Regulation Of Anthocyanin Biosynthesis Research Articles

Transcription Factors LvBBX24 and LvbZIP44 Coordinated Anthocyanin Accumulation in Response to Light in Lily Petals

Abstract Lily (Lilium spp.), a horticultural crop serving both ornamental and edible functions, derives its coloration primarily from anthocyanins. However, limited studies have been conducted on the accumulation of anthocyanins within lilies. In this study, we cloned a light-induced transcription factor named as LvBBX24 in lilies. Through genetic and biochemical analysis, we determined that LvBBX24 could upregulate the transcription of LvMYB5 and facilitate anthocyanin synthesis. Moreover, we identified that darkness promoted the degradation of LvBBX24 protein. Through screening a yeast library, we identified LvbZIP44 acts as its interacting partner. Genetic testing confirmed that LvbZIP44 also plays a role in promoting lily anthocyanin synthesis. This indicates a potential synergistic regulatory effect between LvBBX24 and LvbZIP44. Our study indicates that LvBBX24 and LvbZIP44 cooperate to regulate anthocyanin accumulation in lily petals. These findings provide compelling evidence supporting the idea that LvBBX24 and LvbZIP44 may form a looped helix surrounding the LvMYB5 promoter region to regulate anthocyanin biosynthesis.

Decoding anthocyanin biosynthesis regulation in Asparagus officinalis peel coloration: Insights from integrated metabolomic and transcriptomic analyses

Asparagus is a key global vegetable crop with significant economic importance. Purple asparagus, rich in anthocyanins, stands out for its nutritional value. Despite its prominence, the molecular mechanisms driving purple peel coloration in asparagus remain unclear. This study focuses on three asparagus varieties with distinct peel colors to analyze anthocyanins in both the metabolome and transcriptome, unraveling the regulatory mechanisms. Our findings identify 30 anthocyanins, categorized into five major anthocyanin aglycones across diverse asparagus peel colors. Notably, among the 30 differentially expressed metabolites (DEMs), 18 anthocyanins displayed significantly up-regulated expression in the ‘Purple Passion’ variety. Key contributors include Cyanidin-3-O-rutinoside-5-O-glucoside and Cyanidin-3-O-sophoroside. Cyanidin-3-O-glucoside is most abundant in ‘Purple Passion’, while Petunidin-glucoside-galactoside is the least. Analysis of differentially expressed genes (DEGs) displayed 21 structural genes in anthocyanin synthesis, with F3H, DFR, ANS, and one of three UFGTs showing significantly higher expression in the ‘Purple Passion’ compared to ‘Grande’ and ‘Erasmus’. Additionally, transcription factors (TFs), including 38 MYB, 33 bHLH, and 13 bZIP, also display differential expression in this variety. Validation through real-time qPCR supports the idea that increased expression of anthocyanin structural genes contribute to anthocyanin accumulation. Transient overexpression of AoMYB17 in tobacco further showed that it had the vital function of increasing anthocyanin content. This study sheds light on the mechanisms behind anthocyanin coloration in three distinct asparagus peels. Therefore, it lays the foundation for potential genetic enhancements, aiming to develop new purple-fleshed asparagus germplasms with heightened anthocyanin content.

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.

ERF transcription factors govern anthocyanin biosynthesis in litchi pericarp by modulating the expression of anthocyanin biosynthesis genes

Anthocyanins are the most prevalent phytochemicals that color the fruits. Ethylene promotes fruit ripening and modulates anthocyanin biosynthesis in plants, however, the underlying molecular mechanism in litchi is unclear. Herein, the slow-reddening cultivar ‘Feizixiao’ (FXZ) and swift-reddening cultivar ‘Guiwei’ (GW) were selected to study the regulation of anthocyanin biosynthesis in litchi. Total anthocyanin contents were found to be higher in ‘GW’ and lower in ‘FZX’. We then comprehensively investigated ethylene response factor (ERF) subfamily genes and their expression patterns in different tissues and fruit developmental stages. Using transcriptome data, eight LcERF genes were identified as putative key genes associated with litchi pericarp coloration and anthocyanin accumulation. To test whether these genes are expressed under ethephon treatment, we applied 100 and 200 µL L−1 ethephon 4 weeks before harvest. Ethephon treatment dramatically increased anthocyanin accumulation as well as the expression of targeted ERF genes. LcERF1/22/25/37 were significantly upregulated in response to ethephon treatment, while LcERF64 was significantly downregulated. Anthocyanin content and expression of anthocyanin-related genes were considerably reduced in LcERF1/22/25/37-silenced fruits, but the opposite trend was observed in LcERF64-silenced fruits. Yeast-one-hybrid and dual luciferase assays revealed that the tested ERFs bind to the promoters of key anthocyanin biosynthesis genes and alter their expression. These findings suggested that ethylene promotes anthocyanin accumulation and consequently fruit coloration by stimulating the positive anthocyanin biosynthesis regulators LcERF1/22/25/37, or by suppressing the negative regulator LcERF64, which regulates the expression LcDFR and LcMYB1 by directly binding to their promoters.

