R2R3-MYB Transcription Factor NtMYB330 Regulates Proanthocyanidin Biosynthesis and Seed Germination in Tobacco (Nicotiana tabacum L.)

Proanthocyanidins (PAs) are important phenolic compounds and PA biosynthesis is regulated by a ternary MBW complex consisting of a R2R3-MYB regulator, a bHLH factor and a WDR protein. In this study, a tobacco R2R3-MYB factor NtMYB330 was characterized as the PA-specific regulator in which the PA biosynthesis was promoted in the flowers of NtMYB330-overexpressing lines while decreased in the flowers of ntmyb330 mutants. NtMYB330 can interact with flavonoid-related bHLH partner NtAn1b and WDR protein NtAn11-1, and the NtMYB330-NtAn1b complex is required to achieve strong transcriptional activation of the PA-related structural genes NtDFR1, NtANS1, NtLAR1 and NtANR1. Our data reveal that NtMYB330 regulates PA biosynthesis in seeds and affects seed germination, in which NtMYB330-overexpressing lines showed higher PA accumulations in seed coats and inhibited germination, while ntmyb330 mutants had reduced seed coat PAs and improved germination. NtMYB330 affects seed germination possibly through two mechanisms: modulating seed coat PAs to affect coat-imposed dormancy. In addition, NtMYB330 regulates the expressions of abscisic acid (ABA) and gibberellin acid (GA) signaling-related genes, affecting ABA-GA crosstalk and seed germination. This study reveals that NtMYB330 specifically regulates PA biosynthesis via formation of the MBW complex in tobacco flowers and affects germination through adjustment of PA concentrations and ABA/GA signaling in tobacco seeds.

Regulation of flavonoid biosynthesis in representatives of the tribe Phaseoleae DC.

Flavonoids play a crucial role in plant metabolism. Many of them have antioxidant activity, and they are also pigments that render a variety of colors to plant tissues. Foods rich in flavonoid compounds are considered as functional components of a healthy diet. Currently, there is an increased interest in studying genetic mechanisms underlying the coloration of plants. Flavonoid biosynthesis pathways are controlled by two groups of genes. Structural genes encode enzymes, while regulatory genes are responsible for transcription factors that activate the expression of structural genes. Transcription factors that belong to R2R3-Myb, bHLH-Myc and WDR families form the ternary MBW complex, which is involved in regulating the expression of structural genes of flavonoid biosynthesis. The mechanisms of regulation of the anthocyanins and proanthocyanidin biosynthesis by the MBW complex are described in detail for the model plant Arabidopsis thaliana L. This review summarizes data on the regulation of phenolic pigment biosynthesis and the features of phenolic pigment accumulation in plant tissues in the main representatives of the Phaseoleae tribe: soybean Glycine max (L.) Merr., common bean Phaseolus vulgaris L., adzuki bean Vigna angularis (Willd.) Ohwi & Ohashi, and cowpea V. unguiculata (L.) Walp. The species discussed in this review are the most important food legumes in many countries of the world and they comprise the staple food in diets of millions of people. Identification and characterization of the genes controlling the flavonoid biosynthesis pathways are necessary for successful breeding of modern varieties with an increased dietary value. Identification of the flavonoid accumulation patterns is essential for solving the problem of broadening the diversity of plant products.

Identification of MBW Complex Components Implicated in the Biosynthesis of Flavonoids in Woodland Strawberry

Flavonoids belong to the family of polyphenolic secondary metabolites and contribute to fruit quality traits. It has been shown that MBW complexes (MYB-bHLH-WD40) regulate the flavonoids biosynthesis in different plants, but only a limited number of MBW complexes have been identified in strawberry species in general. In this study, we identified 112 R2R3-MYB proteins in woodland strawberry; 12 of them were found to have potential functions in regulating flavonoids biosynthesis by phylogenetic analysis. qRT-PCR assays showed that FvMYB3, FvMYB9, FvMYB11, FvMYB22, FvMYB64, and FvMYB105 mostly expressed at green stage of fruit development, aligned with proanthocyanidins accumulation; FvMYB10 and FvMYB41 showed higher expression levels at turning and ripe stages, aligned with anthocyanins accumulation. These results suggest that different MYBs might be involved in flavonoids biosynthesis at specific stages. Furthermore, FvMYB proteins were demonstrated to interact with FvbHLH proteins and induce expression from the promoters of CHS2 and DFR2 genes, which encode key enzymes in flavonoids biosynthesis. The co-expression of FvMYB and FvbHLH proteins in strawberry fruits also promoted the accumulation of proanthocyanidins. These findings confirmed and provided insights into the biofunction of MBW components in the regulation of flavonoid biosynthesis in woodland strawberry.

