Induced Anthocyanin Accumulation Research Articles

MYB transcription factors encoded by diversified tandem gene clusters cause varied Morella rubra fruit color.

Chinese bayberry (Morella rubra) is a fruit tree with a remarkable variation in fruit color, ranging from white to dark red as determined by anthocyanin content. In dark red "Biqi" (BQ), red "Dongkui" (DK), pink "Fenhong" (FH), and white "Shuijing" (SJ), we identified an anthocyanin-related MYB transcription factor-encoding gene cluster of four members, i.e. MrMYB1.1, MrMYB1.2, MrMYB1.3, and MrMYB2. Collinear analysis revealed that the MYB tandem cluster may have occurred in a highly conserved region of many eudicot genomes. Two alleles of MrMYB1.1 were observed; MrMYB1.1-1 (MrMYB1.1n) was a full-length allele and homozygous in "BQ", MrMYB1.1-2 (MrMYB1.1d) was a nonfunctional allele with a single base deletion and homozygous in "SJ", and MrMYB1.1n/MrMYB1.1d were heterozygous in "DK" and "FH". In these four cultivars, expression of MrMYB1.1, MrMYB1.2, and MrMYB2 was enhanced during ripening. Both alleles were equally expressed in MrMYB1.1n/MrMYB1.1d heterozygous cultivars as revealed by a cleaved amplified polymorphic sequence marker. Expression of MrMYB1.3 was restricted to some dark red cultivars only. Functional characterization revealed that MrMYB1.1n and MrMYB1.3 can induce anthocyanin accumulation while MrMYB1.1d, MrMYB1.2, and MrMYB2 cannot. DNA-protein interaction assays indicated that MrMYB1.1n and MrMYB1.3 can directly bind to and activate the promoters of anthocyanin-related genes via interaction with a MYC-like basic helix-loop-helix protein MrbHLH1. We concluded that the specific genotype of MrMYB1.1 alleles, as well as the exclusive expression of MrMYB1.3 in some dark red cultivars, contributes to fruit color variation. The study provides insights into the mechanisms for regulation of plant anthocyanin accumulation by MYB tandem clusters.

Mutation in BrFLS encoding flavonol synthase induced anthocyanin accumulation in Chinese cabbage.

BrFLS mutation promoted anthocyanin accumulation in Chinese cabbage, which was verified in four allelic mutants. Chinese cabbage is a major vegetable crop in Eastern Asia. Anthocyanin-rich vibrantly colored varieties are increasingly favored by consumers for their higher nutritional and aesthetic value compared to the typical green varieties of Chinese cabbage. Herein, we identified an anthocyanin accumulation mutant aam1 from a mutant library of EMS-mutagenized Chinese cabbage DH line 'FT', which appeared partial purple on leaves, bolting stems and floral buds. This anthocyanin accumulation trait was genetically controlled by a recessive nuclear gene, and through MutMap mapping and KASP genotyping, BraA10g030950.3C was identified as the candidate causal gene with a G202 to A202 non-synonymous SNP variation in exon 1. Three additional mutants allelic to aam1 were obtained via screening of similar-phenotype mutants from the mutant library, namely aam2/3/4, where the causal SNPs reside in the same gene as aam1, corroborating that the mutation of BraA10g030950.3C caused anthocyanin accumulation. BraA10g030950.3C encodes a flavonol synthase that catalyzes dihydroflavonols substrate into flavonols and is homologous to Arabidopsis FLS1 (AT5G08640), named BrFLS. Compared to wildtype, the expression level of BrFLS was significantly reduced in the mutants, while BrDFR, which is involved in the anthocyanin biosynthesis and competes with FLS for the common substrate dihydroflavonols, was increased. The flavonol synthase activity decreased, and dihydroflavonol 4-reductase activity was elevated. Differentially accumulated flavonoid metabolites were detected between wildtype and aam1, which were enriched primarily in flavonol and anthocyanin pathways. Our results revealed that mutations in the BrFLS gene could contribute to anthocyanin accumulation and provide a new target for Chinese cabbage color modification.

