MYB-bHLH-WD40 Research Articles

Intron-retained radish (Raphanus sativus L.) RsMYB1 transcripts found in colored-taproot lines enhance anthocyanin accumulation in transgenic Arabidopsis plants.

Overexpression of the naturally occurring intron-retained (IR) forms of radish RsMYB1 and RsTT8 transcripts in Arabidopsis causes a substantial increase in anthocyanin accumulation. The production of anthocyanins in plants is tightly controlled by the MYB-bHLH-WD40 (MBW) complex. In this study, analysis of four radish (Raphanus sativus L.) inbred lines with different colored taproots revealed that regulatory genes of anthocyanin biosynthesis, RsMYB1 and RsTT8, produce three transcripts, one completely spliced and two intron retention (IR1 and IR2) forms. Transcripts RsMYB1-IR1 and RsMYB1-IR2 retained the 1st (380 nt) and 2nd (149 nt) introns, respectively; RsTT8-IR1 retained the 4th intron (113 nt); RsTT8-IR2 retained both the 3rd (128 nt) and 4th introns. Levels of most IR forms were substantially low in radish samples, but the RsTT8-IR2 level was higher than RsTT8 in red skin/red flesh (RsRf) root. Since all IR forms contained a stop codon within the intron, they were predicted to encode truncated proteins with defective interaction domains, resulting in the inability to form the MBW complex in vivo. However, tobacco leaves transiently co-expressing RsMYB1-IRs and RsTT8-IRs showed substantially higher anthocyanin accumulation than those co-expressing their spliced forms. Consistently, co-expression of constructs encoding truncated proteins with spliced or IR forms of their interaction partner in tobacco leaves did not result in anthocyanin accumulation. Compared with RsMYB1, the overexpression of RsMYB1-IRs in Arabidopsis pap1 mutant increased anthocyanin accumulation by > sevenfold and upregulated the expression of Arabidopsis flavonoid biosynthesis genes including AtTT8. Our results suggest that the stable co-expression of RsMYB1-IRs in fruit trees and vegetable crops could be used to increase their anthocyanin contents.

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

BackgroundTrichome 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.ResultsBased 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.ConclusionThese 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.

OsTTG1, a WD40 repeat gene, regulates anthocyanin biosynthesis in rice.

Anthocyanins play an important role in the growth of plants, and are beneficial to human health. In plants, the MYB-bHLH-WD40 (MBW) complex activates the genes for anthocyanin biosynthesis. However, in rice, the WD40 regulators remain to be conclusively identified. Here, a crucial anthocyanin biosynthesis gene was fine mapped to a 43.4-kb genomic region on chromosome 2, and a WD40 gene OsTTG1 (Oryza sativa TRANSPARENT TESTA GLABRA1) was identified as ideal candidate gene. Subsequently, a homozygous mutant (osttg1) generated by CRISPR/Cas9 showed significantly decreased anthocyanin accumulation in various rice organs. OsTTG1 was highly expressed in various rice tissues after germination, and it was affected by light and temperature. OsTTG1 protein was localized to the nucleus, and can physically interact with Kala4, OsC1, OsDFR and Rc. Furthermore, a total of 59 hub transcription factor genes might affect rice anthocyanin biosynthesis, and LOC_Os01g28680 and LOC_Os02g32430 could have functional redundancy with OsTTG1. Phylogenetic analysis indicated that directional selection has driven the evolutionary divergence of the indica and japonica OsTTG1 alleles. Our results suggest that OsTTG1 is a vital regulator of anthocyanin biosynthesis, and an important gene resource for the genetic engineering of anthocyanin biosynthesis in rice and other plants.

Transcriptional Regulation of Anthocyanin Synthesis by MYB-bHLH-WDR Complexes in Kiwifruit (Actinidia chinensis).