Conserved and non-conserved functions of the rice homologs of the Arabidopsis trichome initiation-regulating MBW complex proteins

Background Trichome initiation in Arabidopsis is regulated by a MYB-bHLH-WD40 (MBW) transcriptional activator complex formed by the R2R3 MYB transcription factor GLABRA1 (GL1), MYB23 or MYB82, the bHLH transcription factor GLABRA3 (GL3), ENHANCER OF GLABRA3 (EGL3) or TRANSPARENT TESTA8 (TT8), and the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1). However, the functions of the rice homologs of the MBW complex proteins remained uncharacterized. Results Based on amino acid sequence identity and similarity, and protein interaction prediction, we identified OsGL1s, OsGL3s and OsTTG1s as rice homologs of the MBW complex proteins. By using protoplast transfection, we show that OsGL1D, OsGL1E, OsGL3B and OsTTG1A were predominantly localized in the nucleus, OsGL3B functions as a transcriptional activator and is able to interact with GL1 and TTG1. By using yeast two-hybrid and protoplast transfection assays, we show that OsGL3B is able to interact with OsGL1E and OsTTG1A, and OsGL1E and OsTTG1A are also able to interact with GL3. On the other hand, we found that OsGL1D functions as a transcription activator, and it can interact with GL3 but not OsGL3B. Furthermore, our results show that expression of OsTTG1A in the ttg1 mutant restored the phenotypes including alternations in trichome and root hair formation, seed color, mucilage production and anthocyanin biosynthesis, indicating that OsTTG1A and TTG1 may have similar functions. Conclusion These results suggest that the rice homologs of the Arabidopsis MBW complex proteins are able to form MBW complexes, but may have conserved and non-conserved functions.

Conserved and None-Conserved Functions of the Rice Homologs of the Arabidopsis Trichome Initiation-Regulating MBW Complex Proteins

Background: Trichome initiation in Arabidopsis is regulated by a MYB-bHLH-WD40 (MBW) transcriptional activator complex formed by the R2R3 MYB transcription factor GLABRA1 (GL1), MYB23 or MYB82, the bHLH transcription factor GLABRA3 (GL3), ENHANCER OF GLABRA3 (EGL3) or TRANSPARENT TESTA8 (TT8), and the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1). However, the functions of the rice homologs of the MBW complex proteins remained uncharacterized. Results: Based on amino acid sequence identity and similarity, and protein interaction prediction, we identified OsGL1s, OsGL3s and OsTTG1s as rice homologs of the MBW complex proteins. By using protoplast transfection, we show that OsGL1D, OsGL1E, OsGL3B and OsTTG1A were predominantly localized in the nucleus, OsGL3B functions as a transcriptional activator and is able to interact with GL1 and TTG1. By using yeast two hybridization and protoplast transfection assays, we show that OsGL3B is able to interact with OsGL1E and OsTTG1A, and OsGL1E and OsTTG1A are also able to interact with GL3. On the other hand, we found that OsGL1D functions as a transcription activator, and it can interact with GL3 but not OsGL3B. These results suggest that rice homologs of the Arabidopsis MBW complex proteins are able to form MBW complexes, but may have conserved and none-conserved functions. Furthermore, we found that expression of OsTTG1A in the ttg1 mutant restored the phenotypes including alternations in trichome and root hair formation, seed color, mucilage production and anthocyanin biosynthesis, indicating that OsTTG1A and TTG1 may have similar functions. Conclusion: These results aid in our understanding of the mechanism of trichome initiation in rice.