The SMXL8-AGL9 module mediates crosstalk between strigolactone and gibberellin to regulate strigolactone-induced anthocyanin biosynthesis in apple.

Although the strigolactone (SL) signaling pathway and SL-mediated anthocyanin biosynthesis have been reported, the molecular association between SL signaling and anthocyanin biosynthesis remains unclear. In this study, we identified the SL signal transduction pathway associated with anthocyanin biosynthesis and the crosstalk between gibberellin (GA) and SL signaling in apple (Malus × domestica). ELONGATED HYPOCOTYL5 (HY5) acts as a key node integrating SL signaling and anthocyanin biosynthesis, and the SL response factor AGAMOUS-LIKE MADS-BOX9 (AGL9) promotes anthocyanin biosynthesis by activating HY5 transcription. The SL signaling repressor SUPPRESSOR OF MAX2 1-LIKE8 (SMXL8) interacts with AGL9 to form a complex that inhibits anthocyanin biosynthesis by downregulating HY5 expression. Moreover, the E3 ubiquitin ligase PROTEOLYSIS1 (PRT1) mediates the ubiquitination-mediated degradation of SMXL8, which is a key part of the SL signal transduction pathway associated with anthocyanin biosynthesis. In addition, the GA signaling repressor REPRESSOR-of-ga1-3-LIKE2a (RGL2a) mediates the crosstalk between GA and SL by disrupting the SMXL8-AGL9 interaction that represses HY5 transcription. Taken together, our study reveals the regulatory mechanism of SL-mediated anthocyanin biosynthesis and uncovers the role of SL-GA crosstalk in regulating anthocyanin biosynthesis in apple.

The MdNAC72-MdABI5 module acts as an interface integrating jasmonic acid and gibberellin signals and undergoes ubiquitination-dependent degradation regulated by MdSINA2 in apple.

Jasmonic acid (JA) and gibberellin (GA) coordinately regulate plant developmental programs and environmental cue responses. However, the fine regulatory network of the cross-interaction between JA and GA remains largely elusive. In this study, we demonstrate that MdNAC72 together with MdABI5 positively regulates anthocyanin biosynthesis through an exquisite MdNAC72-MdABI5-MdbHLH3 transcriptional cascade in apple. MdNAC72 interacts with MdABI5 to promote the transcriptional activation of MdABI5 on its target gene MdbHLH3 and directly activates the transcription of MdABI5. The MdNAC72-MdABI5 module regulates the integration of JA and GA signals in anthocyanin biosynthesis by combining with JA repressor MdJAZ2 and GA repressor MdRGL2a. MdJAZ2 disrupts the MdNAC72-MdABI5 interaction and attenuates the transcriptional activation of MdABI5 by MdNAC72. MdRGL2a sequesters MdJAZ2 from the MdJAZ2-MdNAC72 protein complex, leading to the release of MdNAC72. The E3 ubiquitin ligase MdSINA2 is responsive to JA and GA signals and promotes ubiquitination-dependent degradation of MdNAC72. The MdNAC72-MdABI5 interface fine-regulates the integration of JA and GA signals at the transcriptional and posttranslational levels by combining MdJAZ2, MdRGL2a, and MdSINA2. In summary, our findings elucidate the fine regulatory network connecting JA and GA signals with MdNAC72-MdABI5 as the core in apple.