The anthocyanin synthetic pathway is regulated centrally by an MYB-bHLH-WD40 (MBW) complex. Anthocyanin pigmentation is an important fruit quality trait in red-fleshed kiwifruit; however, the underlying regulatory mechanisms involving the MBW complex are not well understood. In this study, one R2R3MYB (AcMYBF110 expressed in fruit characteristically), one bHLH (AcbHLH1), two upstream regulators of AcbHLH1 (AcbHLH4 and AcbHLH5), and one WDR (AcWDR1) are characterized as being involved in the regulation of anthocyanin synthesis in kiwifruit. AcMYBF110 plays an important role in the regulation of anthocyanin accumulation by specifically activating the promoters of several anthocyanin pathway genes including AcCHS, AcF3'H, AcANS, AcUFGT3a, AcUFGT6b, and AcGST1. Coexpression of AcbHLH1, AcbHLH4, or AcbHLH5 together with AcMYBF110 induces much greater anthocyanin accumulation in both tobacco leaves and in Actinidia arguta fruit compared with AcMYBF110 alone. Moreover, this activation is further enhanced by adding AcWDR1. We found that both AcMYBF110 and AcWDR1 interact with all three AcbHLH factors, while AcMYBF110 also interacts with AcWDR1 to form three different MBW complexes that have different regulatory roles in anthocyanin accumulation of kiwifruit. The AcMYBF110-AcbHLH1-AcWDR1 complex directly targets the promoters of anthocyanin synthetic genes. Other features of the regulatory pathways identified include promotion of AcMYBF110, AcbHLH1,and AcWDR1 activities by this MBW complex, providing for both reinforcement and feedback regulation, whereas the AcMYBF110-AcbHLH4/5-AcWDR1 complex is indirectly involved in the regulation of anthocyanin synthesis by activating the promoters of AcbHLH1 and AcWDR1 to amplify the regulation signals of the first MBW complex.

TCP transcription factors suppress cotyledon trichomes by impeding a cell differentiation-regulating complex.

Trichomes are specialized epidermal cells that act as barriers against biotic and abiotic stresses. Although the formation of trichomes on hairy organs is well studied, the molecular mechanisms of trichome inhibition on smooth organs are still largely unknown. Here, we demonstrate that the CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors inhibit the formation of trichomes on cotyledons in Arabidopsis (Arabidopsis thaliana). The tcp2/3/4/5/10/13/17 septuple mutant produces cotyledons with ectopic trichomes on the adaxial sides. The expression patterns of TCP genes are developmentally regulated during cotyledon development. TCP proteins directly interact with GLABRA3 (GL3), a key component of the MYB transcription factor/basic helix-loop-helix domain protein/WD40-repeat proteins (MYB-bHLH-WD40, MBW) complex essential for trichome formation, to interfere with the transactivation activity of the MBW complex in cotyledons. TCPs also disrupt the MBW complex-R3 MYB negative feedback loop by directly promoting the expression of R3 MYB genes, which enhance the repression of the MBW complex. Our findings reveal a molecular framework in which TCPs suppress trichome formation on adaxial sides of cotyledons by repressing the activity of the MBW complex at the protein level and the transcripts of R3 MYB genes at the transcriptional level.

Maize SRO1e represses anthocyanin synthesis through regulating the MBW complex in response to abiotic stress.

Plants experiencing abiotic stress react by generating reactive oxygen species (ROS), compounds that, if allowed to accumulate to excess, repress plant growth and development. Anthocyanins induced by abiotic stress are strong antioxidants that neutralize ROS, whereas their over-accumulation retards plant growth. Although the mechanism of anthocyanin synthesis has been revealed, how plants balance anthocyanin synthesis under abiotic stress to maintain ROS homeostasis is unknown. Here, ROS-related proteins, SIMILAR TO RCD-ONEs (SROs), were analysed in Zea mays (maize), and all six SRO1 genes were inducible by a variety of abiotic stress agents. The constitutive expression of one of these genes, ZmSRO1e, in maize as well as in Arabidopsis thaliana increased the sensitivity of the plant to abiotic stress, but repressed anthocyanin biosynthesis and ROS scavenging activity. Loss-of-function mutation of ZmSRO1e enhanced ROS tolerance and anthocyanin accumulation. We showed that ZmSRO1e competed with ZmR1 (a core basic helix-loop-helix subunit of the MYB-bHLH-WD40 transcriptional activation complex) for binding with ZmPL1 (a core MYB subunit of the complex). Thus, during the constitutive expression of ZmSRO1e, the formation of the complex was compromised, leading to the repression of genes, such as ZmA4 (encoding dihydroflavonol reductase), associated with anthocyanin synthesis. Overall, the results have revealed a mechanism that allows the products of maize SRO1e to participate in the abiotic stress response.

