Promoted Anthocyanin Accumulation Research Articles

FveDREB1B improves cold tolerance of woodland strawberry by positively regulating FveSCL23 and FveCHS.

Cold stress has seriously inhibited the growth and development of strawberry during production. CBF/DREB1 is a key central transcription factor regulating plant cold tolerance, but its regulatory mechanisms are varied in different plants. Especially in strawberry, the molecular mechanism of CBF/DREB1 regulating cold tolerance is still unclear. In this study, we found that FveDREB1B was most significantly induced by cold stress in CBF/DREB1 family of diploid woodland strawberry. FveDREB1B was localized to the nucleus, and DREB1B sequences were highly conserved in diploid and octoploid strawberry, and even similar in Rosaceae. And FveDREB1B overexpressed strawberry plants showed delayed flowering and increased cold tolerance, while FveDREB1B silenced plants showed early flowering and decreased cold tolerance. Under cold stress, FveDREB1B activated FveSCL23 expression by directly binding to its promoter. Meanwhile, FveDREB1B and FveSCL23 interacted with FveDELLA, respectively. In addition, we also found that FveDREB1B promoted anthocyanin accumulation in strawberry leaves by directly activating FveCHS expression after cold treatment and recovery to 25°C. DREB1B genes were also detected to be highly expressed in cold-tolerant strawberry resources 'Fragaria mandschurica' and 'Fragaria nipponica'. In conclusion, our study reveals the molecular mechanism of FveDREB1B-FveSCL23-FveDELLA module and FveDREB1B-FveCHS module to enhance the cold tolerance of woodland strawberry. It provides a new idea for improving the cold tolerance of cultivated strawberry and evaluating the cold tolerance of strawberry germplasm resources.

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.

ZbMYB111 Expression Positively Regulates ZbUFGT-Mediated Anthocyanin Biosynthesis in Zanthoxylum bungeanum with the Involvement of ZbbHLH2.

Anthocyanin (ACN)-derived pigmentation in the red Zanthoxylum bungeanum peel is an essential commercial trait. Therefore, exploring the metabolic regulatory networks involved in peel ACN levels in this species is crucial for improving its quality. However, its underlying transcriptional regulatory mechanisms are still unknown. This transcriptomic and bioinformatics study not only discovered a new TF (ZbMYB111) as a potential regulator for ACN biosynthesis in Z. bungeanum peel, but also deciphered the underlying molecular mechanisms of ACN biosynthesis. Overexpression of ZbMYB111 and flavonoid 3-O-glucosyltransferase (ZbUFGT) induced ACN accumulation in both Z. bungeanum peels and callus along with Arabidopsis thaliana and tobacco flowers, whereas their silencing impaired ACN biosynthesis. Therefore, the dual-luciferase reporter, yeast-one-hybrid, and electrophoretic mobility shift assays showed that ZbMYB111 directly interacted with the ZbUFGT promoter to activate its expression. This diverted the secondary metabolism toward the ACN pathway, thereby promoting ACN accumulation.

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.

AcMYB96 promotes anthocyanin accumulation in onion (Allium cepa L) without forming the MBW complex

AcMYB96 promotes anthocyanin accumulation in onion (Allium cepa L) without forming the MBW complex

The AnUFGT1 Is Involved in the Anthurium ‘Alabama’ Anthocyanidin Deficiency

Anthurium is the second largest tropical flower crop in the world. The international market has urgent demand for anthurium varieties with different spathe colors, which mainly arises from the types and contents of anthocyanin. The flavonoid 3-O-glycosyltransferase (UF3GT) gene is the key enzyme involved in promoting anthocyanin accumulation through glycosylation downstream of the anthocyanin synthesis pathway (ASP). Abnormal functioning of UFGT usually results in a reduction in or loss of anthocyanins. The aim of this study was to reveal the role of one anthurium UFGT gene (AnUFGT1) in ‘Xueyu’ (X), an anthocyanin-deficient mutant of ‘Alabama’. Metabolome analysis was used to analyze the metabolic products in the ASP to determine the possible key link of the anthocyanin deletion mutation. Agrobacterium-mediated transformation of Arabidopsis UFGT functionally deficient mutant (ufgt) and ‘X’ validated the function of AnUFGT1. The results of comparative metabolome analysis of ‘X’ and ‘Alabama’ showed that there was no significant difference in product levels upstream of ASP. The expression levels of AnUFGT1 were significantly greater in ‘Alabama’ than in ‘X’. The overexpression of AnUFGT1 in ufgt significantly increased its anthocyanin contents. The overexpression of AnUFGT1 in ‘X’, mediated by a new injection method, can only promote the synthesis of trace anthocyanins. These results showed that AnUFGT1 could fully compensate the phenotype of ufgt, but only partially compensate the anthocyanidin-deficient phenotype of anthurium mutant X. This difference suggested that anthocyanin-deletion mutations in anthurium ‘X’ are associated with AnUFGT1, but AnUFGT1 is not the only factor. There should be other factors interacting with AnUFGT1 that cause anthocyanin deficiency.

