Anthocyanin Biosynthetic Genes Research Articles

5-Aminolevulinic acid activates the MdWRKY71-MdMADS1 module to enhance anthocyanin biosynthesis in apple

5-Aminolevulinic acid (ALA), as a natural plant growth regulator, is well known for promoting red fruit coloring by enhancing anthocyanin accumulation. However, the underlying mechanisms remain elusive. In this study, we firstly demonstrated that ALA upregulates gene expression of the transcription factor MdMADS1, which in turn directly binds to and activates transcription of the key anthocyanin biosynthetic genes, MdCHS and MdUFGT. Then, we identified a novel WRKY transcription factor, MdWRKY71, that interacts with MdMADS1. Through gene manipulation, we revealed that MdWRKY71 plays a pivotal role in ALA-induced anthocyanin accumulation, highlighting its regulatory significance in this process. Further investigation unveiled that MdWRKY71 not only activates MdMADS1 transcription but also enhances its transcriptional activation on its target genes, MdCHS and MdUFGT. Additionally, we discovered that MdWRKY71 independently binds to and activates the transcription of two other anthocyanin biosynthetic genes, MdANS and MdDFR. The protein–protein interaction between MdWRKY71 and MdMADS1 amplifies the transcriptional activation of these genes by MdWRKY71. These findings delineate a fine and complex regulatory framework where MdWRKY71 and MdMADS1 coordinately regulate anthocyanin biosynthesis in apples, providing new insights into the molecular control of fruit coloration and offering potential target genes for breeding aimed at enhancing fruit quality.

Genome-wide identification and functional roles relating to anthocyanin biosynthesis analysis in maize

BackgroundAnthocyanin is an important class of water-soluble pigments that are widely distributed in various tissues of plants, and it not only facilitates diverse color changes but also plays important roles in various biological processes. Maize silk, serving as an important reproductive organ and displaying a diverse range of colors, plays an indispensable role in biotic resistance through its possession of anthocyanin. However, the copy numbers, characteristics, and expression patterns of genes involved in maize anthocyanin biosynthesis are not fully understood. In this study, gene numbers, distribution, structure, cis-elements of the anthocyanin synthetic gene family were identified, and then the potential transcriptional factors were predicted by two analyzed methods. Finally, genes involved in maize silk pigment were screened by un-targeted metabolism analysis.ResultsTen gene families involved in the maize anthocyanin biosynthesis pathway were identified, and 142 synthetic genes were obtained. These anthocyanin biosynthetic genes have high copy numbers and are normally clustered on chromosomes. The promoters of these synthetic genes contain various cis-elements and the gene expression patterns and transcriptional regulatory networks were analyzed. These genes are distributed on different chromosomes and gene expression patterns vary across different tissues in maize. Specifically, these genes often exhibit higher expression in the stem, leaves, and seeds. Ten highly expressed genes in silks were identified. Based on un-targeted metabolites detection in the silks of four maize representative inbred lines with different colors, two main differential anthocyanin components were identified. Furthermore, the gene expression patterns of the ten highly expressed genes and their potential interacting transcriptional factors were analyzed across the four inbred lines.ConclusionsThe results in this study show a through picture of maize anthocyanin synthetic genes, and the structure and function of genes related to anthocyanin biosynthesis in maize could be further investigated.

Impact of Hormone on Growth and GA3 Regulation of Anthocyanin Biosynthesis in Suspension-Culture Cells of Cyclocarya paliurus.

Hormones are effective in regulating plant metabolism and, therefore, are often used in plant cell culture to increase the yield of target products. This study investigated the effects of hormones on the growth and anthocyanin biosynthesis of suspension-cultured red Cyclocarya paliurus cells. Additionally, the mechanism by which gibberellin induces anthocyanin biosynthesis was explored through multi-omics integrated analysis and the assay of the dynamic changes in signaling molecule concentration. The results showed that the total anthocyanin content and yield of suspension-cultured cells, when induced by 1.0 mg L⁻¹ Gibberellin A3 (GA3), experienced increases of 1.92- and 1.83-fold, respectively. The application of exogenous GA3 activated the synthesis and transduction of four signaling molecules, that is, nitric oxide (NO), hydrogen peroxide (H2O2), salicylic acid (SA), and jasmonic acid (JA), in the cells and altered the expression patterns of transcription factors. The altered expression of transcription factors upregulated the expression of anthocyanin biosynthetic genes such as anthocyanin-3-O-glucosyl transferase and leucoanthocyanidin dioxygenase, while downregulated the expression of anthocyanin reductase and flavonoid 3',5',-hydroxylase, which activated the anthocyanin biosynthesis pathway, ultimately leading to a significant increase in anthocyanin biosynthesis. This research work establishes a foundation for further research on the role of hormones in regulating anthocyanin biosynthesis in suspension-cultured plant cells.