Interactions of JAZ Repressors with Anthocyanin Biosynthesis-Related Transcription Factors of Fragaria × ananassa

Strawberry fruits are rich in flavonoids like proanthocyanidins and anthocyanins. Their biosynthesis and accumulation are controlled by the MYB-bHLH-WD40 (MBW) transcriptional complex, which is mainly formed by basic helix-loop-helix (bHLH) and MYB transcription factors (TFs). In Arabidopsis thaliana both bHLH and MYB TFs are repressed by JASMONATE ZIM-DOMAIN (JAZ) proteins, the key repressors of the jasmonate-signaling pathway. The aim of this research was the characterization of the FaJAZ1/8.1/9/10 proteins and molecular targets of signaling components and anthocyanin biosynthesis-related TFs of Fragaria × ananassa by protein–protein interactions. For this, domain compositions were studied by multiple alignments and phylogenetic analyses, while interactions were analyzed by yeast two-hybrid (Y2H) assays. We detected high conservation of FaJAZ proteins and jasmonate-signaling components, as well as FabHLHs and FaMYB10 TFs. Moreover, we report the F. × ananassa YABBY1 (FaYAB1) TF, which is related to anthocyanin biosynthesis in Arabidopsis, showed high conservation of functional domains. We demonstrated that FaJAZ repressors interacted with F. × ananassa NOVEL INTERACTOR OF JAZ (FaNINJA), FaMYC2, and JASMONATE ASSOCIATED MYC2-LIKE (FaJAM) proteins. Besides, transcription factors of MBW-complex like FabHLH3, FabHLH33, and FaMYB10, together with FaYAB1, were molecular targets of FaJAZ repressors, exhibiting specificity or redundancy of interaction depending on particular FaJAZ protein. Overall, these results suggest that interactions of jasmonate-signaling components are fully conserved, and anthocyanin biosynthesis might be regulated by JAZ repressors in F. × ananassa.

The proanthocyanin-related transcription factors MYBC1 and WRKY44 regulate branch points in the kiwifruit anthocyanin pathway

The groups of plant flavonoid metabolites termed anthocyanins and proanthocyanins (PA) are responsible for pigmentation in seeds, flowers and fruits. Anthocyanins and PAs are produced by a pathway of enzymes which are transcriptionally regulated by transcription factors (TFs) that form the MYB-bHLH-WD40 (MBW) complex. In this study, transcriptomic analysis of purple-pigmented kiwifruit skin and flesh tissues identified MYBC1, from subgroup 5 of the R2R3 MYB family, and WRKY44 (highly similar to Arabidopsis TTG2) as candidate activators of the anthocyanin pathway. Transient over-expression of MYBC1 and WRKY44 induced anthocyanin accumulation in tobacco leaves. Dual luciferase promoter activation assays revealed that both MYBC1 and WRKY44 were able to strongly activate the promoters of the kiwifruit F3′H and F3′5′H genes. These enzymes are branch points of the pathway which specifies the type of anthocyanin accumulated. Stable over-expression of MYBC1 and WRKY44 in kiwifruit calli activated the expression of F3′5′H and PA-related biosynthetic genes as well as increasing levels of PAs. These results suggest that while previously characterised anthocyanin activator MYBs regulate the overall anthocyanin biosynthesis pathway, the PA-related TFs, MYBC1 and WRKY44, more specifically regulate key branch points. This adds a layer of regulatory control that potentially balances anthocyanin and PA levels.