Tandemly duplicated MYB genes are functionally diverged in the regulation of anthocyanin biosynthesis in soybean.

Gene duplications have long been recognized as a driving force in the evolution of genes, giving rise to novel functions. The soybean (Glycine max) genome is characterized by a large number of duplicated genes. However, the extent and mechanisms of functional divergence among these duplicated genes in soybean remain poorly understood. In this study, we revealed that 4 MYB genes (GmMYBA5, GmMYBA2, GmMYBA1, and Glyma.09g235000)-presumably generated by tandem duplication specifically in the Phaseoleae lineage-exhibited a stronger purifying selection in soybean compared to common bean (Phaseolus vulgaris). To gain insights into the diverse functions of these tandemly duplicated MYB genes in anthocyanin biosynthesis, we examined the expression, transcriptional activity, induced metabolites, and evolutionary history of these 4 MYB genes. Our data revealed that Glyma.09g235000 is a pseudogene, while the remaining 3 MYB genes exhibit strong transcriptional activation activity, promoting anthocyanin biosynthesis in different soybean tissues. GmMYBA5, GmMYBA2, and GmMYBA1 induced anthocyanin accumulation by upregulating the expression of anthocyanin pathway-related genes. Notably, GmMYBA5 showed a lower capacity for gene induction compared to GmMYBA2 and GmMYBA1. Metabolomics analysis further demonstrated that GmMYBA5 induced distinct anthocyanin accumulation in Nicotiana benthamiana leaves and soybean hairy roots compared to GmMYBA2 and GmMYBA1, suggesting their functional divergence leading to the accumulation of different metabolites accumulation following gene duplication. Together, our data provide evidence of functional divergence within the MYB gene cluster following tandem duplication, which sheds light on the potential evolutionary directions of gene duplications during legume evolution.

Enhanced UV-B Radiation in Potato Stems and Leaves Promotes the Accumulation of Anthocyanins in Tubers.

Enhanced ultraviolet-B (UV-B) radiation promotes anthocyanin biosynthesis in leaves, flowers and fruits of plants. However, the effects and underlying mechanisms of enhanced UV-B radiation on the accumulation of anthocyanins in the tubers of potatoes (Solanum tuberosum L.) remain unclear. Herein, reciprocal grafting experiments were first conducted using colored and uncolored potatoes, demonstrating that the anthocyanins in potato tubers were synthesized in situ, and not transported from the leaves to the tubers. Furthermore, the enhanced UV-B radiation (2.5 kJ·m-2·d-1) on potato stems and leaves significantly increased the contents of total anthocyanin and monomeric pelargonidin and peonidin in the red-fleshed potato '21-1' tubers, compared to the untreated control. A comparative transcriptomic analysis showed that there were 2139 differentially expressed genes (DEGs) under UV-B treatment in comparison to the control, including 1724 up-regulated and 415 down-regulated genes. The anthocyanin-related enzymatic genes in the tubers such as PAL, C4H, 4CL, CHS, CHI, F3H, F3'5'H, ANS, UFGTs, and GSTs were up-regulated under UV-B treatment, except for a down-regulated F3'H. A known anthocyanin-related transcription factor StbHLH1 also showed a significantly higher expression level under UV-B treatment. Moreover, six differentially expressed MYB transcription factors were remarkably correlated to almost all anthocyanin-related enzymatic genes. Additionally, a DEGs enrichment analysis suggested that jasmonic acid might be a potential UV-B signaling molecule involved in the UV-B-induced tuber biosynthesis of anthocyanin. These results indicated that enhanced UV-B radiation in potato stems and leaves induced anthocyanin accumulation in the tubers by regulating the enzymatic genes and transcription factors involved in anthocyanin biosynthesis. This study provides novel insights into the mechanisms of enhanced UV-B radiation that regulate the anthocyanin biosynthesis in potato tubers.