Reactive oxygen species act as the key signaling molecules mediating light-induced anthocyanin biosynthesis in Eucalyptus

Light plays a pivotal role in regulating anthocyanin biosynthesis in plants, and the early light-responsive signals that initiate anthocyanin biosynthesis remain to be elucidated. In this study, we showed that the anthocyanin biosynthesis in Eucalyptus is hypersensitive to increased light intensity. The combined transcriptomic and metabolomic analyses were conducted on Eucalyptus leaves after moderate (ML; 100 μmol m−2 s−1) and high (HL; 300 μmol m−2 s−1) light intensity treatments. The results identified 1940, 1096, 1173, and 2756 differentially expressed genes at 6, 12, 24, and 36 h after HL treatment, respectively. The metabolomic results revealed the primary anthocyanin types, and other differentially accumulated flavonoids and phenylpropane intermediates that were produced in response to HL, which well aligned with the transcriptome results. Moreover, biochemical analysis showed that HL inhibited peroxidase activity and increased the ROS level in Eucalyptus leaves. ROS depletion through co-application of the antioxidants rutin, uric acid, and melatonin significantly reduced, and even abolished, anthocyanin biosynthesis induced by HL treatment. Additionally, exogenous application of hydrogen peroxide efficiently induced anthocyanin biosynthesis within 24 h, even under ML conditions, suggesting that ROS played a major role in activating anthocyanin biosynthesis. A HL-responsive MYB transcription factor EgrMYB113 was identified to play an important role in regulating anthocyanin biosynthesis by targeting multiple anthocyanin biosynthesis genes. Additionally, the results demonstrated that gibberellic acid and sugar signaling contributed to HL-induced anthocyanin biosynthesis. Conclusively, these results suggested that HL triggers multiple signaling pathways to induce anthocyanin biosynthesis, with ROS acting as indispensable mediators in Eucalyptus.

Epigenetic regulation of high light-induced anthocyanin biosynthesis by histone demethylase IBM1 in Arabidopsis.

In plant species, anthocyanin accumulation is specifically regulated by light signaling. Although the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) complex is known to control anthocyanin biosynthesis in response to light, the precise mechanism underlying this process remains largely unknown. Here, we report that Increase in BONSAI Methylation 1 (IBM1), a JmjC domain-containing histone demethylase, participates in the regulation of light-induced anthocyanin biosynthesis in Arabidopsis. The expression of IBM1 was induced by high light (HL) stress, and loss-of-function mutations in IBM1 led to accelerated anthocyanin accumulation under HL conditions. We further identified that IBM1 is directly associated with SPA1/3/4 chromatin in vivo to establish a hypomethylation status on H3K9 and DNA non-CG at these loci under HL, thereby releasing their expression. Genetic analysis showed that quadruple mutants of IBM1 and SPA1/3/4 resemble spa134 mutants. Overexpression of SPA1 in ibm1 mutants complements the mutant phenotype. Our results elucidate the significance and mechanism of IBM1 histone demethylase in the epigenetic regulation of anthocyanin biosynthesis in Arabidopsis under HL conditions.

Genome-Wide Analysis of Transcription Factor R2R3-MYB Gene Family and Gene Expression Profiles during Anthocyanin Synthesis in Common Walnut (Juglans regia L.).

The R2R3-MYB gene family, encoding plant transcriptional regulators, participates in many metabolic pathways of plant physiology and development, including flavonoid metabolism and anthocyanin synthesis. This study proceeded as follows: the JrR2R3-MYB gene family was analyzed genome-wide, and the family members were identified and characterized using the high-quality walnut reference genome "Chandler 2.0". All 204 JrR2R3-MYBs were established and categorized into 30 subgroups via phylogenetic analysis. JrR2R3-MYBs were unevenly distributed over 16 chromosomes. Most JrR2R3-MYBs had similar structures and conservative motifs. The cis-acting elements exhibit multiple functions of JrR2R3-MYBs such as light response, metabolite response, and stress response. We found that the expansion of JrR2R3-MYBs was mainly caused by WGD or segmental duplication events. Ka/Ks analysis indicated that these genes were in a state of negative purifying selection. Transcriptome results suggested that JrR2R3-MYBs were widely entangled in the process of walnut organ development and differentially expressed in different colored varieties of walnuts. Subsequently, we identified 17 differentially expressed JrR2R3-MYBs, 9 of which may regulate anthocyanin biosynthesis based on the results of a phylogenetic analysis. These genes were present in greater expression levels in 'Zijing' leaves than in 'Lvling' leaves, as revealed by the results of qRT-PCR experiments. These results contributed to the elucidation of the functions of JrR2R3-MYBs in walnut coloration. Collectively, this work provides a foundation for exploring the functional characteristics of the JrR2R3-MYBs in walnuts and improving the nutritional value and appearance quality of walnuts.