Airborne fungus-induced biosynthesis of anthocyanins in Arabidopsis thaliana via jasmonic acid and salicylic acid signaling

Anthocyanins are plant-specific pigments, the biosynthesis of which is stimulated by pathogen infection in several plant species. A. thaliana seedlings injected with airborne fungi can accumulate a high content of anthocyanins. The mechanism involved in fungus-induced anthocyanin accumulation in plants has not been fully described. In this study, the fungus Penicillium corylophilum (P. corylophilum), isolated from an Arabidopsis culture chamber, triggered jasmonic acid (JA), salicylic acid (SA), and anthocyanin accumulation in A. thaliana. Inhibitors of JA and SA biosynthesis suppressed the anthocyanin accumulation induced by P. corylophilum. The anthocyanin content was minimal in both the null mutant of JA-receptor coi1 and the null mutant of SA-receptor npr1 under P. corylophilum stimulation. The results indicate that JA and SA signaling mediated fungus-induced anthocyanin biosynthesis in A. thaliana. P. corylophilum led to different levels of anthocyanin generation in null mutants for MYB75, bHLH, EGL3, and GL3 transcription factors and WD40 protein, demonstrating that multiple MYB–bHLH–WD40 transcription factor complexes participated in fungus-induced anthocyanin accumulation in A. thaliana. The present study will help further elucidate the mechanism of plant resistance to pathogen infection.

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.

Anthocyanin accumulation is initiated by abscisic acid to enhance fruit color during fig (Ficus carica L.) ripening

Fig fruit is well-known for its attractive flavor, color, and nutritional and medicinal value. Anthocyanin contributes to the fruit's color and constitutes a high percentage of the total antioxidant content of the fig fruit. We quantified the major anthocyanins and characterized the expression levels of anthocyanin-biosynthesis and transcription factor genes in fruit treated on-tree with exogenous abscisic acid (ABA) or ethephon, or the ABA inhibitors nordihydroguaiaretic acid (NDGA) or fluridone. The major anthocyanins cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside were found in significantly higher quantities in exogenous ABA- and ethephon-treated fruit, with early dark purple color compared to the controls. On the other hand, NDGA- and fluridone-treated fruit had significantly lower amounts of anthocyanins, with less purple color coverage than controls. Expression levels of the anthocyanin-biosynthesis genes FcPAL, FcCHS2, FcCHI, FcF3H, FcDFR, FcANS, FcUFGT and Fc3RT were upregulated by exogenous ABA and ethephon treatment, and downregulated by NDGA and fluridone treatment. The MYB–bHLH–WD40 complex-related genes of ripe fig fruit were identified. In particular, FcMYB113 was strongly upregulated by exogenous ABA and ethephon, and strongly downregulated by NDGA and fluridone. In addition, moderate upregulation of FcGL3 and FcWD40 was observed with exogenous ABA and ethephon treatment, and moderate downregulation in NDGA- and fluridone-treated fruit. These results indicate that ABA can initiate anthocyanin biosynthesis, which ultimately improves the color and nutritional value of fig fruit, enhancing their attractiveness to consumers.

Abnormal expression of bHLH3 disrupts a flavonoid homeostasis network, causing differences in pigment composition among mulberry fruits

