesis, whereas those of ?Pinot Blanc? (56 probe sets) were related to disease resistance and unidentified ones. In addition to flavonoid-3?, 5?-hydroxylase and cytochrome b5 DIF-F, the genes specific to and putatively involved in anthocyanin biosynthesis, such as UFGT, gluta-thione S-transferase, O-methyltransferase, and acyltransferase (serine carboxypeptidase like protein), were highly expressed in ?Pinot Noir?, while their expressions were negligible in ?Pinot Blanc?. This result confirmed that the expressions of these genes are induced by VvMYBAs. On the other hand, those involved in the common pathway of flavonoid biosynthesis, such as phenylalanine ammonia-lyase, chalcone synthase 3, flavanone-3-hydroxylase, dihydroflavonol 4-reductase, and leucoanthocyanidin dioxygenase, were also expressed in ?Pinot Blanc? to a certain extent, and their expressions were enhanced in ?Pinot Noir?. This result indicates a possibility that VvMYBAs, in addition to other transcription factor(s), also induce the expression of these genes.

The Malus crabapple transcription factor McMYB10 regulates anthocyanin biosynthesis during petal coloration

Anthocyanins are responsible for the pigmentation of flowers in the Malus crabapple, and anthocyanin accumulation is based on the expression level of both anthocyanin biosynthetic and regulatory genes. Here, we analyzed the chromatic parameters of the petals and the expression levels of anthocyanin biosynthetic pathway genes and the McMYB10 transcription factor in two cultivars with different flower colors during flower development. The results showed that the anthocyanin content and expression levels of anthocyanin-related genes in the cultivar (‘Royalty’) with red petal were much higher than those in the cultivar (‘Flame’) with a lightly pigmented petal. We also overexpressed the McCHS, McANS, and McMYB10 genes in tobacco. The transgenic plants harboring 35S::McCHS or 35S::McANS had an altered flower color, with the petals turned into red, compared with the control with pink petals. Moreover, plants transformed with 35S::McMYB10 appeared to exhibit stronger anthocyanin accumulation, showing deeper red petal color compared with the McCHS- and McANS-overexpressing lines. All tested anthocyanin biosynthetic genes, especially NtDFR and NtANS, showed higher expression levels in the McMYB10-overexpressing lines than in the control lines. These results suggested that the transcription factor McMYB10 plays an important role in anthocyanin accumulation during petal coloration in the Malus crabapple, at least partially through transcriptional regulation of its downstream genes in the anthocyanin biosynthetic pathway.

Characterization of the regulatory network of BoMYB2 in controlling anthocyanin biosynthesis in purple cauliflower

Purple cauliflower (Brassica oleracea L. var. botrytis) Graffiti represents a unique mutant in conferring ectopic anthocyanin biosynthesis, which is caused by the tissue-specific activation of BoMYB2, an ortholog of Arabidopsis PAP2 or MYB113. To gain a better understanding of the regulatory network of anthocyanin biosynthesis, we investigated the interaction among cauliflower MYB-bHLH-WD40 network proteins and examined the interplay of BoMYB2 with various bHLH transcription factors in planta. Yeast two-hybrid studies revealed that cauliflower BoMYBs along with the other regulators formed the MYB-bHLH-WD40 complexes and BobHLH1 acted as a bridge between BoMYB and BoWD40-1 proteins. Different BoMYBs exhibited different binding activity to BobHLH1. Examination of the BoMYB2 transgenic lines in Arabidopsis bHLH mutant backgrounds demonstrated that TT8, EGL3, and GL3 were all involved in the BoMYB2-mediated anthocyanin biosynthesis. Expression of BoMYB2 in Arabidopsis caused up-regulation of AtTT8 and AtEGL3 as well as a subset of anthocyanin structural genes encoding flavonoid 3'-hydroxylase, dihydroflavonol 4-reductase, and leucoanthocyanidin dioxygenase. Taken together, our results show that MYB-bHLH-WD40 network transcription factors regulated the bHLH gene expression, which may represent a critical feature in the control of anthocyanin biosynthesis. BoMYB2 together with various BobHLHs specifically regulated the late anthocyanin biosynthetic pathway genes for anthocyanin biosynthesis. Our findings provide additional information for the complicated regulatory network of anthocyanin biosynthesis and the transcriptional regulation of transcription factors in vegetable crops.

A systems approach to identifying correlated gene targets for the loss of colour pigmentation in plants

BackgroundThe numerous diverse metabolic pathways by which plant compounds can be produced make it difficult to predict how colour pigmentation is lost for different tissues and plants. This study employs mathematical and in silico methods to identify correlated gene targets for the loss of colour pigmentation in plants from a whole cell perspective based on the full metabolic network of Arabidopsis. This involves extracting a self-contained flavonoid subnetwork from the AraCyc database and calculating feasible metabolic routes or elementary modes (EMs) for it. Those EMs leading to anthocyanin compounds are taken to constitute the anthocyanin biosynthetic pathway (ABP) and their interplay with the rest of the EMs is used to study the minimal cut sets (MCSs), which are different combinations of reactions to block for eliminating colour pigmentation. By relating the reactions to their corresponding genes, the MCSs are used to explore the phenotypic roles of the ABP genes, their relevance to the ABP and the impact their eliminations would have on other processes in the cell.ResultsSimulation and prediction results of the effect of different MCSs for eliminating colour pigmentation correspond with existing experimental observations. Two examples are: i) two MCSs which require the simultaneous suppression of genes DFR and ANS to eliminate colour pigmentation, correspond to observational results of the same genes being co-regulated for eliminating floral pigmentation in Aquilegia and; ii) the impact of another MCS requiring CHS suppression, corresponds to findings where the suppression of the early gene CHS eliminated nearly all flavonoids but did not affect the production of volatile benzenoids responsible for floral scent.ConclusionsFrom the various MCSs identified for eliminating colour pigmentation, several correlate to existing experimental observations, indicating that different MCSs are suitable for different plants, different cells, and different conditions and could also be related to regulatory genes. Being able to correlate the predictions with experimental results gives credence to the use of these mathematical and in silico analyses methods in the design of experiments. The methods could be used to prioritize target enzymes for different objectives to achieve desired outcomes, especially for less understood pathways.