Evolutionary analysis of anthocyanin biosynthetic genes: insights into abiotic stress adaptation.

Anthocyanin regulation can be fruitfully explored from a diverse perspective by studying distantly related model organisms. Land plants pioneers faced a huge evolutionary leap, involving substantial physiological and genetic changes. Anthocyanins have evolved alongside these changes, becoming versatile compounds capable of mitigating terrestrial challenges such as drought, salinity, extreme temperatures and high radiation. With the accessibility of whole-genome sequences from ancient plant lineages, deeper insights into the evolution of key metabolic pathways like phenylpropanoids have emerged. Despite understanding the function of anthocyanins under stress, gaps remain in uncovering the precise metabolic and regulatory mechanisms driving their overproduction under stressful conditions. For example, the regulatory effect of reactive oxygen species (ROS) on well-known transcription factors like MYBs is not fully elucidated. This manuscript presents an evolutionary analysis of the anthocyanin biosynthetic pathway to elucidate key genes. CINNAMATE 4-HYDROXYLASE (C4H) and CHALCONE ISOMERASE (CHI2) received particular attention. C4H exposes remarkable differences between aquatic and land plants, while CHI2 demonstrates substantial variation in gene copy number and sequence similarity across species. The role of transcription factors, such as MYB, and the involvement of ROS in the regulation of anthocyanin biosynthesis are discussed. Complementary gene expression analyses under abiotic stress in Arabidopsis thaliana, Selaginella moellendorffii, and Marchantia polymorpha reveal intriguing gene-stress relationships. This study highlights evolutionary trends and the regulatory complexity of anthocyanin production under abiotic stress, providing insights and opening avenues for future research.

A single nucleotide polymorphism affects protein translation and leads to post-anthesis color change variation in closely related Lotus species.

Flower color change, a common phenomenon that is important in pollination ecology, has intrigued scientists for decades. While previous flower color studies have mainly focused on color diversity among different plant species, our focus is on unraveling the mechanism of post-anthesis color change (PACC) and the molecular basis for its presence and absence, respectively, in two closely related species of Lotus, Lotus filicaulis and Lotus japonicus MG20. Metabolomic analysis reveals anthocyanins as the key metabolites responsible for the observed PACC. Differential expression of anthocyanin biosynthetic and transport genes causes the variation in PACC between the two Lotus species. Crucially, the significant upregulation of a functionally characterized MYB regulator, LfPAP1, is linked to the accumulation of anthocyanins and visible color alterations in L. filicaulis flowers. Notably, we uncover a nucleotide polymorphism in the initiation codon of LjPAP1. Although this mutation does not affect transcription, we show that it has a major effect in attenuating protein translation, reducing its capacity to activate anthocyanin biosynthesis, and leading to a failure of PACC in L. japonicus MG20. Our study sheds light on mechanisms of PACC phenomenon and highlights the potential for mutations in initiation sequences to generate phenotypic differences between species in evolution.

Transcriptome-based analysis reveals a role for PpCDF5 in the promotion of anthocyanin accumulation at a low nighttime temperature