Melatonin foliar application promoted anthocyanin accumulation boosted reactive oxygen scavenging by upregulating the key enzymes activities and genes expression of anthocyanin biosynthesis pathway in green and purple mustard under vanadium stress

Heavy metal vanadium (V) is poisonous to both plants and animals. Plant resistance to heavy metals stress can be conferred by the multifunctional signaling molecule melatonin, although the underlying mechanisms are still largely understood. We elucidated the significant contribution of the secondary metabolite anthocyanin to the enhancement of V stress tolerance by melatonin in both green and purple mustard genotypes. The effects of excessive V were leaf yellowing, slowed development, decreased photosynthetic activity, and an increase in the buildup of reactive oxygen species and malondialdehyde in both mustard genotypes. It interesting to note that during V stress, key anthocyanin biosynthetic pathway enzymes activities and leaf anthocyanin concentration increased while exogenous melatonin applied as foliar further boosted activities of key anthocyanin biosynthetic pathway enzymes and leaf anthocyanin concentration in both green and purple mustards. Furthermore, the application of exogenous melatonin increased the levels of endogenous melatonin and mitigated V toxicity. Conversely, the suppression of melatonin intensified V-induced phytotoxicity and delayed the accumulation of anthocyanin by downregulating the transcript levels of essential structural genes. Under V stress conditions, the genes expression related to anthocyanin production, antioxidant enzymes activities, and stress resistance was significantly higher in purple mustard than green mustard genotype. Additionally, melatonin decreased V deposition in roots and leaves. The results recommended that melatonin foliar application to mustard plants inhibited the availability of V to plant and promote V stress tolerance.

JrATHB-12 mediates JrMYB113 and JrMYB27 to control the anthocyanin levels in different types of red walnut

Red walnut has broad market prospects because it is richer in anthocyanins than ordinary walnut. However, the mechanism driving anthocyanin biosynthesis in red walnut is still unknown. We studied two types of red walnut, called red walnut 1 (R1), with a red pericarp and seed coat, and red walnut 2 (R2), with a red seed coat only. R1 mostly contained cyanidin-3-O-galactoside, while R2 contained a various amounts of cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside, and cyanidin-3-O-glucoside. The LDOX-2 (LOC109007163) and LDOX-3 (LOC109010746) genes, which encode leucoanthocyanidin dioxygenase/anthocyanidin synthase (LDOX/ANS), were preliminarily indicated as the crucial genes for anthocyanin biosynthesis in R1 and R2, respectively. The MYB differential genes analysis showed that MYB27 and MYB113 are specifically expressed in the red parts of R1 and R2, respectively, and they are regarded as candidate regulatory genes. Ectopic expression in Arabidopsis and transient injection in walnut showed that both MYB27 and MYB113 were located in the nucleus and promoted anthocyanin accumulation, while MYB27 promoted the expression of LDOX-2, and MYB113 promoted the expression of LDOX-3 and UAGT-3. Yeast one-hybrid and electrophoretic mobility shift assays showed that MYB27 could only bind to the LDOX-2 promoter, while MYB113 could bind to the promoters of both LDOX-3 and UAGT-3. In addition, we also identified an HD-Zip transcription factor, ATHB-12, which is specifically expressed in the pericarp. After silencing the expression of ATHB-12, the R2 pericarp turned red, and MYB113 expression increased. Further experiments showed that ATHB-12 could specifically interact with MYB113 and bind to its promoter. This suggests that MYB27 controls R1 coloration by regulating LDOX-2, while MYB113 controls R2 coloration by regulating LDOX-3 and UAGT-3, but ATHB-12 can specifically bind to and inhibit the MYB113 of the R2 pericarp so that it becomes unpigmented. This study reveals the anthocyanin biosynthetic mechanisms in two different types of red walnut and provides a scientific basis for the selection and breeding of red walnut varieties.

