R2R3-MYB Protein Research Articles

PtrMYB57 contributes to the negative regulation of anthocyanin and proanthocyanidin biosynthesis in poplar.

A novel R2R3 MYB transcription factor PtrMYB57 interacted with bHLH131 and PtrTTG1 to form the MBW complex and negatively regulated the biosynthesis of both anthocyanins and PAs in poplar. R2R3-MYB transcription factors (TFs) are important regulators of secondary metabolite biosynthesis in woody species. A series of R2R3-MYB TFs involved in anthocyanin and proanthocyanidin (PA) biosynthesis have been identified in poplar. In this study, we report the identification and characterization of a subgroup 4 MYB member PtrMYB57, which contains a repressor domain (LxLxL) at the C-terminal end. PtrMYB57 encodes an R2R3 MYB protein localized in the nucleus and is predominantly expressed in mature leaves. Transgenic poplar overexpressing PtrMYB57 showed a reduction in anthocyanin and PA accumulation compared to wild-type plants. By contrast, a high anthocyanin and PA phenotype was observed in Ptrmyb57 mutants generated by the CRISPR/Cas9 system. Furthermore, transient expression assays revealed that PtrMYB57 interacted with bHLH131 (bHLH) and PtrTTG1 (WDR) to form the MBW complex and bound to the flavonoid gene promoters, leading to inhibition of these promoters. Taken together, our results suggest that PtrMYB57 plays a negative role in the regulation of anthocyanin and PA biosynthesis in poplar.

MdMYB4, an R2R3-Type MYB Transcription Factor, Plays a Crucial Role in Cold and Salt Stress in Apple Calli

MYB (v-myb avian myeloblastosis viral oncogene homologs) transcription factors (TFs) are involved in diverse physiological processes, including cell shape determination, cell differentiation, and secondary metabolism, as well as abiotic stress response. In the present study, MdMYB4, an R2R3-MYB protein that is a homolog of Arabidopsis thaliana MYB4, was identified and characterized. Quantitative real-time polymerase chain reaction (qRT-PCR) expression analysis demonstrated that MdMYB4 is extensively expressed in various apple (Malus domestica) tissues and that its expression is induced by cold, osmotic, and salt stress. An MdMYB4-GFP fusion protein was localized in the nucleus of transformed onion (Allium cepa) epidermal cells and had a certain transcriptional activation activity by yeast one-hybrid assay. Overexpression of the MdMYB4 gene remarkably enhanced the tolerance of stably transgenic apple calli to severe salt and cold stress, and both the relative conductivity and malondialdehyde (MDA) accumulation of transgenic calli under salt and cold stress were significantly lower than in the wild type control. Taken together, these results suggest that MdMYB4 may play a positive regulatory role in both cold and salt stress responses.

A sugarcane R2R3-MYB transcription factor gene is alternatively spliced during drought stress

MYB transcription factors of the R2R3-MYB family have been shown to play important roles in many plant processes. A sugarcane R2R3-MYB gene (ScMYB2) and its two alternative forms of transcript (ScMYB2S1 and ScMYB2S2) were identified in this study. The deduced protein of ScMYB2S1 is a typical plant R2R3-MYB protein, while ScMYB2S2 encodes a truncated protein. Real-time qPCR analysis revealed that ScMYB2S1 is suppressed under PEG-simulated drought stress in sugarcane, while ScMYB2S2 is induced at later treatment stage. A senescence symptom was observed when ScMYB2S1 was injected into tobacco leaves mediated by Agrobacterium, but no symptom for ScMYB2S2. Further investigation showed that the expression levels of 4 senescence-associated genes, NtPR-1a, NtNYC1, NtCAT3 and NtABRE, were markedly induced in tobacco leaves after ScMYB2S1-injection, while they were not sensitive to ScMYB2S2-injection. Moreover, MDA and proline were also investigated after injection. Similarly, MDA and proline levels were induced by ABA and ScMYB2S1, while inhibited by ScMYB2S2. We propose that ScMYB2, by alternatively splicing two transcripts (ScMYB2S1 and ScMYB2S2), is involved in an ABA-mediated leaf senescence signaling pathway and play positive role in respond to drought-induced senescence in sugarcane. The results of this study provide information for further research in sugarcane stress processes.