Mulberry fruits with high concentrations of anthocyanins are favored by consumers because of their good taste, bright color, and high nutritional value. However, neither the regulatory mechanism controlling flavonoid biosynthesis in mulberry nor the molecular basis of different mulberry fruit colors is fully understood. Here, we report that a flavonoid homeostasis network comprising activation and feedback regulation mechanisms determines mulberry fruit color. In vitro and in vivo assays showed that MYBA-bHLH3-TTG1 regulates the biosynthesis of anthocyanins, while TT2L1 and TT2L2 work with bHLH3 or GL3 and form a MYB-bHLH-WD40 (MBW) complex with TTG1 to regulate proanthocyanidin (PA) synthesis. Functional and expression analyses showed that bHLH3 is a key regulator of the regulatory network controlling mulberry fruit coloration and that MYB4 is activated by MBW complexes and participates in negative feedback control of the regulatory network to balance the accumulation of anthocyanins and proanthocyanidins. Our research demonstrates that the interaction between bHLH3 and MYB4 in the homeostasis regulatory network ensures that the fruits accumulate desirable flavonoids and that this network is stable in pigment-rich mulberry fruits. However, the abnormal expression of bHLH3 disrupts the balance of the network and redirects flavonoid metabolic flux in pale-colored fruits, resulting in differences in the levels and proportions of anthocyanins, flavones, and flavonols among differently colored mulberry fruits (red, yellow, and white). The results of our study reveal the molecular basis of the diversity of mulberry fruit colors.

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.

Comparative transcriptome analysis of differentially expressed genes between the fruit peel and flesh of the purple tomato cultivar ‘Indigo Rose’

ABSTRACT Anthocyanins are considered health-promoting phytonutrients; however, anthocyanins strictly occurr in the fruit peel of purple tomato cultivars, making the total anthocyanin content limited per tomato fruit. In this study, we performed a transcriptome analysis between the fruit peel and flesh of a purple tomato cultivar ‘Indigo Rose’ at both the mature green stage and breaking stage. In total, 1,945 differently expressed genes, including 165 transcription factors, were detected between the fruit peel and flesh, both at and after the mature green stage. We further analyzed the transcription of anthocyanin biosynthesis genes and the regulatory genes composing the MYB-bHLH-WD40 (MBW) complex between the fruit peel and flesh at both development stages. In addition, several light-sensing genes and other transcription factor genes, including BBX family genes and WRKY genes, showed different expression patterns between the fruit peel and flesh. These findings deepen our understanding of anthocyanin biosynthesis in tomato fruit peels and facilitate the identification of genes limiting the anthocyanin biosynthesis in tomato fruit flesh.

SNP in DFR1 Coding Sequence Is Tightly Associated with Anthocyanin Accumulation in Cabbage (B. oleracea var. capitata f. alba) at Low Temperature

Keeping green leaf color at the time of harvest is one of the important traits for breeding of Brassica oleracea var. capitata f. alba, and this trait is related to low anthocyanin contents. To understand the differential accumulation of anthocyanins in cabbage, we selected high anthocyanin accumulators (HAAs) and low anthocyanin accumulator (LAAs) of cabbages and examined the anthocyanin content and the expression of anthocyanin biosynthesis-related genes. Among many genes investigated, BoDFR1 was found to be closely related to anthocyanin accumulation, even under low temperature (LT) conditions. BoDFR1 sequence analyses between HAAs and LAAs revealed that there is a single nucleotide polymorphism (SNP) (1118T/A) in the coding sequence, which substitutes one amino acid from Leu261 to His261; we named BoDFR1 with His261 substitution as BoDFR1v. This amino acid substitution did not affect dihydroflavonol 4-reductase (DFR) activity and substrate specificity, but the polymorphism showed tight association to the BoDFR1 expression, i.e., high level expression of BoDFR1 and low level expression of BoDFR1v under LT conditions. The high levels of BoDFR1 expression were due to the high levels of BoMYB114 and BobHLH expressions combined with low level expression of BoMYBL2, a repressor MYB. On the other hand, low levels of BoDFR1v expression seemed to be related to very low level expressions of BoMYB114 and BobHLH combined with a high level expression of BoMYBL2. It seems that different expression levels of these regulatory genes for MBW (MYB-bHLH-WD40) complex between HAAs and LAAs regulate BoDFR expression and anthocyanin accumulation. Using a single nucleotide polymorphism (SNP) between BoDFR1 and BoDFR1v, molecular markers for PCR and high resolution melt analyses were developed and validated to distinguish between HAAs and LAAs. Combined use of the BoDFR1 SNP marker with other stress markers, such as a cold tolerant marker, will greatly improve cabbage breeding.