The MpNAC72/MpERF105-MpMYB10b module regulates anthocyanin biosynthesis in Malus 'Profusion' leaves infected with Gymnosporangium yamadae.

Apple rust is a serious fungal disease affecting Malus plants worldwide. Infection with the rust pathogen Gymnosporangium yamadae induces the accumulation of anthocyanins in Malus to resist rust disease. However, the mechanism of anthocyanin biosynthesis regulation in Malus against apple rust is still unclear. Here, we show that MpERF105 and MpNAC72 are key regulators of anthocyanin biosynthesis via the ethylene-dependent pathway in M. 'Profusion' leaves under rust disease stress. Exogenous ethephon treatment promoted high expression of MpERF105 and MpNAC72 and anthocyanin accumulation in G. yamadae-infected M. 'Profusion' leaves. Overexpression of MpERF105 increased the total anthocyanin content of Malus plant material and acted by positively regulating its target gene, MpMYB10b. MpNAC72 physically interacted with MpERF105 in vitro and in planta, and the two form a protein complex. Coexpression of the two leads to higher transcript levels of MpMYB10b and higher anthocyanin accumulation. In addition, overexpression of MpERF105 or MpNAC72 enhanced the resistance of M. 'Profusion' leaves to apple rust. In conclusion, our results elucidate the mechanism by which MpERF105 and MpNAC72 are induced by ethylene in G. yamadae-infected M. 'Profusion' leaves and promote anthocyanin accumulation by mediating the positive regulation of MpMYB10b expression.

Exogenous Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression in Blueberry Leaves

Blueberry (Vaccinium corymbosum) leaves have a positive influence on health because of their phenolic contents, including anthocyanins. Phytohormone abscisic acid (ABA) promotes anthocyanin accumulation, but the underlying mechanisms are unclear in blueberry leaves. In this study, we found that exogenous ABA promotes anthocyanin accumulation in blueberry leaves and we explored the global molecular events involved in these physiological changes by treating in vitro-grown blueberry seedlings with ABA and performing transcriptome deep sequencing (RNA-seq). We identified 6390 differentially expressed genes (DEGs), with 2893 DEGs at 6 h and 4789 at 12 h of ABA treatment compared to the control. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to plant hormone signal transduction and phenylpropanoid and flavonoid biosynthesis were significantly enriched at both stages of the ABA treatment. Analysis of DEGs in plant hormone signal transduction pathways revealed that exogenous ABA affected the expression of genes from other plant hormone signaling pathways, especially brassinosteroid, auxin, and gibberellin signaling. To elucidate the mechanism driving anthocyanin biosynthesis in blueberry in response to ABA treatment, we screened anthocyanin biosynthesis structural genes (ASG) from the phenylpropanoid and flavonoid biosynthetic pathways, MYB transcription factor genes from R2R3-MYB subgroups 5, 6, and 7 and ABRE-binding factor (ABF) genes from the ABA signal transduction pathway. Pearson’s correlation coefficient (r) analysis indicated that the ABFs, MYBs, and structural genes form a network to regulate ABA-induced anthocyanin biosynthesis and MYBA1 is likely to play an important role in this regulatory network. These findings lay the foundation for improving anthocyanin biosynthesis in blueberry leaves.

MdWER interacts with MdERF109 and MdJAZ2 to mediate methyl jasmonate- and light-induced anthocyanin biosynthesis in apple fruit.

Anthocyanin generation in apples (Malus domestica) and the pigmentation that results from it may be caused by irradiation and through administration of methyl jasmonate (MeJA). However, their regulatory interrelationships associated with fruit coloration are not well defined. To determine whether MdERF109, a transcription factor (TF) involved in light-mediated coloration and anthocyanin biosynthesis, has synergistic effects with other proteins, we performed a yeast two-hybrid assessment and identified another TF, MdWER. MdWER was induced by MeJA treatment, and although overexpression of MdWER alone did not promote anthocyanin accumulation co-overexpression with MdERF109 resulted in significantly increase in anthocyanin biosynthesis. MdWER may form a protein complex with MdERF109 to promote anthocyanin accumulation by enhancing combinations between the proteins and their corresponding genes. In addition, MdWER, as a MeJA responsive protein, interacts with the anthocyanin repressor MdJAZ2. Transient co-expression in apple fruit and protein interaction assays allowed us to conclude that MdERF109 and MdJAZ2 interact with MdWER and take part in the production of anthocyanins upon MeJA treatment and irradiation. Our findings validate a role for the MdERF109-MdWER-MdJAZ2 module in anthocyanin biosynthesis and uncover a novel mechanism for how light and MeJA signals are coordinated anthocyanin biosynthesis in apple fruit.