Identification of HpMYB1 inducing anthocyanin accumulation in Hippeastrum Hybridum tepals by RNA-seq

BackgroundCultivated Hippeastrum × hybridum is a popular ornamental plant with large and colorful flowers, long flowering duration, and high commercial value. As its main ornamental feature, its flower color is related to the anthocyanin content in the tepals. However, the molecular regulatory mechanisms of anthocyanin biosynthesis in H. × hybridum have not yet been elucidated.ResultsIn the present study, 12 cDNA libraries of four stages of H.× hybridum ‘Royal Velvet’ tepal development were used for RNA-seq, obtaining 79.83 gigabases (GB) of clean data. The data were assembled into 148,453 unigenes, and 11,262 differentially expressed genes were identified. Forty key enzymes participating in anthocyanin biosynthesis were investigated, and the results showed that most of the anthocyanin structural genes were expressed at low levels in S1 and were markedly upregulated in S2 and S3. The expression profiles of 12 selected genes were verified by qRT-PCR. Furthermore, the R2R3-MYB transcription factor (TF), HpMYB1, involved in the regulation of anthocyanin biosynthesis was identified by sequence, expression pattern, and subcellular localization analyses. Its overexpression in tobacco significantly increased the anthocyanin levels in various tissues and activated anthocyanin-related genes.ConclusionsUsing RNA-seq technology, we successfully identified a potential R2R3-MYB gene, HpMYB1, that regulates anthocyanin biosynthesis in H.× hybridum ‘Royal Velvet’. Our findings provide basic transcript information and valuable transcriptome data for further identification of key genes involved in anthocyanin biosynthesis and can be applied in the artificial breeding of new H. × hybridum cultivars with enhanced ornamental value.

Transcriptome analysis reveals the mechanism for blue-light-induced biosynthesis of delphinidin derivatives in harvested purple pepper fruit.

Anthocyanins are the main pigments that affect the color and quality of purple-fruited sweet pepper (Capsicum annuum). Our previous study indicated that blue light can induce anthocyanin accumulation in purple pepper. In view of its underlying mechanism that is unclear, here, anthocyanin content was determined, and transcriptome analysis was performed on pepper fruits harvested from different light treatments. As a result, among the identified anthocyanin metabolites, the levels of delphinidin (Dp) glycosides, including Dp-3-O-rhamnoside, Dp-3-O-rutinoside, and Dp-3-O-glucoside, were highly accumulated in blue-light-treated fruit, which are mainly responsible for the appearance color of purple pepper. Correlation between anthocyanin content and transcriptomic analysis indicated a total of 1,619 upregulated genes were found, of which six structural and 12 transcription factor (TF) genes were involved in the anthocyanin biosynthetic pathway. Structural gene, for instance, CaUFGT as well as TFs such as CaMYC2-like and CaERF113, which were highly expressed under blue light and presented similar expression patterns consistent with Dp glycoside accumulation, may be candidate genes for anthocyanin synthesis in response to blue-light signal.

Potential regulatory genes of light induced anthocyanin accumulation in sweet cherry identified by combining transcriptome and metabolome analysis.

Anthocyanins exist widely in various plant tissues and organs, and they play an important role in plant reproduction, disease resistance, stress resistance, and protection of human vision. Most fruit anthocyanins can be induced to accumulate by light. Here, we shaded the "Hong Deng" sweet cherry and performed an integrated analysis of its transcriptome and metabolome to explore the role of light in anthocyanin accumulation. The total anthocyanin content of the fruit and two of its anthocyanin components were significantly reduced after the shading. Transcriptome and metabolomics analysis revealed that PAL, 4CL, HCT, ANS and other structural genes of the anthocyanin pathway and cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, and other metabolites were significantly affected by shading. Weighted total gene network analysis and correlation analysis showed that the upstream and middle structural genes 4CL2, 4CL3, and HCT2 of anthocyanin biosynthesis may be the key genes affecting the anthocyanin content variations in fruits after light shading. Their expression levels may be regulated by transcription factors such as LBD, ERF4, NAC2, NAC3, FKF1, LHY, RVE1, and RVE2. This study revealed for the first time the possible role of LBD, FKF1, and other transcription factors in the light-induced anthocyanin accumulation of sweet cherry, thereby laying a preliminary foundation for further research on the role of light in anthocyanin accumulation of deep red fruit varieties and the genetic breeding of sweet cherry.