A wheat R2R3-MYB protein PURPLE PLANT1 (TaPL1) functions as a positive regulator of anthocyanin biosynthesis

Transcriptional activation of anthocyanin biosynthesis genes in vegetative tissues of monocotyledonous plants is mediated by cooperative activity of one component from each of the following two transcription factor families: MYB encoded by PURPLE PLANT1/COLORED ALEURONE1 (PL1/C1), and basic helix-loop-helix (bHLH) encoded by RED/BOOSTER (R1/B1). In the present study, putative PL cDNA was cloned from the wheat (Triticum aestivum) cultivar Iksan370, which preferentially expresses anthocyanins in coleoptiles. Phylogenetic tree analysis of deduced amino acid sequences showed that a putative TaPL1 is highly homologous to barley (Hordeum vulgare) HvPL1, but is distinct from wheat TaC1. Transgenic Arabidopsis thaliana stably expressing putative TaPL1 accumulated anthocyanin pigments in leaves and up-regulated structural genes involved in both early and late anthocyanin biosynthesis steps. TaPL1 transcript levels in Iksan370 were more prominent in vegetative tissues such as young coleoptiles than in reproductive tissues such as spikelets. TaPL1 expression was significantly up-regulated by environmental stresses including cold, salt, and light, which are known to induce anthocyanin accumulation. These combined results suggest that TaPL1 is an active positive regulator of anthocyanin biosynthesis in wheat coleoptiles.

CrMYB73, a PH-like gene, contributes to citric acid accumulation in citrus fruit

Organic acids strongly influence the organoleptic properties of fruit, and in citrus the major organic acid is citric acid. The pH-associated genes (PH) have been widely studied in various fruit and their expression has been suggested to be linked to citric acid accumulation in citrus fruit, but in planta functional evidence is lacking. A novel PH-like gene, named CrMYB73, was isolated from ‘Ponkan’ (Citrus reticulate Blanco) fruit, and the amino acid sequence analysis suggested that CrMYB73 was an R2R3 MYB protein. Expression of CrMYB73 was positively correlated with citric acid accumulation and its role was tested directly by overexpression in both wild type and anthocyanin-enriched transgenic Nicotiana tabacum. The results indicated that transient overexpression of CrMYB73 leads to citric acid accumulation.

Identification and expression analyses of MYB and WRKY transcription factor genes in Papaver somniferum L.

Papaver somniferum L. is an herbaceous, annual and diploid plant that is important from pharmacological and strategic point of view. The cDNA clones of two putative MYB and WRKY genes were isolated (GeneBank accession numbers KP411870 and KP203854, respectively) from this plant, via the nested-PCR method, and characterized. The MYB transcription factor (TF) comprises 342 amino acids, and exhibits the structural features of the R2R3MYB protein family. The WRKY TF, a 326 amino acid-long polypeptide, falls structurally into the group II of WRKY protein family. Quantitative real-time PCR (qRT-PCR) analyses indicate the presence of these TFs in all organs of P. somniferum L. and Papaver bracteatum L. Highest expression levels of these two TFswere observed in the leaf tissues ofP. somniferum L. while in P. bracteatumL. the espression levels were highest in the root tissues. Promoter analysis of the 10 co-expressed gene clustered involved in noscapine biosynthesis pathway in P. somniferum L. suggested that not only these 10 genes are co-expressed, but also share common regulatory motifs and TFs including MYB and WRKY TFs, and that may explain their common regulation.