A Fruit-Expressed MYB Transcription Factor Regulates Anthocyanin Biosynthesis in Atropa belladonna.

Anthocyanins are water-soluble flavonoid pigments that play a crucial role in plant growth and metabolism. They serve as attractants for animals by providing plants with red, blue, and purple pigments, facilitating pollination and seed dispersal. The fruits of solanaceous plants, tomato (Solanum lycopersicum) and eggplant (Solanum melongena), primarily accumulate anthocyanins in the fruit peels, while the ripe fruits of Atropa belladonna (Ab) have a dark purple flesh due to anthocyanin accumulation. In this study, an R2R3-MYB transcription factor (TF), AbMYB1, was identified through association analysis of gene expression and anthocyanin accumulation in different tissues of A. belladonna. Its role in regulating anthocyanin biosynthesis was investigated through gene overexpression and RNA interference (RNAi). Overexpression of AbMYB1 significantly enhanced the expression of anthocyanin biosynthesis genes, such as AbF3H, AbF3'5'H, AbDFR, AbANS, and Ab3GT, leading to increased anthocyanin production. Conversely, RNAi-mediated suppression of AbMYB1 resulted in decreased expression of most anthocyanin biosynthesis genes, as well as reduced anthocyanin contents in A. belladonna. Overall, AbMYB1 was identified as a fruit-expressed R2R3-MYB TF that positively regulated anthocyanin biosynthesis in A. belladonna. This study provides valuable insights into the regulation of anthocyanin biosynthesis in Solanaceae plants, laying the foundation for understanding anthocyanin accumulation especially in the whole fruits of solanaceous plants.

Laser capture microdissection transcriptome (LCM RNA-seq) reveals BcDFR is a key gene in anthocyanin synthesis of non-heading Chinese cabbage

BackgroundPurple non-heading Chinese cabbage [Brassica campestris (syn. Brassica rapa) ssp. chinensis] has become popular because of its richness in anthocyanin. However, anthocyanin only accumulates in the upper epidermis of leaves. Further studies are needed to investigate the molecular mechanisms underlying the specific accumulation of it.ResultsIn this study, we used the laser capture frozen section method (LCM) to divide purple (ZBC) and green (LBC) non-heading Chinese cabbage leaves into upper and lower epidermis parts (Pup represents the purple upper epidermis, Plow represents the purple lower epidermis, Gup represents the green upper epidermis, Glow represents the green lower epidermis). Through transcriptome sequencing, we found that the DIHYDROFLAVONOL 4-REDUCTASE-encoding gene BcDFR, is strongly expressed in Pup but hardly in others (Plow, Gup, Glow). Further, a deletion and insertion in the promoter of BcDFR in LBC were found, which may interfere with BcDFR expression. Subsequent analysis of gene structure and conserved structural domains showed that BcDFR is highly conserved in Brassica species. The predicted protein-protein interaction network of BcDFR suggests that it interacts with almost all functional proteins in the anthocyanin biosynthesis pathway. Finally, the results of the tobacco transient expression also demonstrated that BcDFR promotes the synthesis and accumulation of anthocyanin.ConclusionsBcDFR is specifically highly expressed on the upper epidermis of purple non-heading Chinese cabbage leaves and regulates anthocyanin biosynthesis and accumulation. Our study provides new insights into the functional analysis and transcriptional regulatory network of anthocyanin-related genes in purple non-heading Chinese cabbage.

Genome-Wide Analysis of MYB Gene Family in Chrysanthemum ×morifolium Provides Insights into Flower Color Regulation.