For red pear, the anthocyanin content is a crucial factor determining the fruit skin color, which affects consumer preferences. Low overnight temperatures promote anthocyanin accumulation, but the molecular mechanism responsible is unclear. In this study, ‘Hongzaosu’ pear (Pyrus pyrifolia × Pyrus communis) fruit were treated with a low nighttime temperature (LNT, 16 °C) or a warm nighttime temperature (WNT, 26 °C), with sampling conducted within two diurnal cycles. The results showed that LNT promoted anthocyanin accumulation in the fruit skin. The structural anthocyanin biosynthetic genes PpCHS, PpF3H, and PpUFGT exhibited a rhythmic increase in expression at night under LNT. To examine the underlying mechanism, RNA sequencing was conducted using pear calli exposed to LNT and WNT for different durations (24, 48, 72, or 96 h). Transcriptome analysis revealed 285 differentially expressed genes (DEGs) common to all pairwise comparisons of LNT- and WNT-treated calli of ‘Clapp's Favorite’ (P. communis) at the sampling time points. KEGG pathway and gene ontology enrichment analyses indicated that the common DEGs were enriched in secondary metabolic processes and phenylpropanoid metabolic processes, which are associated with anthocyanin biosynthesis. The transcription factor PpCDF5, which was responsive to LNT, was selected for further study. Dual-luciferase assays showed that PpCDF5 activated the transcription of anthocyanin biosynthetic genes PpMYB10, PpCHS, PpF3H, PpDFR, PpANS, and PpUFGT. The yeast one-hybrid and EMSA assays demonstrated that PpCDF5 directly binds to the PpF3H promoter, which contains an AAAG motif. Overexpression of PpCDF5 in pear calli and transient overexpression in pear fruit both increased anthocyanin accumulation. The results indicate that PpCDF5 is involved in LNT-induced anthocyanin biosynthesis in pear fruit and provide insights into the molecular regulation of commercial fruit coloration.

A Key R2R3-MYB Transcription Factor Activates Anthocyanin Biosynthesis and Leads to Leaf Reddening in Poplar Mutants.

Colorful woody plants are highly valued for their ornamental qualities, and are commonly used in garden landscape design. We previously cultivated several ornamental poplar varieties from bud mutants of Populus sp. Linn. '2025' (ZL2025), each with different leaf colors. Based on transcriptome data from these varieties with varying anthocyanin pigmentation, we identified and named an R2R3-MYB gene, PdMYB113. The mRNA of PdMYB113 accumulated in the leaves of the red-leaf mutants 'QHY' and 'LHY', but barely expressed in the leaves of 'ZL2025'. The anthocyanin biosynthesis genes were upregulated, resulting in high levels of red anthocyanins (particularly Peonidin-3-O-rutinoside, Cyanidin-3-O-rutinoside, and Cyanidin-3-O-glucoside) in both OE-PdMYB113 tobacco and poplar plants. This upregulation caused a color change in the tissues from green to red or dark purple. Yeast one-hybrid and luciferase assays demonstrated that PdMYB113 activates the expression of anthocyanin biosynthesis genes, including the early anthocyanin biosynthetic gene PdCHS and the late anthocynin biosynthetic gene PdANS. Consequently, PdMYB113 is identified as a key regulator of red coloration in poplar. Additionally, PdMYB113 does not dwarf transgenic plants under normal lighting conditions. This study elucidates the regulatory mechanisms of color change in ZL2025 and highlights a crucial gene for breeding new varieties of woody plants.

BZIP Transcription Factor PavbZIP6 Regulates Anthocyanin Accumulation by Increasing Abscisic Acid in Sweet Cherry.

Basic leucine zipper (bZIP) transcription factors (TFs) play a crucial role in anthocyanin accumulation in plants. In addition to bZIP TFs, abscisic acid (ABA) increases anthocyanin biosynthesis. Therefore, this study aimed to investigate whether bZIP TFs are involved in ABA-induced anthocyanin accumulation in sweet cherry and elucidate the underlying molecular mechanisms. Specifically, the BLAST method was used to identify bZIP genes in sweet cherry. Additionally, we examined the expression of ABA- and anthocyanin-related genes in sweet cherry following the overexpression or knockdown of a bZIP candidate gene. In total, we identified 54 bZIP-encoding genes in the sweet cherry genome. Basic leucine zipper 6 (bZIP6) showed significantly increased expression, along with increased anthocyanin accumulation in sweet cherry. Additionally, yeast one-hybrid and dual-luciferase assays indicated that PavbZIP6 enhanced the expression of anthocyanin biosynthetic genes (PavDFR, PavANS, and PavUFGT), thereby increasing anthocyanin accumulation. Moreover, PavbZIP6 interacted directly with the PavBBX6 promoter, thereby regulating PavNCED1 to promote abscisic acid (ABA) synthesis and enhance anthocyanin accumulation in sweet cherry fruit. Conclusively, this study reveals a novel mechanism by which PavbZIP6 mediates anthocyanin biosynthesis in response to ABA and contributes to our understanding of the mechanism of bZIP genes in the regulation of anthocyanin biosynthesis in sweet cherry.