AeWRKY32 from okra regulates anthocyanin accumulation and cold tolerance in Arabidopsis

Okra (Abelmoschus esculentus L.) is a tropical crop species, and its growth and development are severely affected by cold stress. Recent studies have identified a potential association between WRKY transcription factors and the cold response mechanism of crops. In this study, the AeWRKY32 transcription factor that encodes 482 amino acids was amplified from A. esculentus, and its expression level was found to be the highest in the okra flower. AeWRKY32 localized to the nucleus and displayed transcriptional activation capability. Under normal conditions, overexpression of AeWRKY32 induced anthocyanin accumulation, with higher expression levels of AtCHS1, AtCHI4, AtF3H1, and AtDFR2 in transgenic Arabidopsis. Under cold stress, anthocyanin levels were further elevated in transgenic Arabidopsis plants. At the same time, AeWRKY32 overexpression promoted ABA biosynthesis, inhibited H2O2 and O2− generation, induced stomatal closure, reduced electrolyte leakage, and thus improved the cold resistance of transgenic Arabidopsis. Furthermore, under cold stress, the expression profiles of AtCOR413, AtCOR15B, AtCBF1, and AtCBF2 were upregulated in transgenic Arabidopsis. Overall, our study provides evidence that AeWRKY32 serves as a crucial regulator in both anthocyanin accumulation and cold tolerance of transgenic Arabidopsis. Our findings could provide insights into the molecular mechanism linking AeWRKYs to plant cold tolerance.

The E3 ubiquitin ligase SINA1 and the protein kinase BIN2 cooperatively regulate PHR1 in apple anthocyanin biosynthesis.

PHR1 (PHOSPHATE STARVATION RESPONSE1) plays key roles in the phosphate (Pi) starvation response and in Pi-deficiency-induced anthocyanin biosynthesis in plants. However, the post-translational regulation of PHR1 is unclear, and the molecular basis of PHR1-mediated anthocyanin biosynthesis remains elusive. In this study, we determined that MdPHR1 was essential for Pi-deficiency-induced anthocyanin accumulation in apple (Malus × domestica). MdPHR1 interacted with MdWRKY75, a positive regulator of anthocyanin biosynthesis, to enhance the MdWRKY75-activated transcription of MdMYB1, leading to anthocyanin accumulation. In addition, the E3 ubiquitin ligase SEVEN IN ABSENTIA1 (MdSINA1) negatively regulated MdPHR1-promoted anthocyanin biosynthesis via the ubiquitination-mediated degradation of MdPHR1. Moreover, the protein kinase MdBIN2 phosphorylated MdPHR1 and positively regulated MdPHR1-mediated anthocyanin accumulation by attenuating the MdSINA1-mediated ubiquitination degradation of MdPHR1. Taken together, these findings not only demonstrate the regulatory role of MdPHR1 in Pi-starvation induced anthocyanin accumulation, but also provide an insight into the post-translational regulation of PHR1. This article is protected by copyright. All rights reserved.

Transcription factor NnMYB5 controls petal color by regulating GLUTATHIONE S-TRANSFERASE2 in Nelumbo nucifera.

Lotus (Nelumbo spp.) is an important aquatic ornamental genus in the family Nelumbonaceae comprising only two species: N. lutea with yellow flowers and N. nucifera with red or white flowers. The petal color variations between these two species have previously been associated with the potential activities of FLAVONOL SYNTHASE (FLS) and MYB5. However, the underlying genetic mechanisms of flower color divergence within the N. nucifera species remains unclear. Here, quantitative trait locus mapping led to the identification of MYB5, a candidate gene controlling petal color in N. nucifera. Genotyping of 213 natural lotus accessions revealed an 80-kb presence/absence variant (PAV) of the NnMYB5 gene that is associated with petal color variation. Transcriptome analysis, dual-luciferase and yeast one-hybrid assays showed that NnMYB5 could directly activate the anthocyanin transporter gene GLUTATHIONE S-TRANSFERASE2 (NnGST2). Heterologous expression of NnGST2 in Arabidopsis (Arabidopsis thaliana) and its overexpression in lotus petals induced anthocyanin accumulation. Deletion of the 80-kb PAV within NnMYB5 inactivated NnGST2 expression and blocked anthocyanin accumulation in white N. nucifera petals. In contrast, the anthocyanin deficiency of N. lutea occurred due to pseudogenized NlMYB5 alleles. Our results establish a regulatory link between NnMYB5 and NnGST2 in petal anthocyanin accumulation and demonstrate the independent mechanisms controlling flower coloration in Nelumbo.