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.

The Impact of Different Nitrogen Levels on the Tuber Yield and Anthocyanin Synthesis of Purple Potatoes

The biosynthesis of anthocyanins is influenced by external environmental conditions such as light, temperature, and nitrogen level, with nitrogen level being a key factor in anthocyanin synthesis and accumulation. Nitrogen level regulates the transcription factors involved in the anthocyanin synthesis pathway, with low nitrogen levels promoting anthocyanin accumulation, while high nitrogen levels have the opposite effect. Purple potatoes are a type of cultivated crop that is rich in anthocyanins and has unique economic value. Nitrogen fertilizer is crucial to improve the agronomic traits, yield, quality, and anthocyanin content of purple potatoes. In this study, the impact of four different nitrogen concentrations—0 kg/hm2 (N0), 90 kg/hm2 (N1), 225 kg/hm2 (N2) and 360 kg/hm2 (N3)—on the agronomic traits, yield, quality, and anthocyanin content of purple potatoes, ‘Huasong 66’, at different stages were investigated by using physiological index measurement and RNA-seq technology. It was found that the purple potato ‘Huasong 66’ was more sensitive to low nitrogen (N1). Under N1 level of nitrogen fertilization, ‘Huasong 66’ possessed the finest agronomic traits, yield, and quality, and the total anthocyanins in the tubers were significantly increased. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that nitrogen levels in purple potato tubers primarily affect genes related to nutrient transport and metabolism by regulating carbon and nitrogen metabolism, enzyme catalysis and binding, and signal transduction. In addition, nine candidate genes related to the anthocyanin synthesis pathway had been preliminarily screened. These results provide a basis to understand the impact of different nitrogen levels on the tuber yield and anthocyanin synthesis of purple potatoes.

MiR156b-targeted VvSBP8/13 functions downstream of the abscisic acid signal to regulate anthocyanins biosynthesis in grapevine fruit under drought.

Anthocyanins are the primary color components of grapevine berries and wines. In cultivation practices, a moderate water deficit can promote anthocyanin accumulation in red grape skins. Our previous study showed that abscisic acid (ABA) plays a key role in this process. Herein, we identified a microRNA, vv-miR156b, that is generated in grapevine berries in response to drought stress, along with increasing anthocyanin content and biosynthetic structural gene transcripts. In contrast, vv-miR156b short tandem target mimic (STTM) function-loss callus exhibits the opposite phenotype. Results from in vivo and in vitro experiments revealed that the ABA-signaling-regulated transcription factor VvAREB2 binds directly to the ABA-responsive element (ABRE) of the MIR156b promoter and activates miR156b expression. Furthermore, two miR156b downstream targets, VvSBP8 and VvSBP13, exhibited reduced grape anthocyanin content in their overexpressors but there was a contrary result in their CRISPR-edited lines, the decrease in anthocyanin content was rescued in miR156b and SBP8/13 double overexpressors. We further demonstrated that both VvSBP8 and VvSBP13, encoding transcriptional repressors, displayed sufficient ability to interact with VvMYC1 and VvMYBA1, thereby interfering with MYB-bHLH-WD (MBW) repeat transcriptional complex formation, resulting in the repression of anthocyanin biosynthesis. Our findings demonstrate a direct functional relationship between ABA signaling and the miR156-SBP-MBW complex regulatory module in driving drought-induced anthocyanin accumulation in grape berries.

A light responsive transcription factor CsbHLH89 positively regulates anthocyanidin synthesis in tea (Camellia sinensis)

Anthocyanins are a class of secondary metabolites with rich physiological functions and many benefits for the human body. Although there have been numerous reports on anthocyanin synthesis in tea plants, the mechanism by which bHLH transcription factors regulate anthocyanin synthesis in tea plants is still unclear. In this study, we identified CsbHLH89, a basic helix–loop–helix (bHLH) transcription factor that is located in the nucleus and cytoplasm and has transcriptional activation activity. CsbHLH89 directly binds to G-box elements and activates the CsCHS, CsFLS, and CsDFR promoters in vitro and in vivo. Further experiments demonstrated that CsbHLH89 could be involved in the synthesis of tea plant anthocyanins as homodimers or heterodimers. Our research results show that CsbHLH89 can positively regulate anthocyanin synthesis in tea. In addition, it was found that CsHY5, a light signal transduction factor, could bind to the promoter of CsbHLH89 to indirectly promote anthocyanin accumulation. Silencing of CsHY5 and CsbHLH89 by antisense oligonucleotide, respectively, revealed that the expression of structural genes and transcription factors were down-regulated. Shading treatment of tea plants revealed a significant reduction in anthocyanin content and down-regulation of the expression of genes involved in anthocyanin synthesis. Our research provides new insight into the regulatory network of anthocyanin synthesis in tea and a new strategy for breeding rich anthocyanin.