The soybean gene, GmMYBJ2, encodes a R2R3-type transcription factor involved in drought stress tolerance in Arabidopsis thaliana

MYB genes are extensively distributed in higher plants and constitute one of the largest transcription factors (TFs) families. These TFs have been proved to be implicated in the regulation of plant growth, development, metabolism, and multiple abiotic stress responses. In the present study, a new soybean MYB gene, denoted GmMYBJ2, was isolated and its function was characterized. The GmMYBJ2 cDNA is 1428 bp in length with an open reading frame (ORF) of 960 bp encoding 319 amino acids. Sequence and yeast one-hybrid analyses showed GmMYBJ2 contains two MYB domains and belongs to R2R3-MYB protein with transactivation activity. Transient expression analysis using the GmMYBJ2-GFP fusion gene in onion epidermal cells showed GmMYBJ2 protein is targeted to the nucleus. GmMYBJ2 was induced by drought, cold, salt, and exogenous abscisic acid (ABA). Arabidopsis overexpressing GmMYBJ2 exhibited a higher seed germination rates (GRs), a notable increase in the soluble sugar content under water-deficit stress, and a lower water loss rate (WLR) when water is sufficient. These results indicated the overexpression of GmMYBJ2 make transgenic Arabidopsis more tolerant to drought stress than wild-type (WT) plants, and GmMYBJ2 may be useful for improving drought stress tolerance in transgenic plant breeding.

Physiological changes in fruit ripening caused by overexpression of tomato SlAN2, an R2R3-MYB factor

The R2R3-MYB protein SlAN2 has long been thought to be a positive regulator of anthocyanin accumulation. To investigate the role of SlAN2, we have previously overexpressed the gene in tomato. In this work, we analysed physiological characters of the transgenic plants during the fruit ripening. We show that fruits of transformants overexpressing SlAN2 displayed an orange colour, fast softening and elevated ethylene production. Overexpression of SlAN2 resulted in reduction of carotenoid levels via alteration of flux through the carotenoid pathway, elevated ethylene synthesis mainly via upregulation of ethylene biosynthesis genes, and early softening of fruits. We also found that the transcript level of SlRIN, an important ripening-related gene, was up-regulated in transgenic fruits. These results suggest that SlAN2 acts as an important regulator of fruit ripening.

A Scutellaria baicalensis R2R3-MYB gene, SbMYB8, regulates flavonoid biosynthesis and improves drought stress tolerance in transgenic tobacco

R2R3-MYB proteins are involved in the primary and secondary metabolism, developmental processes and the responses to biotic and abiotic stresses. Little is known about the functions of R2R3-MYB proteins in Scutellaria baicalensis Georgi which is a traditional Chinese medicinal plants. In this study, the function of a S. baicalensis R2R3-MYB protein, SbMYB8, was investigated. SbMYB8 had similar expression pattern with SbC4H and SbCHS in ABA-treated S. baicalensis, indicating that SbMYB8 might be involved in the flavonoid metabolism. SbMYB8 protein could bind to the GmMYB92 BS3 sequence of SbCHS promoter region, regulating the expression of SbCHS. The SbMYB8 protein was localized to the nucleus where it activated transcription. The transgenic tobacco plants over-expressing SbMYB8 had higher caffeoylquinic acid contents, compared to that in wild type plants. Overexpression of SbMYB8 also changed the expression level of some flavonoid biosynthesis-related genes. It was found that overexpression of SbMYB8 can improve stress tolerance of transgenic plants, and can alter the activity and expression levels of some antioxidant enzymes. These results indicate that SbMYB8 plays important roles in flavonoid biosynthesis and stress tolerance of plant.

An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis.

Trichomes are specialized epidermal cells located on aerial parts of plants and are associated with a wide array of biological processes. Trichomes protect plants from adverse conditions including UV light and herbivore attack and are also an important source of a number of phytochemicals. The simple unicellular trichomes of Arabidopsis serve as an excellent model to study molecular mechanism of cell differentiation and pattern formation in plants. The emerging picture suggests that the developmental process is controlled by a transcriptional network involving three major groups of transcription factors (TFs): the R2R3 MYB, basic helix-loop-helix (bHLH), and WD40 repeat (WDR) protein. These regulatory proteins form a trimeric activator complex that positively regulates trichome development. The single repeat R3 MYBs act as negative regulators of trichome development. They compete with the R2R3 MYBs to bind the bHLH factor and form a repressor complex. In addition to activator–repressor mechanism, a depletion mechanism may operate in parallel during trichome development. In this mechanism, the bHLH factor traps the WDR protein which results in depletion of WDR protein in neighboring cells. Consequently, the cells with high levels of bHLH and WDR proteins are developed into trichomes. A group of C2H2 zinc finger TFs has also been implicated in trichome development. Phytohormones, including gibberellins and jasmonic acid, play significant roles in this developmental process. Recently, microRNAs have been shown to be involved in trichome development. Furthermore, it has been demonstrated that the activities of the key regulatory proteins involved in trichome development are controlled by the 26S/ubiquitin proteasome system (UPS), highlighting the complexity of the regulatory network controlling this developmental process. To complement several excellent recent relevant reviews, this review focuses on the transcriptional network and hormonal interplay controlling trichome development in Arabidopsis.