Transcriptomic and Metabolic Analysis Reveals Genes and Pathways Associated with Flesh Pigmentation in Potato (Solanum tuberosum) Tubers.

Anthocyanins, flavonoid pigments, are responsible for the purple and red hues in potato tubers. This study analyzed tubers from four potato cultivars-red RR, purple HJG, yellow QS9, and white JZS8-to elucidate the genetic mechanisms underlying tuber pigmentation. Our transcriptomic analysis identified over 2400 differentially expressed genes between these varieties. Notably, genes within the flavonoid biosynthesis pathway were enriched in HJG and RR compared to the non-pigmented JZS8, correlating with their higher levels of anthocyanin precursors and related substances. Hierarchical clustering revealed inverse expression patterns for the key genes involved in anthocyanin metabolism between pigmented and non-pigmented varieties. Among these, several MYB transcription factors displayed strong co-expression with anthocyanin biosynthetic genes, suggesting a regulatory role. Specifically, the expression of 16 MYB genes was validated using qRT-PCR to be markedly higher in pigmented HJG and RR versus JZS8, suggesting that these MYB genes might be involved in tuber pigmentation. This study comprehensively analyzed the transcriptome of diverse potato cultivars, highlighting specific genes and metabolic pathways involved in tuber pigmentation. These findings provide potential molecular targets for breeding programs focused on enhancing tuber color.

Transcription factors BZR1 and PAP1 cooperate to promote anthocyanin biosynthesis in Arabidopsis shoots.

Anthocyanins play critical roles in protecting plant tissues against diverse stresses. The complicated regulatory networks induced by various environmental factors modulate the homeostatic level of anthocyanins. Here, we show that anthocyanin accumulation is induced by brassinosteroids (BRs) in Arabidopsis (Arabidopsis thaliana) shoots and shed light on the underlying regulatory mechanism. We observed that anthocyanin levels are altered considerably in BR-related mutants, and BRs induce anthocyanin accumulation by upregulating the expression of anthocyanin biosynthetic genes. Our genetic analysis indicated that BRASSINAZOLE RESISTANT 1 (BZR1) and PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) are essential for BR-induced anthocyanin accumulation. The BR-responsive transcription factor BZR1 directly binds to the PAP1 promoter, regulating its expression. In addition, we found that intense anthocyanin accumulation caused by the pap1-D-dominant mutation is significantly reduced in BR mutants, implying that BR activity is required for PAP1 function after PAP1 transcription. Moreover, we demonstrated that BZR1 physically interacts with PAP1 to cooperatively regulate the expression of PAP1-target genes, such as TRANSPARENT TESTA 8, DIHYDROFLAVONOL 4-REDUCTASE, and LEUKOANTHOCYANIDIN DIOXYGENASE. Our findings indicate that BZR1 functions as an integral component of the PAP1-containing transcription factor complex, contributing to increased anthocyanin biosynthesis. Notably, we also show that functional interaction of BZR1 with PAP1 is required for anthocyanin accumulation induced by low nitrogen stress. Taken together, our results demonstrate that BR-regulated BZR1 promotes anthocyanin biosynthesis through cooperative interaction with PAP1 of the MBW complex.

Role of Rubus chingii BBX gene family in anthocyanin accumulation during fruit ripening.

The B-box (BBX) family, which is a class of zinc finger transcription factors, exhibits special roles in plant growth and development as well as in plants' ability to cope with various stresses. Even though Rubus chingii is an important traditional medicinally edible plant in east Asia, there are no comprehensive studies of BBX members in R. chingii. In this study, 32 RcBBX members were identified, and these were divided into five groups. A collinearity analysis showed that gene duplication events were common, and when combined with a motif analysis of the RcBBX genes, it was concluded that group V genes might have undergone deletion of gene fragments or mutations. Analysis of cis-acting elements revealed that each RcBBX gene contained hormone-, light-, and stress-related elements. Expression patterns of the 32 RcBBX genes during fruit ripening revealed that highest expression occurred at the small green fruit stage. Of note, the expression of several RcBBX genes increased rapidly as fruit developed. These findings, combined with the expression profiles of anthocyanin biosynthetic genes during fruit ripening, allowed us to identify the nuclear-targeted RcBBX26, which positively promoted anthocyanin production in R. chingii. The collective findings of this study shed light on the function of RcBBX genes in different tissues, developmental stages, and in response to two abiotic stresses.