Metabolomic and Transcriptomic Insights into Anthocyanin Biosynthesis in ‘Ziyan’ Tea Plants under Varied Photoperiod and Temperature Conditions

(1) Background: Anthocyanins, the main pigments in plants, are influenced by both temperature and photoperiod. However, the specific mechanisms underlying anthocyanin accumulation in tea plants in response to these two environmental factors remain unclear. (2) Methods: This study subjected potted ‘Ziyan’ tea plants to four types of day-length and temperature treatments (HL (28 °C, 16 h), HS (28 °C, 8 h), LL (18 °C, 16 h), and LS (18 °C, 8 h)), and then conducted targeted metabolomic and transcriptomic analyses of the samples. (3) Results: Long photoperiods and low temperatures both promoted anthocyanin accumulation in the new shoots of the tea plants. Furthermore, the enhancing effects of these two environmental factors on anthocyanin accumulation are additive and exhibit interactive effects. Through a combined analysis of metabolomics and transcriptomics, five key differentially accumulated metabolites (DAMs) and twenty-two key differentially expressed genes (DEGs) were identified, the latter being found to participate in the regulation of anthocyanin biosynthesis pathways under varying light and temperature conditions. In summary, extended photoperiods primarily increase the content levels of ten metabolites, including cyanidin and naringenin-7-O-glucoside, by upregulating CHS, F3H, and ANS genes. In contrast, low temperatures primarily enhance the synthesis of seven anthocyanins, including cyanidin and cyanidin-3-O-rutinoside, by upregulating the ANS and UFGT genes. (4) Conclusions: Collectively, the differences in the expression levels of CHS, F3H, ANS, and UFGT might be responsible for ‘Ziyan’ tea plants’ purple shoot coloration, providing important data towards the discovery of candidate genes and molecular bases controlling the purple leaves of these tea plants under varied photoperiods and temperatures.

Transcription factor MdNAC33 is involved in ALA-induced anthocyanin accumulation in apples

5-Aminolevulinic acid (ALA), as a new natural plant growth regulator, has a significant function in promoting anthocyanin accumulation in many species of fruits. However, the mechanisms underlying remain obscure. In a transcriptome study of our group, it was found that many transcription factors (TFs) including NACs responsive to ALA treatment during anthocyanin accumulation. In the present study, we found a NAC of apple, MdNAC33 was coordinatively expressed with anthocyanin accumulation after ALA treatment in the apple fruits and leaves, suggesting that this TF may be involved in anthocyanin accumulation induced by ALA. We found that the MdNAC33 protein was localized in the nucleus and exhibited strong transcriptional activity in both yeast cells and plants, where its C-terminal contributed to the transcriptional activity. Functional analysis showed that overexpression of MdNAC33 promoted the accumulation of anthocyanin, while the silencing vector of MdNAC33 (RNAi) significantly impaired the anthocyanin accumulation induced by ALA. Yeast one-hybrid (Y1H), luciferase assay and electrophoretic mobility shift assay (EMSA) indicated that MdNAC33 could bind to promoters of MdbHLH3, MdDFR and MdANS to activate the gene expressions. In addition, MdNAC33 specifically interacts with MdMYB1, a positive regulator of anthocyanin biosynthesis, which was then in turn binding to its target genes MdUFGT and MdGSTF12, to promote anthocyanin accumulation in apples. Taken together, our data indicate that MdNAC33 plays multiple roles in ALA-induced anthocyanin biosynthesis. It provides new insights into the mechanisms of anthocyanin accumulation induced by ALA.

The Characterization of R2R3-MYB Genes in Ammopiptanthus nanus Uncovers That the miR858-AnaMYB87 Module Mediates the Accumulation of Anthocyanin under Osmotic Stress.