Why do plants blush when they are hungry?

Foliar anthocyanins, as well as other secondary metabolites, accumulate transiently under nutritional stress. A misconception that only nitrogen or phosphorus deficiency induces leaf purpling/reddening has led to overuse of fertilizers that burden the environment. Here, we emphasize that several other nutritional imbalances induce anthocyanin accumulation, and nutrient-specific differences in this response have been reported for some deficiencies. A range of ecophysiological functions have been attributed to anthocyanins. We discuss the proposed functions and signalling pathways that elicit anthocyanin synthesis in nutrient-stressed leaves. Knowledge from the fields of genetics, molecular biology, ecophysiology and plant nutrition is combined to deduce how and why anthocyanins accumulate under nutritional stress. Future research to fully understand the mechanisms and nuances of foliar anthocyanin accumulation in nutrient-stressed crops could be utilized to allow these leaf pigments to act as bioindicators for demand-oriented application of fertilizers. This would benefit the environment, being timely due to the increasing impact of the climate crisis on crop performance.

Modulation of anthocyanin accumulation in storage roots of sweetpotato by transcription factor IbMYB1-2 through direct binding to anthocyanin biosynthetic gene promoters

The storage roots of purple-fleshed sweetpotato rich in anthocyanins are considered nutrient-rich foods with health effects. However, the molecular mechanism underlying anthocyanin biosynthesis and regulation remains to be revealed. In this study, IbMYB1-2 was isolated from purple-fleshed sweetpotato “Xuzishu8”. The phylogenetic and sequence analysis indicated that IbMYB1-2 belongs to the SG6 subfamily with a conserved bHLH motif. Subcellular localization analysis and transcriptional activity assay revealed that IbMYB1-2 is a key transcriptional activator and is specific to the nucleus. Agrobacterium rhizogenes-mediated overexpression of IbMYB1-2 in sweetpotato through in vivo root transgenic system led to an increase in anthocyanins in the root of sweetpotato. qRT-PCR and transcriptome analysis depicted that the transcript levels of IbMYB1-2, IbbHLH42, and eight structural genes that are associated with the synthesis of anthocyanin were upregulated in overexpressed IbMYB1-2 transgenic roots. Dual-luciferase reporter (DLR) assay and yeast one-hybrid (Y1H) assay demonstrated IbMYB1-2 binding to the promoter regions of IbbHLH42 and other anthocyanin biosynthetic genes, including IbCHS, IbCHI, IbF3H, IbDFR, IbANS, IbGSTF12, IbUGT78D2, and IbUF3GT. Moreover, IbbHLH42 was shown to be an active enhancer for the formation of MYB-bHLH-WD40 (MBW) complex, which strongly supports the promoter activities of the IbCHS, IbANS, IbUGT78D2, and IbGSTF12 genes to induce anthocyanin accumulation. Taken together, our findings not only revealed the underlying regulatory molecular mechanism of IbMYB1-2 for anthocyanin accumulation in the storage roots of sweetpotato but also uncovered a potential mechanism by which IbbHLH42 modulated anthocyanin biosynthesis through a positive feedback regulatory loop.