A viroid-derived small interfering RNA targets bHLH transcription factor MdPIF1 to regulate anthocyanin biosynthesis in Malus domestica.

Fruit colour is a critical determinant for the appearance quality and commercial value of apple fruits. Viroid-induced dapple symptom severely affects the fruit coloration, however, the underlying mechanism remains unknown. In this study, we identified an apple dimple fruit viroid (ADFVd)-derived small interfering RNA, named vsiR693, which targeted the mRNA coding for a bHLH transcription factor MdPIF1 (PHYTOCHROME-INTERACTING FACTOR 1) to regulate anthocyanin biosynthesis in apple. 5' RLM-RACE and artificial microRNA transient expression system proved that vsiR693 directly targeted the mRNA of MdPIF1 for cleavage. MdPIF1 positively regulated anthocyanin biosynthesis in both apple calli and fruits, and it directly bound to G-box element in the promoter of MdPAL and MdF3H, two anthocyanin biosynthetic genes, to promote their transcription. Expression of vsiR693 negatively regulated anthocyanin biosynthesis in both apple calli and fruits. Furthermore, co-expression of vsiR693 and MdPIF1 suppressed MdPIF1-promoted anthocyanin biosynthesis in apple fruits. Infiltration of ADFVd infectious clone suppressed coloration surrounding the injection sites in apple fruits, while a mutated version of ADFVd, in which the vsiR693 producing region was mutated, failed to repress fruit coloration around the injection sites. These data provide evidence that a viroid-derived small interfering RNA targets host transcription factor to regulate anthocyanin biosynthesis in apple.

De novo transcriptome analysis identifies RpMYB1 as an activator of anthocyanin biosynthesis in Rehmannia piasezkii

Rehmannia piasezkii is a kind of medicinal plants, of the Orobanchaceae family, and well known for its large pink or purple corolla. However, no research on the molecular mechanism of flower color formation in R. piasezkii has been conducted so far. In this study, we investigated the transcriptome of root, stem, leaf and corollas of R. piasezkii using transcriptome sequencing technology and assembled 144,582 unigenes. A total of 58 anthocyanin biosynthetic genes were identified in the R. piasezkii transcriptome, fourteen of which were highly correlated with anthocyanin content, especially RpF3H2, RpDFR2, RpANS1, RpANS2 and RpUFGT. Totally, 35 MYB genes with FPKM values greater than 5 were identified in the R. piasezkii transcriptome, including an R2R3 MYB transcriptional factor RpMYB1, which belongs to subgroup 6 of the R2R3 MYB family. Agrobacterium-mediated transient expression of Nicotiana benthamiana revealed that overexpression of RpMYB1 could activate the expression of structural genes in anthocyanin synthesis pathway and promote the accumulation of anthocyanins in N. benthamiana leaves, indicating that RpMYB1 is a positive regulator of anthocyanin synthesis. Furthermore, combined transient overexpression of RpMYB1 with RpANS1, RpMYB1+RpANS1 with other structural genes all could further enhance the accumulation of anthocyanins in N. benthamiana leaves. Permanent overexpression of RpMYB1 in R. glutinosa promoted anthocyanin accumulation and expression levels of RgCHS, RgF3H, RgDFR and RgANS. Further evidence from dual-luciferase assay suggested that RpMYB1 could bind to the promoter of RpDFR2 and hence activating its expression. These findings provide insight into the molecular regulation in anthocyanin biosynthesis in R. piasezkii and provide valuable genetic resources for the genetic improvement of flower color.

CaLAP1 and CaLAP2 orchestrate anthocyanin biosynthesis in the seed coat of Cicer arietinum.

