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Wounding stress leads to anthocyanin accumulation. However, the underlying molecular mechanism remains elusive. In this study, MdWRKY40 was found to promote wounding-induced anthocyanin biosynthesis in association with MdMYB1 and undergo MdBT2-mediated degradation in apple. We found that MdMYB1, a positive regulator of anthocyanin biosynthesis, was essential for the wounding-induced anthocyanin biosynthesis in apple. MdWRKY40 was identified as an MdMYB1-interacting protein, and enhanced the binding of MdMYB1 to its target genes in response to wounding. We found that MdBT2 interacted physically with MdWRKY40 and was involved in its degradation through the 26S proteasome pathway. Our results demonstrate that MdWRKY40 is a key modulator in the wounding-induced anthocyanin biosynthesis, which provides new insights into the regulation of wounding-induced anthocyanin biosynthesis at both the transcriptional and post-translational levels in apple.

Key messageSunlight enhanced peel color and significantly up-regulated the expression of PyMYB10 and PybHLH genes. MYB-bHLH-WD40 transcriptional complex forms in the light and is involved in regulating anthocyanin accumulation in the peel.Anthocyanin is the major pigment in the peel of Yunnan red pear (Pyrus pyrifolia (Burm.) Nak.). A transcriptional activation protein complex, involving members of the transcription factor classes of MYB, bHLH and WD40, regulates anthocyanin biosynthesis. This complex was examined in the peel of red pear. In order to clarify the interaction of PyMYB10, PybHLH and PyWD40, fruit were bagged then peel samples collected 0, 3, 5, and 7 days after bag removal. Samples were used for Western blotting and protein interaction analysis. The results showed that sunlight enhanced peel color and significantly up-regulated the expression of both PyMYB10 and PybHLH genes. Co-immunoprecipitation (Co-IP) analysis showed that PybHLH interacted with PyMYB10 or PyWD40, and PyMYB10 interacted with PyWD40. Using onion cells as a model system, bimolecular fluorescence complementation (BiFC) confirmed these interactions and showed that the interaction localized to the nuclei. GST Pull down and Far-Western blotting assays demonstrated that PybHLH interacted with PyMYB10 or PyWD40, respectively, and PyMYB10 interacted with PyWD40 in vitro. In addition, EMSA assay showed that PyMYB10 can directly bind to the promoter of the gene encoding the anthocyanin biosynthesis enzyme anthocyanidin synthase (PyANS). Taken together, these results showed that the ternary complex of PyMYB10, PybHLH and PyWD40 transcription factors forms to regulate anthocyanin biosynthesis and accumulation in Yunnan red pear.

Main conclusion Our work strongly suggests that microRNA858 regulates anthocyanin biosynthesis in tomato by modulating the expression of two R2R3 MYB transcription factors, underscoring the importance of microRNAs in the gene regulatory network controlling specialized metabolism in plants. The biological functions of microRNA858 (miR858), a recently identified small RNA, are not well understood. Here, we identified miR858 as a negative regulator of anthocyanin biosynthesis in tomato (Solarium lycopersicum). RNA ligase-mediated 5'RACE cleavage assay showed that miR858 mediates the cleavage of SlMYB7-like and SlMYB48-like transcripts in tomato. Expression analysis revealed an inverse correlation between the accumulation of miR858 and its target SlMYB7-like mRNA, in different tissues of tomato. Subsequently, a small tandem target mimic construct for blocking miR858 (STTM858) was generated and transformed into tomato. The majority of endogenous miR858 was blocked in STTM858 over-expressing tomato plants, whereas SlMYB7-like transcripts increased significantly. Concomitantly, upregulated expression was detected for several anthocyanin biosynthetic genes, including PAL, CHS, DFR, ANS and 3GT. As a result, anthocyanins were highly accumulated in young seedlings, leaves, stems and leaf buds of the transgenic plants under normal growth conditions. In addition, overexpression of STTM858 in tomato activated another MYB transcription factor, S1MYB48, implicating the possible involvement of S1MYB48 in anthocyanin biosynthesis.