MYBs constitute the second largest transcription factor (TF) superfamily in flowering plants with substantial structural and functional diversity, which have been brought into focus because they affect flower colors by regulating anthocyanin biosynthesis. Up to now, the genomic data of several Chrysanthemum species have been released, which provides us with abundant genomic resources for revealing the evolution of the MYB gene family in Chrysanthemum species. In the present study, comparative analyses of the MYB gene family in six representative species, including C. lavandulifolium, C. seticuspe, C. ×morifolium, Helianthus annuus, Lactuca sativa, and Arabidopsis thaliana, were performed. A total of 1104 MYBs, which were classified into four subfamilies and 35 lineages, were identified in the three Chrysanthemum species (C. lavandulifolium, C. seticuspe, and C. ×morifolium). We found that whole-genome duplication and tandem duplication are the main duplication mechanisms that drove the occurrence of duplicates in CmMYBs (particularly in the R2R3-MYB subfamily) during the evolution of the cultivated chrysanthemums. Sequence structure and selective pressure analyses of the MYB gene family revealed that some of R2R3-MYBs were subjected to positive selection, which are mostly located on the distal telomere segments of the chromosomes and contain motifs 7 and 8. In addition, the gene expression analysis of CmMYBs in different organs and at various capitulum developmental stages of C. ×morifolium indicated that CmMYBS2, CmMYB96, and CmMYB109 might be the negative regulators for anthocyanin biosynthesis. Our results provide the phylogenetic context for research on the genetic and functional evolution of the MYB gene family in Chrysanthemum species and deepen our understanding of the regulatory mechanism of MYB TFs on the flower color of C. ×morifolium.

The R2R3-MYB transcription factor ZeMYB32 negatively regulates anthocyanin biosynthesis in Zinnia elegans.

This study identified an R2R3-MYB from Zinnia elegans, ZeMYB32, which negatively regulates anthocyanin biosynthesis.

Multifaceted Regulation of Anthocyanin Biosynthesis in Plants: A Comprehensive Review

Multifaceted Regulation of Anthocyanin Biosynthesis in Plants: A Comprehensive Review

Genome-wide characterization of SmZHD gene family and the role of SmZHD12 in regulating anthocyanin biosynthesis in eggplant (Solanum melongena L.).

SmZHDs was highly expressed in anthocyanin-rich parts of eggplant. SmZHD12 can activate the expression of SmCHS, SmANS, SmDFR and SmF3H. Overexpression of SmZHD12 promotes anthocyanin biosynthesis in Arabidopsis. The Zinc finger-homeodomain (ZHD) proteins family genes are known to play a significant role in plant development and physiological processes. However, the evolutionary history and function of the ZHD gene family in eggplant remain largely unexplored. This study categorizes a total of 15 SmZHD genes into SmMIF and SmZHD subfamilies based on conserved domains. The phylogeny, gene structure, conserved motifs, promoter elements, and chromosomal locations of the SmZHD genes were comprehensively analyzed. Tissue expression profiles indicate that the majority of SmZHD genes are expressed in anthocyanin-rich areas. qRT-PCR assays revealed distinct expression patterns of SmZHD genes in response to various treatments, indicating their potential involvement in multiple signaling pathways. Analysis of transcriptomic data from light-treated eggplant peel identified SmZHD12 as the most light-responsive gene among the 15 SmZHD genes. Consequently, this study provides further evidence that SmZHD12 facilitates anthocyanin accumulation in Arabidopsis leaves by upregulating the expression of anthocyanin biosynthesis structural genes, as confirmed by dual-luciferase assays and Arabidopsis genetic transformation. Our study will lay a solid foundation for the in-depth study of the involvement of SmZHD genes in the regulation of anthocyanin biosynthesis.

Fine mapping and candidate gene analysis of qSRC3 controlling the silk color in maize (Zea mays L.).