Comparative genomic analysis of the R2R3 MYB secondary cell wall regulators of Arabidopsis, poplar, rice, maize, and switchgrass

BackgroundR2R3 MYB proteins constitute one of the largest plant transcription factor clades and regulate diverse plant-specific processes. Several R2R3 MYB proteins act as regulators of secondary cell wall (SCW) biosynthesis in Arabidopsis thaliana (At), a dicotyledenous plant. Relatively few studies have examined SCW R2R3 MYB function in grasses, which may have diverged from dicots in terms of SCW regulatory mechanisms, as they have in cell wall composition and patterning. Understanding cell wall regulation is especially important for improving lignocellulosic bioenergy crops, such as switchgrass.ResultsHere, we describe the results of applying phylogenic, OrthoMCL, and sequence identity analyses to classify the R2R3 MYB family proteins from the annotated proteomes of Arabidposis, poplar, rice, maize and the initial genome (v0.0) and translated transcriptome of switchgrass (Panicum virgatum). We find that the R2R3 MYB proteins of the five species fall into 48 subgroups, including three dicot-specific, six grass-specific, and two panicoid grass-expanded subgroups. We observe four classes of phylogenetic relationships within the subgroups of known SCW-regulating MYB proteins between Arabidopsis and rice, ranging from likely one-to-one orthology (for AtMYB26, AtMYB103, AtMYB69) to no homologs identifiable (for AtMYB75). Microarray data for putative switchgrass SCW MYBs indicate that many maintain similar expression patterns with the Arabidopsis SCW regulators. However, some of the switchgrass-expanded candidate SCW MYBs exhibit differences in gene expression patterns among paralogs, consistent with subfunctionalization. Furthermore, some switchgrass representatives of grass-expanded clades have gene expression patterns consistent with regulating SCW development.ConclusionsOur analysis suggests that no single comparative genomics tool is able to provide a complete picture of the R2R3 MYB protein family without leaving ambiguities, and establishing likely false-negative and -positive relationships, but that used together a relatively clear view emerges. Generally, we find that most R2R3 MYBs that regulate SCW in Arabidopsis are likely conserved in the grasses. This comparative analysis of the R2R3 MYB family will facilitate transfer of understanding of regulatory mechanisms among species and enable control of SCW biosynthesis in switchgrass toward improving its biomass quality.

A negative MYB regulator of proanthocyanidin accumulation, identified through expression quantitative locus mapping in the grape berry

Flavonoids are secondary metabolites with multiple functions. In grape (Vitis vinifera), the most abundant flavonoids are proanthocyanidins (PAs), major quality determinants for fruit and wine. However, knowledge about the regulation of PA composition is sparse. Thus, we aimed to identify novel genomic regions involved in this mechanism. Expression quantitative trait locus (eQTL) mapping was performed on the transcript abundance of five downstream PA synthesis genes (dihydroflavonol reductase (VvDFR), leucoanthocyanidin dioxygenase (VvLDOX), leucoanthocyanidin reductase (VvLAR1), VvLAR2 and anthocyanidin reductase (VvANR)) measured by real-time quantitative PCR on a pseudo F1 population in two growing seasons. Twenty-one eQTLs were identified; 17 of them did not overlap with known candidate transcription factors or cis-regulatory sequences. These novel loci and the presence of digenic epistasis support the previous hypothesis of a polygenic regulatory mechanism for PA biosynthesis. In a genomic region co-locating eQTLs for VvDFR, VvLDOX and VvLAR1, gene annotation and a transcriptomic survey suggested that VvMYBC2-L1, a gene coding for an R2R3-MYB protein, is involved in regulating PA synthesis. Phylogenetic analysis showed its high similarity to characterized negative MYB factors. Its spatiotemporal expression profile in grape coincided with PA synthesis. Its functional characterization via overexpression in grapevine hairy roots demonstrated its ability to reduce the amount of PA and to down-regulate expression of PA genes.