MYB repressors and MBW activation complex collaborate to fine-tune flower coloration in Freesia hybrida

Floral anthocyanin has multiple ecological and economic values, its biosynthesis largely depends on the conserved MYB-bHLH-WD40 (MBW) activation complex and MYB repressors hierarchically with the MBW complex. In contrast to eudicots, the MBW regulatory network model has not been addressed in monocots because of the lack of a suitable system, as grass plants exhibit monotonous floral pigmentation patterns. Presently, the MBW regulatory network was investigated in a non-grass monocot plant, Freesia hybrida. FhMYB27 and FhMYBx with different functional manners were confirmed to be anthocyanin related R2R3 and R3 MYB repressors, respectively. Particularly, FhMYBx could obstruct the formation of positive MBW complex by titrating bHLH proteins, whereas FhMYB27 mainly defected the activator complex into suppressor via its repression domains in C-terminus. Furthermore, the hierarchical and feedback regulatory loop was verified, indicating the synergistic and sophisticated regulatory network underlying Freesia anthocyanin biosynthesis was quite similar to that reported in eudicot plants.

The Conserved and Particular Roles of the R2R3-MYB Regulator FhPAP1 from Freesia hybrida in Flower Anthocyanin Biosynthesis

Anthocyanin biosynthesis is mainly controlled by MYB–bHLH–WD40 (MBW) complexes that modulate the expression of anthocyanin biosynthetic genes (ABGs). The MYB regulators involved in anthocyanin biosynthesis arose early during plant evolution and thus might function divergently in different evolutionary lineages. Although the anthocyanin-promoting R2R3-MYB regulators in eudicots have been comprehensively explored, little consensus has been reached about functional discrepancies versus conservation among MYB regulators from different plant lineages. Here, we integrated transcriptome analysis, gene expression profiles, gain-of-function experiments and transient protoplast transfection assays to functionally characterize the monocot Freesia hybrida anthocyanin MYB regulator gene FhPAP1, which showed correlations with late ABGs. FhPAP1 could activate ABGs as well as TT8-clade genes FhTT8L, AtTT8 and NtAN1 when overexpressed in Freesia, Arabidopsis and tobacco, respectively. Consistently, FhPAP1 could interact with FhTT8L and FhTTG1 to form the conserved MBW complex and shared similar target genes with its orthologs from Arabidopsis. Most prominently, FhPAP1 displayed higher transactivation capacity than its homologs in Arabidopsis and tobacco, which was instantiated in its powerful regulation on ABGs. Moreover, we found that FhPAP1 might be the selected gene during the domestication and rapid evolution of the wild Freesia species to generate intensive flower pigmentation. These results showed that while the MBW complex was highly evolutionarily conserved between tested monocot and core eudicot plants, participating MYB regulators showed functional differences in transactivation capacity according to their activation domain and played important roles in the flower coloration domestication and evolution of angiosperms.

Transcriptome and metabolome reveal redirection of flavonoids in a white testa peanut mutant

Background: Coat color determines both appearance and nutrient quality of peanut. White seed coat in peanut can enhance the processing efficiency and quality of peanut oil.An integrative analysis of transcriptomes, metabolomes and histocytology was performed on wsc mutant and its wild type to investigate the regulatory mechanisms underlying color pigmentation. Result：Metabolomes revealed flavonoids were redirected in wsc, while multi-omics analyses of wsc mutant seeds and testae uncovered WSC influenced the flavonoids biosynthesis in testa as well as suberin formation, glycolysis, the TCA cycle and amino acid metabolism. The mutation also enhanced BR, GA, and JA biosynthesis as well as ABA, AUX, BR and JA signaling. Further, co-expression analysis showed that FLS genes co-expressed with MBW complex member genes. Combining tissue expression patterns, genetic analyses, and the annotation of common DEGs for these three stages revealed that three testa specific expressed candidate genes, Araip.M7RY3, Aradu.R8PMF and Araip.MHR6K were likely responsible for the white testa phenotype. WSC might be regulated expression competition between FLS and DRF by controlling hormone synthesis and signaling as well as the MBW complex. Conclusions: The results of this study therefore provide both candidate genes and novel approaches that can be applied to improve peanut with desirable seed coat color and flavonoid quality.