Fine mapping of the maize QTL qSRC3, responsible for red silk, uncovered the candidate gene ZmMYB20, which encodes an R2R3-MYB transcription factor, has light-sensitive expression, and putatively regulates genes expression associated with anthocyanin biosynthesis. Colorless silk is a key characteristic contributing to the visual quality of fresh corn intended for market distribution. Nonetheless, the identification of Mendelian trait loci and associated genes that control silk color has been scarce. In this study, a F2 population arising from the hybridization of the single-segment substitution line qSRC3MT1 with red silk, carrying an introgressed allele from teosinte (Zea mays ssp. mexicana), and the recurrent maize inbred line Mo17, characterized by light green silk, was utilized for fine mapping. We found that the red silk trait is controlled by a semi-dominant genetic locus known as qSRC3, and its expression is susceptible to light-mediated inhibition. Moreover, qSRC3 explained 68.78% of the phenotypic variance and was delimited to a 133.2kb region, which includes three genes. Subsequent expression analyses revealed that ZmMYB20 (Zm00001d039700), which encodes an R2R3-MYB transcription factor, was the key candidate gene within qSRC3. Yeast one-hybrid and dual-luciferase reporter assays provided evidence that ZmMYB20 suppresses the expression of two crucial anthocyanin biosynthesis genes, namely ZmF3H and ZmUFGT, by directly binding to their respective promoter regions. Our findings underscore the significance of light-inhibited ZmMYB20 in orchestrating the spatial and temporal regulation of anthocyanin biosynthesis. These results advance the production of colorless silk in fresh corn, responding to the misconception that fresh corn with withered colored silk is not fresh and providing valuable genetic resources for the improvement of sweet and waxy maize.

Integrated transcriptome and metabolome analysis reveals anthocyanin biosynthesis mechanisms in pepper (Capsicum annuum L.) leaves under continuous blue light irradiation

BackgroundDifferent metabolic compounds give pepper leaves and fruits their diverse colors. Anthocyanin accumulation is the main cause of the purple color of pepper leaves. The light environment is a critical factor affecting anthocyanin biosynthesis. It is essential that we understand how to use light to regulate anthocyanin biosynthesis in plants.ResultPepper leaves were significantly blue–purple only in continuous blue light or white light (with a blue light component) irradiation treatments, and the anthocyanin content of pepper leaves increased significantly after continuous blue light irradiation. This green-to-purple phenotype change in pepper leaves was due to the expression of different genes. We found that the anthocyanin synthesis precursor-related genes PAL and 4CL, as well as the structural genes F3H, DFR, ANS, BZ1, and F3’5’H in the anthocyanin synthesis pathway, had high expression under continuous blue light irradiation. Similarly, the expression of transcription factors MYB1R1-like, MYB48, MYB4-like isoform X1, bHLH143-like, and bHLH92-like isoform X3, and circadian rhythm-related genes LHY and COP1, were significantly increased after continuous blue light irradiation. A correlation network analysis revealed that these transcription factors and circadian rhythm-related genes were positively correlated with structural genes in the anthocyanin synthesis pathway. Metabolomic analysis showed that delphinidin-3-O-glucoside and delphinidin-3-O-rutinoside were significantly higher under continuous blue light irradiation relative to other light treatments. We selected 12 genes involved in anthocyanin synthesis in pepper leaves for qRT-PCR analysis, and the accuracy of the RNA-seq results was confirmed.ConclusionsIn this study, we found that blue light and 24-hour irradiation together induced the expression of key genes and the accumulation of metabolites in the anthocyanin synthesis pathway, thus promoting anthocyanin biosynthesis in pepper leaves. These results provide a basis for future study of the mechanisms of light quality and photoperiod in anthocyanin synthesis and metabolism, and our study may serve as a valuable reference for screening light ratios that regulate anthocyanin biosynthesis in plants.

Transcriptome profiling of MYB-overexpressed transgenic lines provides crucial molecular insights into anthocyanin and remodel the biosynthesis regulatory network in Nicotiana tabacum

Anthocyanin is an important pigment in plants and one of the most powerful antioxidants for the food industry. In plants, anthocyanin biosynthesis is controlled by several structural genes and regulated by MYB transcription factors. However, the molecular mechanism and regulation of anthocyanin biosynthesis and the explicit role of MYB in pigmentation and stress responses have not been elucidated yet. In this study, we conducted transcriptomic profiling of MYB overexpressed transgenic lines, unveiling differentially expressed genes (DEGs) likely involved in anthocyanin biosynthesis and abiotic stress responses. Over-expression of two peanut MYB genes, Arahy.J3K16K (JK) and Arahy.CD1PG8 (C8), individually, as well as their co-expression (CJ), led to alterations in anthocyanin content in tobacco. Notably, JK over-expression line exhibited superior maintenance of growth indices, chlorophyll pigments, and anthocyanin content. Transcriptome profiling identified thousands of DEGs across JK, C8 and CJ transgenic plants. Functional annotation analysis revealed enrichment in metabolic pathways such as flavonoid biosynthesis, MAPK signaling, and hormone signaling transduction among these DEGs. Specifically, the expression of DEGs involved in the anthocyanin pathway, such as PAL, CHS, CHI, F3H, DFR, and ANR, was upregulated in JK lines, suggesting their regulatory role in pigmentation. Additionally, DEGs related to MAPK cascade, ethylene, auxin, and abscisic acid were preferentially expressed in JK lines, indicating their potential involvement in abiotic stress responses. The co-expression complex of MYB, bHLH, and WD40 also revealed the transcriptional activation model of anthocyanin biosynthesis in JK lines. In conclusion, these findings provide crucial molecular insights into MYB-induced regulation of anthocyanin biosynthesis and abiotic stress tolerance in tobacco, offering potential strategies for enhancing anthocyanin content and improving resistance.