Interactions between the R2R3-MYB transcription factor, AtMYB61, and target DNA binding sites.

Despite the prominent roles played by R2R3-MYB transcription factors in the regulation of plant gene expression, little is known about the details of how these proteins interact with their DNA targets. For example, while Arabidopsis thaliana R2R3-MYB protein AtMYB61 is known to alter transcript abundance of a specific set of target genes, little is known about the specific DNA sequences to which AtMYB61 binds. To address this gap in knowledge, DNA sequences bound by AtMYB61 were identified using cyclic amplification and selection of targets (CASTing). The DNA targets identified using this approach corresponded to AC elements, sequences enriched in adenosine and cytosine nucleotides. The preferred target sequence that bound with the greatest affinity to AtMYB61 recombinant protein was ACCTAC, the AC-I element. Mutational analyses based on the AC-I element showed that ACC nucleotides in the AC-I element served as the core recognition motif, critical for AtMYB61 binding. Molecular modelling predicted interactions between AtMYB61 amino acid residues and corresponding nucleotides in the DNA targets. The affinity between AtMYB61 and specific target DNA sequences did not correlate with AtMYB61-driven transcriptional activation with each of the target sequences. CASTing-selected motifs were found in the regulatory regions of genes previously shown to be regulated by AtMYB61. Taken together, these findings are consistent with the hypothesis that AtMYB61 regulates transcription from specific cis-acting AC elements in vivo. The results shed light on the specifics of DNA binding by an important family of plant-specific transcriptional regulators.

TRANSPARENT TESTA1 interacts with R2R3‐MYB factors and affects early and late steps of flavonoid biosynthesis in the endothelium of Arabidopsis thaliana seeds

Wild type seed coats of Arabidopsis thaliana are brown due to the accumulation of proanthocyanidin pigments (PAs). The pigmentation requires activation of phenylpropanoid biosynthesis genes and mutations in some of these genes cause a yellow appearance of seeds, termed transparent testa (tt) phenotype. The TT1 gene encodes a WIP-type zinc finger protein and is expressed in the seed coat endothelium where most of the PAs accumulate in wild type plants. In this study we show that TT1 is not only required for correct expression of PA-specific genes in the seed coat, but also affects CHS, encoding the first enzyme of flavonoid biosynthesis. Many steps of this pathway are controlled by complexes of MYB and BHLH proteins with the WD40 factor TTG1. We demonstrate that TT1 can interact with the R2R3 MYB protein TT2 and that ectopic expression of TT2 can partially restore the lack in PA production in tt1. Reduced seed coat pigmentation was obtained using a TT1 variant lacking nuclear localisation signals. Based on our results we propose that the TT2/TT8/TTG1 regulon may also comprise early genes like CHS and discuss steps to further unravel the regulatory network controlling flavonoid accumulation in endothelium cells during A.thaliana seed development.