Anthocyanins are natural pigments with antioxidant effects that exist in various fruits and vegetables. The accumulation of anthocyanins is induced by environmental signals and regulated by transcription factors in plants. Numerous evidence has indicated that among the environmental factors, light is one of the most signal regulatory factors involved in the anthocyanin biosynthesis pathway. However, the signal transduction of light and molecular regulation of anthocyanin synthesis remains to be explored. Here, we focus on the research progress of signal transduction factors for positive and negative regulation in light-dependent and light-independent anthocyanin biosynthesis. In particular, we will discuss light-induced regulatory pathways and related specific regulators of anthocyanin biosynthesis in plants. In addition, an integrated regulatory network of anthocyanin biosynthesis controlled by transcription factors is discussed based on the significant progress.

Background Anthocyanin synthesis is affected by many factors, among which temperature is an important environmental factor. Eggplant is usually exposed to high temperatures during the cultivation season in Shanghai, China. Therefore,RNA -seq analysis was used to determine the effects of high-temperature stress on gene expression in the anthocyanin biosynthetic pathway of eggplant ( Solanum melongena L.). Results We tested the heat-resistant cultivar ‘Tewangda’. The plants were incubated at 38 °C and 45 °C, and the suitable temperature for eggplant growth was used as a control. The treatment times were 3 h and 6 h. The skin of the eggplant was taken for transcriptome sequencing, qRT-PCR assays and bioinformatic analysis. The results showed that 770 genes were differentially expressed between different treatments. Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analyses identified 16 genes related to anthocyanin biosynthesis, among which CHSB was upregulated. Other genes, including BHLH62, MYB380, CHI3, CHI, CCOAOMT, AN3, ACT-2, HST, 5MA-T1, CYP75A2, ANT17, RT, PAL2, and anthocyanin 5-aromatic acyltransferase were downregulated. In addition, the Myb family transcription factor PHL11 was upregulated in the CK 3 h vs 45 °C 3 h, CK 3 h vs 38 °C 3 h, and CK 6 h vs 38 °C 6 h comparisons, and the transcription factor bHLH35 was upregulated in the CK 3 h vs 38 °C 3 h and CK 6 h vs 38 °C 6 h comparisons. Conclusion These results indicated that high temperature will downregulate most of the genes in the anthocyanin biosynthetic pathway of eggplant. Our data have a reference value for the heat resistance mechanism of eggplant and can provide directions for molecular breeding of heat-resistant germplasm with anthocyanin content in eggplant.

Background Anthocyanins, which have important biological functions and have a beneficial effect on human health, notably account for pigmentation in purple-fleshed sweet potato tuberous roots. Individual regulatory factors of anthocyanin biosynthesis have been identified; however, the regulatory network of anthocyanin biosynthesis in purple-fleshed sweet potato is unclear. Results We functionally determined that IbMYB340 cotransformed with IbbHLH2 in tobacco and strawberry receptacles induced anthocyanin accumulation, and the addition of IbNAC56a or IbNAC56b caused increased pigmentation. Furthermore, we confirmed the interaction of IbMYB340 with IbbHLH2 and IbNAC56a or IbNAC56b via yeast two-hybrid and firefly luciferase complementation assays; these proteins could form a MYB340-bHLH2-NAC56a or MYB340-bHLH2-NAC56b transcriptional complex to regulate anthocyanin biosynthesis by binding to the IbANS promoter rather than the IbUFGT promoter. Furthermore, it was found by a transient expression system in tobacco leaves that IbMYB44 could decrease anthocyanin accumulation. Moreover, the interaction of IbMYB44 with IbMYB340 and IbNAC56a or IbNAC56b was verified. This result suggested that IbMYB44 acts as a repressor of anthocyanin in sweet potato. Conclusions The repressor IbMYB44 affected anthocyanin biosynthesis by competitively inhibiting the IbMYB340 - IbbHLH2 - IbNAC56a or IbMYB340 - IbbHLH2 - IbNAC56b regulatory complex formation. Overall, the present study proposed a novel regulatory network whereby several vital TFs play key roles in regulating anthocyanin biosynthesis, and it provides strong insight into the potential mechanism underlying anthocyanin biosynthesis in sweet potato tuberous roots with purple color.