Anthocyanin biosynthesis in goji berry is inactivated by deletion in a bHLH transcription factor LrLAN1b promoter.

Black goji berry (Lycium ruthenicum Murray) contains a rich source of health-promoting anthocyanins which are used in herbal medicine and nutraceutical foods in China. A natural variant producing white berries allowed us to identify two key genes involved in the regulation of anthocyanin biosynthesis in goji berries: one encoding a MYB transcription factor (LrAN2-like) and one encoding a basic helix-loop-helix (bHLH) transcription factor (LrAN1b). We previously found that LrAN1b expression was lost in the white berry variant, but the molecular basis for this phenotype was unknown. Here, we identified the molecular mechanism for loss of anthocyanins in white goji berries. In white goji, the LrAN1b promoter region has a 229 bp deletion that removes three MYB-binding elements and one bHLH-binding element, which are key to its expression. Complementation of the white goji berry LrAN1b allele with the LrAN1b promoter restored pigmentation. Virus-induced gene silencing of LrAN1b in black goji berry reduced fruit anthocyanin biosynthesis. Molecular analyses showed that LrAN2-like and another bHLH transcription factor LrJAF13 can activate LrAN1b by binding directly to the MYB-recognizing element and bHLH-recognizing element of its promoter-deletion region. LrAN1b expression is enhanced by the interaction of LrAN2-like with LrJAF13 and the WD40 protein LrAN11. LrAN2-like and LrAN11 interact with either LrJAF13 or LrAN1b to form two MYB-bHLH-WD40 complexes, which hierarchically regulate anthocyanin biosynthesis in black goji berry. This study on a natural variant builds a comprehensive anthocyanin regulatory network that may be manipulated to tailor goji berry traits.

Chromosome-scale genome, together with transcriptome and metabolome, provides insights into the evolution and anthocyanin biosynthesis of Rubus rosaefolius Sm. (Rosaceae).

Rubus rosaefolius is a kind of red raspberry possessing high nutritional and pharmaceutical value. Here we present a chromosome-level draft genome of R. rosaefolius. Of the total 131 assembled scaffolds, 70 with a total size of 219.02Mb, accounting for 99.33% of the estimated genome size, were anchored to seven pseudochromosomes. We traced a whole-genome duplication (WGD) event shared among members of the Rosaceae family, from which were derived 5090 currently detectable duplicated gene pairs (dgps). Of the WGD-dgps 75.09% underwent purifying selection, and approximately three-quarters of informative WGD-dgps expressed their two paralogs with significant differences. We detected a wide variety of anthocyanins in the berries of R. rosaefolius, and their total concentration remained relatively stable during berry development but increased rapidly during the ripening stage, mainly because of the contributions of pelargonidin-3-O-glucoside and pelargonidin-3-O-(6″-O-malonyl)glucoside. We identified many structural genes that encode enzymes, such as RrDFR, RrF3H, RrANS, and RrBZ1, and play key roles in anthocyanin biosynthesis. The expression of some of these genes significantly increased or decreased with the accumulation of pelargonidin-3-O-glucoside and pelargonidin-3-O-(6″-O-malonyl)glucoside. We also identified some transcription factors and specific methylase-encoding genes that may play a role in regulating anthocyanin biosynthesis by targeting structural genes. In conclusion, our findings provide deeper insights into the genomic evolution and molecular mechanisms underlying anthocyanin biosynthesis in berries of R. rosaefolius. This knowledge may significantly contribute to the targeted domestication and breeding of Rubus species.