Novel TTG1 Mutants Modify Root-Hair Pattern Formation in Arabidopsis.

The patterning of root-hair and non-hair epidermal cells in the Arabidopsis root is governed by a network of transcriptional regulators. The central MYB-bHLH-WD40 (MBW) transcriptional complex includes the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1). To clarify the role of TTG1, we describe the identification and analysis of two new ttg1 mutants. Each of these mutants contains a single nucleotide change in the TTG1 gene, which causes a single amino-acid substitution in the predicted TTG1 protein and alters root-hair pattern formation. Surprisingly, these new ttg1 mutants exhibit decreased root-hair formation, particularly in the caprice (cpc) mutant background, rather than increased root-hair formation as reported for strong ttg1 mutants. We show that the unique phenotype of these mutants is due to differential effects of the altered TTG1 proteins on target gene expression, associated with a weakened ability to interact with its GLABRA3 bHLH partner. These findings demonstrate the crucial role of TTG1 for the appropriate balance of target gene activation to achieve the proper pattern of epidermal cell types during Arabidopsis root development.

Identification of MYB Transcription Factors Regulating Theanine Biosynthesis in Tea Plant Using Omics-Based Gene Coexpression Analysis.

Theanine (thea) is the most abundant free amino acid in tea plant (Camellia sinensis) and one of the most important secondary metabolites conferring tea quality and health benefits. Great effort has recently been made to functionally dissect enzyme genes (e.g., GS, GDH, GOGAT) responsible for in vivo thea accumulation. However, the transcriptional regulation of its biosynthesis remains to be explored. Starting from publicly available (condition-independent) tea transcriptome data, we performed an exhaustive coexpression analysis between transcription factor (TF) genes and thea enzyme genes in tea plant. Our results showed that two typical plant-specialized (secondary) metabolites related TF families, such as MYB, bHLH, together with WD40 domain proteins, were prominently involved, suggesting a potential MYB-bHLH-WD40 (MBW) complex-mediated regulatory pattern in thea pathway. Aiming at the most involved MYB family, we screened seven MYB genes as thea candidate regulators through a stringent multistep selection (e.g., filtering with condition-specific nitrogen-treated transcriptome data). The control of MYB regulators in thea biosynthesis was further demonstrated using an integrated analysis of thea accumulation and MYB expression in several major tea tissues, including leave, bud, root, and stem. Our investigation aided tea researchers in having a comprehensive view of transcriptional regulatory landscape in thea biosynthesis, serving as the first platform for studying molecular regulation in thea pathway and a paradigm for understanding the characteristic components biosynthesis in nonmodel plants.

Transcriptome analysis reveals the molecular mechanisms of mucilage biosynthesis during Artemisia sphaerocephala seed development

Seed mucilage is a polysaccharide with significant physiological, ecological, and economic value; however, both its biosynthesis pathway and molecular regulatory mechanisms remain unclear. Artemisia sphaerocephala is an important sand-fixed windbreak plant that is widely distributed across the deserts of Northwest China, with seeds that are rich in mucilage. For this research, seven different developmental stages of A. sphaerocephala seeds were employed as samples. The mucilage accumulation patterns were analyzed, and the differentially expressed genes (DEGs) involved in the biosynthesis of mucilage were identified via RNA sequencing. The results revealed that during the rapid accumulation period (10–30 days after flowering), the mucilage content increased from 0 to 49.42 %. Up-regulated expressed phosphoglucomutase (pgm) and uridine diphosphate glucose pyrophosphorylase (UGPase), and down-regulated expressed UDP-glucose 4-epimerase (GALE), UDP-glucose 6-dehydrogenase (UGDH), and UDP-glucose 4,6-dehydratase (RHM) led to the accumulation of UDP-glucose (UDP-Glc). Additionally, highly expressed UDP-apiose/xylose synthase (AXS) and minimally expressed UDP-arabinose 4-epimerase (UXE) led to the accumulation of UDP-xylose (UDP-Xyl). The MYB-bHLH-WD40 (MBW) complex and APETALA2 (AP2) were the primary transcription factors that regulated the biosynthesis of mucilage in A. sphaerocephala seeds. This study might assist with elucidating the molecular regulatory mechanisms of the seed mucilage biosynthesis process, while establishing a foundation for the application of mucilage associated genes in genetic breeding.