Our findings shed light on the regulation of anthocyanin and proanthocyanidin biosynthesis in chickpea seed coats. Expression of R2R3-MYB transcription factors CaLAP1 and CaLAP2 enhanced the anthocyanins and proanthocyanidins content in chickpea. The seed coat color is a major economic trait in leguminous crop chickpea (Cicer arietinum). Anthocyanins and proanthocyanidins (PAs) are two classes of flavonoids that mainly contribute to the flower, seed coat and color of Desi chickpea cultivars. Throughout the land plant lineage, the accumulation of anthocyanins and PAs is regulated by MYB and bHLH transcription factors (TFs), which form an MBW (MYB, bHLH, and WD40) complex. Here, we report two R2R3-MYB TFs in chickpea belonging to the anthocyanin-specific subgroup-6, CaLAP1 (Legume Anthocyanin Production 1), and CaLAP2 (Legume Anthocyanin Production 2), which are mainly expressed in the flowers and developmental stages of the seeds. CaLAP1 and CaLAP2 interact with TT8-like CabHLH1 and WD40, forming the MBW complex, and bind to the promoter sequences of anthocyanin- and PA biosynthetic genes CaCHS6, CaDFR2, CaANS, and CaANR, leading to anthocyanins and PA accumulation in the seed coat of chickpea. Moreover, these CaLAPs partially complement the anthocyanin-deficient phenotype in the Arabidopsis thaliana sextuple mutant seedlings. Overexpression of CaLAPs in chickpea resulted in significantly higher expression of anthocyanin and PA biosynthetic genes leading to a darker seed coat color with higher accumulation of anthocyanin and PA. Our findings show that CaLAPs positively modulate anthocyanin and PA content in seed coats, which might influence plant development and resistance to various biotic and abiotic stresses.

Painted flowers: Eluta generates pigment patterning in Antirrhinum.

In the early 1900s, Erwin Baur established Antirrhinum majus as a model system, identifying and characterising numerous flower colour variants. This included Picturatum/Eluta, which restricts the accumulation of magenta anthocyanin pigments, forming bullseye markings on the flower face. We identified the gene underlying the Eluta locus by transposon-tagging, using an Antirrhinum line that spontaneously lost the nonsuppressive el phenotype. A candidate MYB repressor gene at this locus contained a CACTA transposable element. We subsequently identified plants where this element excised, reverting to a suppressive Eluta phenotype. El alleles inhibit expression of anthocyanin biosynthetic genes, confirming it to be a regulatory locus. The modes of action of Eluta were investigated by generating stable transgenic tobacco lines, biolistic transformation of Antirrhinum petals and promoter activation/repression assays. Eluta competes with MYB activators for promoter cis-elements, and also by titrating essential cofactors (bHLH proteins) to reduce transcription of target genes. Eluta restricts the pigmentation established by the R2R3-MYB factors, Rosea and Venosa, with the greatest repression on those parts of the petals where Eluta is most highly expressed. Baur questioned the origin of heredity units determining flower colour variation in cultivated A. majus. Our findings support introgression from wild species into cultivated varieties.

Fungal invasion-induced accumulation of salicylic acid promotes anthocyanin biosynthesis through MdNPR1-MdTGA2.2 module in apple fruits.

In the field, necrosis area induced by pathogens is usually surrounded by a red circle in apple fruits. However, the underlying molecular mechanism of this phenomenon remains unclear. In this study, we demonstrated that accumulated salicylic acid (SA) induced by fungal infection promoted anthocyanin biosynthesis through MdNPR1-MdTGA2.2 module in apple (Malus domestica). Inoculating apple fruits with Valsa mali or Botryosphaeria dothidea induced a red circle surrounding the necrosis area, which mimicked the phenotype observed in the field. The red circle accumulated a high level of anthocyanins, which was positively correlated with SA accumulation stimulated by fungal invasion. Further analysis showed that SA promoted anthocyanin biosynthesis in a dose-dependent manner in both apple calli and fruits. We next demonstrated that MdNPR1, a master regulator of SA signaling, positively regulated anthocyanin biosynthesis in both apple and Arabidopsis. Moreover, MdNPR1 functioned as a co-activator to interact with and enhance the transactivation activity of MdTGA2.2, which could directly bind to the promoters of anthocyanin biosynthetic and regulatory genes to promote their transcription. Suppressing expression of either MdNPR1 or MdTGA2.2 inhibited coloration of apple fruits, while overexpressing either of them significantly promoted fruit coloration. Finally, we revealed that silencing either MdNPR1 or MdTGA2.2 in apple fruits repressed SA-induced fruit coloration. Therefore, our data determined that fungal-induced SA promoted anthocyanin biosynthesis through MdNPR1-MdTGA2.2 module, resulting in a red circle surrounding the necrosis area in apple fruits.