Molecular mechanism of phosphorous signaling inducing anthocyanin accumulation in Arabidopsis

Anthocyanins, flavonoid compounds derived from secondary metabolic pathways, play important roles in various biological processes. Phosphorus (P) is an essential macroelement for plant growth and development, and P-starvation usually results in anthocyanin accumulation. However, the molecular mechanism of P deficiency promotes anthocyanin biosynthesis has not been well characterized. Here, we provided evidence that the P signaling core protein PHOSPHATE STARVATION RESPONSE1 (PHR1) is physically associate with transcription factors (TFs) involved in anthocyanidin biosynthesis, including PRODUCTION OF ANTHOCYANIN PIGMENTS1 (PAP1/MYB75), MYB DOMAIN PROTEIN 113 (MYB113) and TRANSPARENT TESTA 8 (TT8). PHR1 and its homologies positively regulated anthocyanin accumulation in Arabidopsis seedlings under P-deficient conditions. Disruption of PHR1 simultaneously rendered seedlings hyposensitive to limiting P, whereas the overexpression of PHR1 enhanced P- deficiency-induced anthocyanin accumulation. Genetic analysis demonstrated that 35S:PHR1-2HA-5 seedlings partially recovers the P deficiency insensitive phenotype of myb-RNAi and tt8 mutants. In summary, our study indicated that protein complexes formed by PHR1 and MBW complex directly mediate the process of P-deficiency-induced anthocyanin accumulation, providing a new mechanistic understanding of how P-deficient signaling depends on the endogenous anthocyanin synthesis pathway to promote anthocyanin accumulation in Arabidopsis.

Promoter replacement of ANT1 induces anthocyanin accumulation and triggers the shade avoidance response through developmental, physiological and metabolic reprogramming in tomato.

The accumulation of anthocyanins is a well-known response to abiotic stresses in many plant species. However, the effects of anthocyanin accumulation on light absorbance and photosynthesis are unknown . Here, we addressed this question using a promoter replacement line of tomato constitutively expressing a MYB transcription factor (ANTHOCYANIN1, ANT1) that leads to anthocyanin accumulation. ANT1-overexpressing plants displayed traits associated with shade avoidance response: thinner leaves, lower seed germination rate, suppressed side branching, increased chlorophyll concentration, and lower photosynthesis rates than the wild type. Anthocyanin-rich leaves exhibited higher absorbance of light in the blue and red ends of the spectrum, while higher anthocyanin content in leaves provided photoprotection to high irradiance. Analyses of gene expression and primary metabolites content showed that anthocyanin accumulation produces a reconfiguration of transcriptional and metabolic networks that is consistent with, but not identical to those described for the shade avoidance response. Our results provide novel insights about how anthocyanins accumulation affects the trade-off between photoprotection and growth.

ZeMYB9 regulates cyanidin synthesis by activating the expression of flavonoid 3'-hydroxylase gene in Zinnia elegans.

Petal color in Zinnia elegans is characterized mainly by anthocyanin accumulation. The difference in the content of anthocyanins, especially cyanidins, affects petal coloration in Z. elegans, but the underlying regulatory mechanism remains elusive. Here, we report one R2R3-MYB transcription factor from subgroup 6, ZeMYB9, acting as a positive regulator of anthocyanin accumulation in Z. elegans. Up-regulated expression of ZeMYB9 and flavonoid 3'-hydroxylase gene (ZeF3'H) was detected in the cultivar with higher cyanidin content. ZeMYB9 could specifically activate the promoter of ZeF3'H, and over-expression of ZeMYB9 induces much greater anthocyanin accumulation and higher expression level of anthocyanin biosynthetic genes in both petunia and tobacco. And then, ZeMYB9 was demonstrated to interact with ZeGL3, a bHLH transcription factor belonging to IIIf subgroup. Promoter activity of ZeF3'H was significantly promoted by co-expressing ZeMYB9 and ZeGL3 compared with expressing ZeMYB9 alone. Moreover, transient co-expression of ZeMYB9 and ZeGL3 induced anthocyanin accumulation in tobacco leaves. Our results suggest that ZeMYB9 could enhance cyanidin synthesis and regulate petal color in Z. elegans though activating the expression of ZeF3'H, by itself or interacting with ZeGL3.