The R2R3 Myb protein family in Entamoeba histolytica

The MYB DNA-binding domain is conserved in vertebrates, plants, and fungi. This domain mediates the DNA-binding activity of proteins (that have transcription factor activity) in a sequence-specific manner and is also used for the protection of telomeric regions. The MYB DNA-binding domain contains three imperfect conserved repeats of 52 amino acids (R1, R2, and R3). Within each repeat, there are three tryptophans that are separated by 18 or 19 amino acids. In order to understand the role of Myb transcription factors in Entamoeba histolytica, we searched for MYB DNA-binding domain containing proteins using the amino acid sequence of human c-Myb as the query. We found 34 putative MYB DNA-binding domain containing proteins, which clustered into three monophyletic groups. Family I members conserve only the R2 and R3 repeats in their MYB DNA-binding domain and were dubbed in this report as EhMybR2R3. Family II includes single-repeat proteins related to human telomeric binding proteins. Family III is predicted to comprise proteins with one single repeat where the region corresponding to the conserved tryptophan of the third alpha helix is replaced by a S/THAQKY/FF motif; this family was named EhMybSHAQKYF. In this work, we focused on proteins that belong to the EhMybR2R3 family. RT-PCR analysis showed that EhMybR2R3 genes were differentially expressed in trophozoites grown in basal culture conditions. Purified rEhMyb10 protein, belonging to the EhMybR2R3 family, was able to bind a consensus Myb recognition element in vitro. In addition, using nuclear extracts from trophozoites of E. histolytica, we were able to detect Myb DNA-binding activity to this sequence. Our in silico surveys demonstrated that this consensus sequence is present in E. histolytica gene promoters. Interestingly, these promoters include different families of genes that are related to signal transduction, vesicular transport, heat shock response, and virulence. Thus, Myb putative transcription factors in E. histolytica could be involved in the transcriptional regulation of genes participating in several different pathways.

A dynamic DUO of regulatory proteins coordinates gamete specification and germ cell mitosis in the angiosperm male germline

The production of two functional sperm cells within each male gametophyte is essential for double fertilization in flowering plants and involves a single mitotic division of the male germ cell and cell specification to produce functional gametes. Several proteins that are important regulators of male germ cell division have been identified as well as the R2R3 MYB protein DUO1 that has a dual role in cell division and cell specification. We recently identified a novel regulatory protein DUO3, that has overlapping roles with DUO1 in cell division and specification and shows similarity to GON4 related cell lineage regulators in animals. DUO3 also has important roles outside the germline and is required for embryo patterning and meristem function. We outline the regulatory roles of DUO3 in male germline development and its possible mechanisms of action as a lineage regulator in current models that link germ cell cycle control and gamete specification.

Interactions of two transcriptional repressors and two transcriptional activators in modulating gibberellin signaling in aleurone cells.

Gibberellins (GAs) regulate many aspects of plant development, such as germination, growth, and flowering. The barley (Hordeum vulgare) Amy32b alpha-amylase promoter contains at least five cis-acting elements that govern its GA-induced expression. Our previous studies indicate that a barley WRKY gene, HvWRKY38, and its rice (Oryza sativa) ortholog, OsWRKY71, block GA-induced expression of Amy32b-GUS. In this work, we investigated the functional and physical interactions of HvWRKY38 with another repressor and two activators in barley. HvWRKY38 blocks the inductive activities of SAD (a DOF protein) and HvGAMYB (a R2R3 MYB protein) when either of these proteins is present individually. However, SAD and HvGAMYB together overcome the inhibitory effect of HvWRKY38. Yet, the combination of HvWRKY38 and BPBF (another DOF protein) almost diminishes the synergistic effect of SAD and HvGAMYB transcriptional activators. Electrophoretic mobility shift assays indicate that HvWRKY38 blocks the GA-induced expression of Amy32b by interfering with the binding of HvGAMYB to the cis-acting elements in the alpha-amylase promoter. The physical interaction of HvWRKY38 and BPBF repressors is demonstrated via bimolecular fluorescence complementation assays. These data suggest that the expression of Amy32b is modulated by protein complexes that contain either activators (e.g. HvGAMYB and SAD) or repressors (e.g. HvWRKY38 and BPBF). The relative amounts of the repressor or activator complexes binding to the Amy32b promoter regulate its expression level in barley aleurone cells.

Upregulation of the promoter activity of the carrot (Daucus carota) phenylalanine ammonia-lyase gene (DcPAL3) is caused by new members of the transcriptional regulatory proteins, DcERF1 and DcERF2, which bind to the GCC-box homolog and act as an activator to the DcPAL3 promoter