The R2R3 MYB Ruby1 is activated by two cold responsive ethylene response factors, via the retrotransposon in its promoter, to positively regulate anthocyanin biosynthesis in citrus.

Anthocyanins are natural pigments and dietary antioxidants that play multiple biological roles in plants and are important in animal and human nutrition. Low temperature (LT) promotes anthocyanin biosynthesis in many species including blood orange. A retrotransposon in the promoter of Ruby1, which encodes an R2R3 MYB transcription factor, controls cold-induced anthocyanin accumulation in blood orange flesh. However, the specific mechanism remains unclear. In this study, we characterized two LT-induced ETHYLENE RESPONSE FACTORS (CsERF054 and CsERF061). Both CsERF054 and CsERF061 can activate the expression of CsRuby1 by directly binding to a DRE/CRT cis-element within the retrotransposon in the promoter of CsRuby1, thereby positively regulating anthocyanin biosynthesis. Further investigation indicated that CsERF061 also forms a protein complex with CsRuby1 to co-activate the expression of anthocyanin biosynthetic genes, providing a dual mechanism for the upregulation of the anthocyanin pathway. These results provide insights into how LT mediates anthocyanin biosynthesis and increase the understanding of the regulatory network of anthocyanin biosynthesis in blood orange.

PagMYB128 regulates anthocyanin biosynthesis by directly activating PagMYB116 in Populus alba × Populus glandulosa

MYB transcription factors regulate the biosynthesis of anthocyanin, a crucial flavonoid in plants. In this research, we identified PagMYB128 as a new activator of anthocyanin production in the hybrid poplar (Populus alba × Populus glandulosa). The PagMYB128 protein was located in the nucleus and has higher expression level in stems. Dominant repression of PagMYB128 led to a reduction of anthocyanin content in the petiole and stems. Overexpression of PagMYB128 resulted in the opposite phenotype. We found that PagMYB128 promoted anthocyanin biosynthesis by activating PagCHS1, PagF3’5’H1, PagDFR2, and PagANR1, which encoded the essential enzymes of anthocyanin biosynthesis. Further, PagMYB116 was identified as a potential direct target of PagMYB128 by ChIP-seq. Through ChIP-qPCR, Y1H, and Split-LUC assays, it was demonstrated that PagMYB128 could directly interact with the MYB-site located in the promoter region of PagMYB116, thereby exerting control over the transcription of PagMYB116. Moreover, through the poplar transient transformation method, we found that PagMYB116 could activate anthocyanin biosynthetic enzyme genes PagF3’5’H1 and PagANR1, through direct binding to their promoters. These findings indicate that the PagMYB128-MYB116 module for transcriptional regulation plays a crucial part in the synthesis of anthocyanin, enriching our comprehension of the networks responsible for regulating anthocyanin transcription.

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.

Exogenous melatonin delays oxidative browning in litchi during cold storage by regulating biochemical attributes and gene expression.

Oxidative damage leading to loss of nutritional quality and pericarp discoloration of harvested litchi fruits drastically limits consumer acceptance and marketability. In the present investigation, the impact of postharvest melatonin application at different concentrations, i.e., 0.1 mM, 0.25 mM, and 0.5 mM, on fruit quality and shelf life of litchi fruits under cold storage conditions was studied. The results revealed the positive effect of melatonin application at all concentrations on fruit quality and shelf life. However, treatment with 0.5 mM concentration of melatonin resulted in minimum weight loss, decay loss, pericarp discoloration, and also retained higher levels of TSS, acidity, total sugar, ascorbic acid, anthocyanin, antioxidant, and phenolics content during cold storage. Melatonin administration also restricted the enzymatic activity of the polyphenol oxidase (PPO) and peroxidase (POD) enzymes in the fruit pericarp and maintained freshness of the fruits up to 30 days in cold storage. At the molecular level, a similar reduction in the expression of browning-associated genes, LcPPO, LcPOD, and Laccase, was detected in preserved litchi fruits treated with melatonin. Anthocyanin biosynthetic genes, LcUFGT and LcDFR, on the other hand showed enhanced expression in melatonin treated fruits compared to untreated fruits. Melatonin, owing to its antioxidant properties, when applied to harvested litchi fruits retained taste, nutritional quality and red color pericarp up till 30 days in cold storage.