DNA methylation reprogramming provides insights into light-induced anthocyanin biosynthesis in red pear

DNA methylation, an epigenetic mark, is proposed to regulate plant anthocyanin biosynthesis. It well known that light induces anthocyanin accumulation, with bagging treatments commonly used to investigate light-controlled anthocyanin biosynthesis. We studied the DNA methylome landscape during pear skin coloration under various conditions (fruits re-exposed to sunlight after bag removal). The DNA methylation level in gene body/TE and its flanking sequence was generally similar between debagged and bagged treatments, however differentially methylated regions (DMRs) were re-modelled after light-exposure. Both DNA demethylase homologs and the RNA-directed DNA methylation (RdDM) pathways contributed to this re-distribution. A total of 310 DEGs were DMR-associated during light-induced anthocyanin biosynthesis between debagged and bagged treatments. The hypomethylated mCHH context was seen within the promoter of PyUFGT, together with other anthocyanin biosynthesis genes (PyPAL, PyDFR and PyANS). This enhanced transcriptional activation and promoted anthocyanin accumulation after light re-exposure. Unlike previous reports on bud sports, we did not detect DMRs within the MYB10 promoter. Instead, we observed the genome-wide re-distribution of methylation patterns, suggesting different mechanisms underlying methylation patterns of differentially accumulated anthocyanins caused by either bud mutation or environment change. We investigate the dynamic landscape of genome-scale DNA methylation, which is the combined effect of DNA demethylation and RdDM pathway, in the process of light-induced fruit colour formation in pear. This process is regulated by methylation changes on promoter regions of several DEGs. These results provide a DMR-associated DEGs set and new insight into the mechanism of DNA methylation involved in light-induced anthocyanin biosynthesis.

De Novo Assembly of a Sarcocarp Transcriptome Set Identifies AaMYB1 as a Regulator of Anthocyanin Biosynthesis in Actinidia arguta var. purpurea.

The kiwifruit (Actinidia arguta var. purpurea) produces oval shaped fruits containing a slightly green or mauve outer exocarp and a purple-flesh endocarp with rows of tiny black seeds. The flesh color of the fruit results from a range of anthocyanin compounds, and is an important trait for kiwifruit consumers. To elucidate the molecular mechanisms involved in anthocyanin biosynthesis of the sarcocarp during A. arguta fruit development, de novo assembly and transcriptomic profile analyses were performed. Based on significant Gene Ontology (GO) biological terms, differentially expressed genes were identified in flavonoid biosynthetic and metabolic processes, pigment biosynthesis, carbohydrate metabolic processes, and amino acid metabolic processes. The genes closely related to anthocyanin biosynthesis, such as phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and anthocyanidin synthase (ANS), displayed significant up-regulation during fruit development according to the transcriptomic data, which was further confirmed by qRT-PCR. Meanwhile, a series of transcription factor genes were identified among the DEGs. Through a correlation analysis. AaMYB1 was found to be significantly correlated with key genes of anthocyanin biosynthesis, especially with CHS. Through a transient expression assay, AaMYB1 induced anthocyanin accumulation in tobacco leaves. These data provide an important basis for exploring the related mechanisms of sarcocarp anthocyanin biosynthesis in A. arguta. This study will provide a strong foundation for functional studies on A. arguta and will facilitate improved breeding of A. arguta fruit.

PHR1 positively regulates phosphate starvation-induced anthocyanin accumulation through direct upregulation of genes F3'H and LDOX in Arabidopsis.