Background MYB transcription factors regulate anthocyanin biosynthesis across numerous plant species. However, comprehensive genome-wide investigations regarding the R2R3-MYB gene family and its involvement in regulating anthocyanin biosynthesis in the red and white fruit color morphs of Fragaria pentaphylla remain scarce. Results A total of 101 FpR2R3-MYB genes were identified from the F. pentaphylla genome and were divided into 34 subgroups based on phylogenetic analysis. Gene structure (exon/intron) and protein motifs were particularly conserved among the FpR2R3-MYB genes, especially members within the same subgroup. The FpR2R3-MYB genes were distributed over seven F. pentaphylla chromosomes. Analysis of gene duplication events revealed five pairs of tandem duplication genes and 16 pairs of segmental duplication genes, suggesting that segmental duplications are the major pattern for expansion of the FpR2R3-MYB gene family expansion in F. pentaphylla . Cis-regulatory elements of the FpR2R3-MYB promoters were involved in cellular development, phytohormones, environmental stress and photoresponse. Based on the analysis of the FpR2R3-MYB gene family and transcriptome sequencing (RNA-seq) data, FpMYB9 was identified as a key transcription factor involved in the regulation of anthocyanin synthesis in F. pentaphylla fruits. The expression of FpMYB9 increases significantly during the ripening stage of red fruits, as confirmed by reverse transcription quantitative real-time PCR. In addition, subcellular localization experiments further confirmed the nuclear presence of FpMYB9, supporting its role as a transcription factor involved in anthocyanin biosynthesis. Conclusion Our results showed that the FpR2R3-MYB genes are highly conserved and play important roles in the anthocyanin biosynthesis in F. pentaphylla fruits. Our results also provide a compelling basis for further understanding of the regulatory mechanism underlying the role of FpMYB9 in anthocyanin formation in F. pentaphylla fruits.

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

Background Chinese cherry [ Cerasus pseudocerasus (Lindl.) G.Don] (syn. Prunus pseudocerasus Lindl.) is an economically important fruiting cherry species with a diverse range of attractive colors, spanning from the lightest yellow to the darkest black purple. However, the MYB transcription factors involved in anthocyanin biosynthesis underlying fruit color variation in Chinese cherry remain unknown. Results In this study, we characterized the R2R3-MYB gene family of Chinese cherry by genome-wide identification and compared it with those of 10 Rosaceae relatives and Arabidopsis thaliana . A total of 1490 R2R3-MYBs were classified into 43 subfamilies, which included 29 subfamilies containing both Rosaceae MYBs and AtMYBs . One subfamily (S45) contained only Rosaceae MYBs , while three subfamilies (S12, S75, and S77) contained only AtMYBs . The variation in gene numbers within identical subfamilies among different species and the absence of certain subfamilies in some species indicated the species-specific expansion within MYB gene family in Chinese cherry and its relatives. Segmental and tandem duplication events primarily contributed to the expansion of Chinese cherry R2R3-CpMYBs . The duplicated gene pairs underwent purifying selection during evolution after duplication events. Phylogenetic relationships and transcript profiling revealed that CpMYB10 and CpMYB4 are involved in the regulation of anthocyanin biosynthesis in Chinese cherry fruits. Expression patterns, transient overexpression and VIGS results confirmed that CpMYB10 promotes anthocyanin accumulation in the fruit skin, while CpMYB4 acts as a repressor, inhibiting anthocyanin biosynthesis of Chinese cherry. Conclusions This study provides a comprehensive and systematic analysis of R2R3-MYB gene family in Chinese cherry and Rosaceae relatives, and identifies two regulators, CpMYB10 and CpMYB4 , involved in anthocyanin biosynthesis in Chinese cherry. These results help to develop and utilize the potential functions of anthocyanins in Chinese cherry.