GLABRA2, A Common Regulator for Epidermal Cell Fate Determination and Anthocyanin Biosynthesis in Arabidopsis.

Epidermal cell fate determination—including trichome initiation, root hair formation, and flavonoid and mucilage biosynthesis in Arabidopsis (Arabidopsis thaliana)—are controlled by a similar transcriptional regulatory network. In the network, it has been proposed that the MYB-bHLH-WD40 (MBW) activator complexes formed by an R2R3 MYB transcription factor, a bHLH transcription factor and the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) regulate the expression of downstream genes required for cell fate determination, flavonoid or mucilage biosynthesis, respectively. In epidermal cell fate determination and mucilage biosynthesis, the MBW activator complexes activate the expression of GLABRA2 (GL2). GL2 is a homeodomain transcription factor that promotes trichome initiation in shoots, mucilage biosynthesis in seeds, and inhibits root hair formation in roots. The MBW activator complexes also activate several R3 MYB genes. The R3 MYB proteins, in turn, competing with the R2R3 MYBs for binding bHLH transcription factors, therefore inhibiting the formation of the MBW activator complexes, lead to the inhibition of trichome initiation in shoots, and promotion of root hair formation in roots. In flavonoid biosynthesis, the MBW activator complexes activate the expression of the late biosynthesis genes in the flavonoid pathway, resulting in the production of anthocyanins or proanthocyanidins. Research progress in recent years suggests that the transcriptional regulatory network that controls epidermal cell fate determination and anthocyanin biosynthesis in Arabidopsis is far more complicated than previously thought. In particular, more regulators of GL2 have been identified, and GL2 has been shown to be involved in the regulation of anthocyanin biosynthesis. This review focuses on the research progress on the regulation of GL2 expression, and the roles of GL2 in the regulation of epidermal cell fate determination and anthocyanin biosynthesis in Arabidopsis.

An R3-MYB gene of Phalaenopsis, MYBx1, represses anthocyanin accumulation

Anthocyanins are secondary metabolites that are involved in providing flower petal pigmentation. In plants, anthocyanin synthesis is regulated positively by a conserved MYB-bHLH-WD40 (MBW) complex and negatively by repressors. We isolated a repressor of anthocyanin synthesis, MYBx1, from a moth orchid Phalaenopsis cv. Big Chili. MYBx1 encodes a typical R3-MYB protein and exhibited diverse expression patterns in four Phalaenopsis cultivars, Big Chili, Fuller’s Sunset, Sogo Yukidian “V3”, and Sogo Lit-Sunny. A yeast two-hybrid assay analysis indicated that MYBx1 can also participate in the MBW complex by interacting with the transcription factors ANTHOCYANIN (AN) 1 and AN11. Over-expressing MYBx1 in the petunia hybrid M1 × R27 resulted in reduced accumulations of flower petal pigments through the repression of anthocyanin activities. A quantitative real-time PCR analysis of the expression patterns of anthocyanin synthesis-related structural genes in the transgenic petunia flower petals showed that the expression levels of chalcone synthase, flavanone 3-hydroxylase, and dihydroflavonol reductase genes were considerably down-regulated. However, no significant changes in the expression levels of the MBW complex members, AN1, AN2, and AN11, were observed. This study improves our understanding of anthocyanin synthesis-related regulatory networks in plants and also provides an important foundation for the further genetic engineering of ornamental plant flower colors.