R2R3-MYB transcription factors (TFs) participate in the modulation of plant development, secondary metabolism, and responses to environmental stresses. Ammopiptanthus nanus, a leguminous dryland shrub, tolerates a high degree of environmental stress, including drought and low-temperature stress. The systematic identification, structural analysis, evolutionary analysis, and gene profiling of R2R3-MYB TFs under cold and osmotic stress in A. nanus were performed. Up to 137 R2R3-MYB TFs were identified and clustered into nine clades, with most A. nanus R2R3-MYB members belonging to clade VIII. Tandem and segmental duplication events drove the expansion of the A. nanus R2R3-MYB family. Expression profiling revealed that multiple R2R3-MYB genes significantly changed under osmotic and cold stress conditions. MiR858 and miR159 targeted 88 R2R3-MYB genes. AnaMYB87, an miR858-targeted clade VIII R2R3-MYB TF, was up-regulated under both osmotic and cold stress. A transient expression assay in apples showed that the overexpression of AnaMYB87 promoted anthocyanin accumulation. A luciferase reporter assay in tobacco demonstrated that AnaMYB87 positively affected the transactivation of the dihydroflavonol reductase gene, indicating that the miR858-MYB87 module mediates anthocyanin accumulation under osmotic stress by regulating the dihydroflavonol reductase gene in A. nanus. This study provides new data to understand the roles of R2R3-MYB in plant stress responses.

MdMYB66 Is Associated with Anthocyanin Biosynthesis via the Activation of the MdF3H Promoter in the Fruit Skin of an Apple Bud Mutant

Skin color is an important trait that is mainly determined by the content and composition of anthocyanins in apples. In this study, a new bud mutant (RM) from ‘Oregon Spur II’ (OS) of Red Delicious apple was obtained to reveal the mechanism underlying red color formation. Results showed that the total anthocyanin content in RM was significantly higher than that in OS with the development of fruit. Through widely-targeted metabolomics, we found that cyanidin-3-O-galactoside was significantly accumulated in the fruit skin of RM. Transcriptome analysis revealed that the structural gene MdF3H and MdMYB66 transcription factor were significantly up-regulated in the mutant. Overexpression of MdMYB66 in apple fruit and apple callus significantly promoted anthocyanin accumulation and significantly increased the expression level of MdMYB66 and structural genes related to anthocyanin synthesis. Y1H and LUC analysis verified that MdMYB66 could specifically bind to the promoter of MdF3H. The results of the double luciferase activity test showed that MdMYB66 activated MdF3H 3.8 times, which led to increased anthocyanin contents. This might explain the phenotype of red color in RM at the early stage. Taken together, these results suggested that MdMYB66 was involved in regulating the anthocyanin metabolic pathways through precise regulation of gene expression. The functional characterization of MdMYB66 provides insight into the biosynthesis and regulation of anthocyanins.

DlMYB113 mutation affects anthocyanin accumulation in red pericarp longan (Dimocarpus longan Lour.)

Fruit color influences fruit quality and commodity value. Most longan (Dimocarpus longan Lour.) varieties have a yellowish-brown or grayish-yellow pericarp, and the discovery of red pericarp (RP) longan expanded the color varieties of longan fruit. Our previous research showed that the red pericarp of RP fruit was mainly caused by anthocyanin accumulation; however, its underlying regulatory mechanism remains unknown. Herein, DlMYB113, an R2R3-MYB transcription factor, was discovered by examining differentially expressed genes in two longan cultivars. Dimocarpus longan MYB (v-myb avian myeloblastosis viral) 113 (DlMYB113) expression was significantly higher in the pericarp and leaves of RP longan than in ‘Shixia’ longan. Sequence alignment analysis revealed two amino acid substitutions in the R3 domain between DlMYB113rp in RP longan and DlMYB113sx in ‘Shixia’ longan. Transient expression of DlMYB113rp significantly increased anthocyanin accumulation in tobacco leaves, whereas DlMYB113sx had negligible effect. Meanwhile, DlMYB113 overexpression promotes anthocyanin accumulation in Arabidopsis and longan calli. Site-directed mutation detection revealed divergence in DlMYB113 function when the R3 repeat 197-position base T was replaced with G, and the 317- and 318-position AT bases were replaced with GA. Our findings indicate that DlMYB113 can regulate anthocyanin production in RP longan, and three mutations in its nucleic acid sequence lead to anthocyanin accumulation, thereby developing molecular markers associated with the anthocyanin accumulation trait in RP longan. This study will facilitate early screening of longan hybrids with desirable fruit color and be significant for breeding new characteristic varieties.