The phenylalanine ammonia-lyase (PAL) gene, DcPAL3, was expressed during the synthesis of anthocyanin in suspension-cultured cells of carrot (Daucus carota). There were two putative cis-elements in the DcPAL3 promoter region: the box-L and GCC-box homologs. Both of these are committed to the upregulation of promoter activity. Although box-L is known as the conserved cis-element present in the promoter region of most PAL genes of many plant species targeted by the R2R3-MYB protein, among PAL genes, the GCC-box homolog is unique to the promoter region of the DcPAL3 gene. We have isolated two proteins belonging to the ethylene-responsive element-binding factor (ERF) family, DcERF1 and DcERF2, from two different cDNA libraries prepared from anthocyanin-synthesizing cells of different cultured cell lines of carrot. The methodology employed was yeast one-hybrid screening with the GCC-box homolog as a bait. Both DcERF1 and DcERF2 bound to the GCC-box homolog sequence in vitro. Transient expression analysis showed that, in carrot protoplasts, DcERF1 was able bind to the GCC-box homolog and act as an activator of the DcPAL3 promoter. In contrast, DcERF2 itself had no ability to activate DcPAL3 promoter activity, possibly because transiently expressed DcERF2 may not be exported into the nucleus. These results suggest that DcERF1 and DcERF2 may function in different ways in committing to the upregulation of the DcPAL3 promoter activity in anthocyanin-synthesizing cells of carrot.

The TTG1-bHLH-MYB complex controls trichome cell fate and patterning through direct targeting of regulatory loci

A network of three classes of proteins consisting of bHLH and MYB transcription factors, and a WD40 repeat protein, TRANSPARENT TESTA GLABRA1 (TTG1), act in concert to activate trichome initiation and patterning. Using YFP-TTG1 translational fusions, we show that TTG1 is expressed ubiquitously in Arabidopsis leaves and is preferentially localized in the nuclei of trichomes at all developmental stages. Using a conditional transgenic allele, we demonstrate that TTG1 directly targets the same genes as the bHLH protein GLABRA3 (GL3). In vivo binding of the R2R3-MYB protein GLABRA1 (GL1) to the promoters of GLABRA2 (GL2), TRANSPARENT TESTA GLABRA2 (TTG2), CAPRICE (CPC) and ENHANCER OF TRIPTYCHON AND CAPRICE1 (ETC1) establishes that these genes are major transcriptional targets for the TTG1-bHLH-MYB regulatory complex. By co-precipitation, we confirm that TTG1 associates with GL3 and GL1 in vivo, forming a complex. The loss of TTG1 and GL1 through mutation, affects the subcellular distribution of GL3. Using particle bombardment, we show that TTG1, GL3, GL1 and the homeodomain protein GL2 do not move between adjacent epidermal cells, while the R3-MYB, CPC, does move to neighboring cells. These data support a model for the TTG1 complex directly regulating activators and repressors and the movement of repressors to affect trichome patterning on the Arabidopsis leaf.

Transcription factor AtMYB103 is required for anther development by regulating tapetum development, callose dissolution and exine formation in Arabidopsis

Downregulation of the transcription factor AtMYB103 using transgenic technology results in early tapetal degeneration and pollen aberration during anther development in Arabidopsis thaliana. This paper describes the functional analysis of the AtMYB103 gene in three knock-out mutants. Two male sterile mutants, ms188-1 and ms188-2, were generated by ethyl-methane sulfonate (EMS) mutagenesis. A map-based cloning approach was used, and ms188 was mapped to a 95.8-kb region on chromosome 5 containing an AtMYB103 transcription factor. Sequence analysis revealed that ms188-1 had a pre-mature stop codon in the AtMYB103 coding region, whereas ms188-2 had a CCT-->CTT base-pair change in the first exon of AtMYB103, which resulted in the replacement of a proline by a leucine residue in the R2R3 domain. The third mutant, an AtMYB103 transposon-tagging line, also showed a male sterile phenotype. Allelism tests indicated that MS188 and AtMYB103 belong to the same locus. Cytological observation revealed defective tapetum development and altered callose dissolution in ms188 plants. Additionally, most of the microspores in mature anthers were degraded and surviving microspores lacked exine. AtMYB103 encoded an R2R3 MYB protein that is predominantly located in the nucleus. Real-time RT-PCR analysis indicated that the callase-related gene A6 was regulated by AtMYB103. Expression of the exine formation gene MS2 was not detected in mutant anthers. These results implicate that AtMYB103 plays an important role in tapetum development, callose dissolution and exine formation in A. thaliana anthers.