Phosphate deficiency promotes anthocyanin accumulation in Arabidopsis through direct binding of PHR1 to the P1BS motifs on the promoters of F3'H and LDOX and thereby upregulating their expression. Phosphorus is one of the essential elements for plants, and plants mainly absorb inorganic phosphate (Pi) from soil. But Pi deficiency is a common factor limiting plant growth and development. Anthocyanin accumulation in green tissues (such as leaves) is one of the characteristics of many plants in response to Pi starvation. However, little is known about the mechanism by which Pi starvation induces anthocyanin accumulation. Here, we found that the mutation of the gene PHOSPHATE STARVATION RESPONSE1 (PHR1), which encodes a key factor involved in Pi starvation signaling in Arabidopsis, significantly attenuates anthocyanin accumulation under Pi-limiting conditions. Moreover, the expression of several Pi deficiency-upregulated genes that are involved in anthocyanin biosyntheses, such as flavanone 3'-hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), and production of anthocyanin pigment 1 (PAP1), was significantly lower in the phr1-1 mutant than in the wild type (WT). Both yeast one-hybrid (Y1H) analysis and chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) showed that PHR1 can interact with the promoters of F3'H and LDOX, but not DFR and PAP1. By electrophoretic mobility shift assay (EMSA), it was further confirmed that the PHR1-binding sequence (P1BS) motifs located on the F3'H and LDOX promoters are required for the PHR1 bindings. Also, in Arabidopsis protoplasts, PHR1 enhanced the transcriptional activity of the F3'H and LDOX promoters, but these effects were markedly impaired when the P1BS motifs were mutated. Taken together, these results indicate that PHR1 positively regulates Pi starvation-induced anthocyanin accumulation in Arabidopsis, at least in part, by directly binding the P1BS motifs located on the promoters to upregulate the transcription of anthocyanin biosynthetic genes F3'H and LDOX.

Bagging Strategy and Identification of Coloring Mode of 'Xinqihong' Pear.

‘Xinqihong’ is a recently selected and well-colored red pear (Pyrus bretschneideri Rehd.) cultivar that is popular in the marketplace owing to the bright red color and high quality of the fruit. The red pigmentation is strongly associated with the light signal. However, its responses to bagging treatment and to light exposure after shading are unknown. In this study, the fruit were treated with three types of fruit bags. ’Xinqihong’ fruit colored rapidly in response to light stimulation. A white fruit bag was optimal for bagging of ‘Xinqihong’ fruit. To ensure satisfactory red pigmentation, the fruit required exposure to 30 days of light after bag removal. A transcriptome analysis was conducted to screen light-signal-related genes and identify their possible functions. PbCRY1 activated the promoter of PbHY5.2 and enhanced its expression. PbHY5.2 activated the promoter activity of PbUFGT and induced anthocyanin synthesis, and also showed self-activation characteristics. Both PbCRY2 and PbPHY1 induced anthocyanin accumulation. Thus, blue-light receptors played an important role in anthocyanin synthesis. This study provides a theoretical basis for the bagging cultivation of new varieties of ‘Xinqihong’, and lays a foundation for the study of the mechanisms of red pear fruit coloring in response to light signals.

The bHLH transcription factor AcB2 regulates anthocyanin biosynthesis in onion (Allium cepa L.).

Onion bulb color is a key breeding trait. The red bulb color is caused by the presence of anthocyanins, which are products of the flavonoid synthesis pathway. Research on flavonoid regulation in onion is lagging compared with that in other crops. AcB2 encodes a basic helix–loop–helix (bHLH) transcription factor, and its transcription is positively associated with anthocyanin accumulation and correlated with the expression of AcMYB1, which is an activator in the flavonoid biosynthetic pathway in onion. Phylogenetic analysis showed that AcB2 was grouped into the TRANSPARENT TESTA 8 (TT8) clade of the bHLH IIIf subgroup. The AcB2 protein contained an MYB-interacting region and physically interacted with AcMYB1 in yeast and tobacco leaves. AcMYB1 directly bound to the promoters of anthocyanidin synthase (AcANS) and flavonoid 3-hydroxylase 1 (AcF3H1) and activated their expression. The coexpression of AcB2 with AcMYB1 in Arabidopsis thaliana protoplasts dramatically increased the expression of AcANS and AcF3H1 compared with that under the expression of AcMYB1 alone. Transient co-overexpression of AcB2 with AcMYB1 induced anthocyanin accumulation in the epithelial cells of onion bulbs. Complementation of the Arabidopsis tt8-1 mutant with AcB2 restored pigmentation defects in tt8-1. In addition, AcB2 physically interacted with AtTT2 in yeast cells and tobacco leaves, indicating that the functions of AcB2 were similar to those of AtTT8. Together, these results demonstrated that AcB2 enhanced the function of AcMYB1 in upregulating anthocyanin biosynthesis in onion, which provides a theoretical basis for breeding onions with higher anthocyanin contents.