R3 MYB Transcription Factors Research Articles

BoMYBL2b, an R3-MYB transcription factor, inhibits anthocyanin accumulation via directly repressing BoDFR1 gene transcription in kale

Kale is a globally popular edible and ornamental plant with colorful inner leaves. Anthocyanin is one of the most major pigments for kale. However, the underlying regulatory mechanism for the accumulation of anthocyanin in kale has not been well understood to date. Here, we characterized an R3-MYB transcription factor, BoMYBL2b, whose mRNA was missing in the anthocyanin-rich kale inbred line, but abundant in the anthocyanin-less line. Knockdown of BoMYBL2b enhanced the anthocyanin accumulation. Y1H and DLR assays showed that BoMYBL2b directly inhibited the BoDFR1 gene expression by binding to the MRE sites on the ProBoDFR1. Besides, we found that BoMYBL2b did not interact with the bHLH proteins, indicating that the BoMYBL2b might not be part of the MBW complex. BoMYBL2b expression was significantly repressed by BoMYB1R1 under high light and cold stimulus, suggesting the possibility that BoMYB1R1-BoMYBL2b module might act as a cross-node for integrating light and temperature signals to regulate anthocyanin accumulation in kale. These findings provided a theoretical basis for molecular breeding of new kale cultivars and a reference for understanding the regulatory mechanism of anthocyanin accumulation in Brassica.

CRISPR/Cas editing of a <i>CPC gene in Arabidopsis thaliana</i>

BACKGROUND: Identification of target genes responsible for visible phenotypic effect may contribute to the development of transgene-free bioengineering strategies and application of crop varieties with edited genome. CAPRICE (CPC) is a single-repeat R3 MYB transcription factor, involved in anthocyanin biosynthesis and trichome formation. It is assumed that CPC controls the expression of Dihydroflavonol-4-reductase (DFR), a key gene of anthocyanin biosynthesis. AIM: The aim of the study was to determine whether knockout of the CPC gene using CRISPR/Cas9 results in visible anthocyanin accumulation. MATERIALS AND METHODS: Three guide RNAs were designed to excise a MYB domain from the CPC gene of Arabidopsis thaliana. Anthocyanin content and expression of CPC and DFR genes were studied in edited plants. RESULTS: The expected 662 bp deletion was detected in 2,7% of glufosinate-resistant plants, however none of the mutations were homozygous. Four edited lines were studied in four generations. An upregulation of the DFR gene was observed in edited lines, however CPC gene expression, anthocyanin content and trichome development were not significantly different from those in control plants. Moreover, in A. thaliana pigmentation did not directly depend on DFR or CPC gene expression. CONCLUSIONS: Our results suggest that CPC gene is involved in regulation of DFR gene expression and anthocyanin biosynthesis pathway, however in case of mutations plants might utilize other transcription factors to maintain homeostasis. Therefore, CPC gene is not a suitable target for CRISPR/Cas studies in Arabidopsis.

Effect of shading on trichome formation and CAPRICE-like gene expression in tea (Camellia sinensis var. sinensis) leaves

Tea (Camellia sinensis var. sinensis) is one of the most popular beverages worldwide and is highly regarded for its unique taste and health benefits. The traditional practice of shading tea plants before harvest is employed to ensure the production of high-quality Japanese green tea. However, the precise effects of shading on trichome formation, leaf tissue structure, and the expression of related genes remain largely unknown. In the present study, we conducted a comparative analysis to investigate the effect of shading on the aforementioned aspects of tea leaves. Our findings revealed a significant decrease in the number of trichomes in the youngest 1st stage leaves under shaded conditions compared with that under full-light conditions. Histological analyses revealed that epidermal cells in tea leaves under shaded conditions were thinner, further indicating the influence of light on leaf structures. To delve deeper into the regulatory mechanisms, the expression levels of six Camellia sinensis CAPRICE (CsCPC) genes encoding R3 MYB transcription factors, which have been implicated in trichome formation in tea plants, were examined using real-time reverse transcription PCR. Among the six genes, CsCPC-1, Camellia sinensis ENHANCER OF TRY AND CPC 1–2 (CsETC1–2), and CsETC3 exhibited increased expression under shaded conditions, indicating their potential involvement in the light-mediated regulation of tea leaf characteristics. The expression levels of the phenylpropanoid pathway structural genes Camellia sinensis cinnamic acid 4-hydroxylase (CsC4H), Camellia sinensis cinnamoyl CoA reductase (CsCCR), and Camellia sinensis flavanone 3′ hydroxylase (CsF3′H), were significantly reduced under shaded conditions, suggesting the regulation of phenylpropanoid content. Our study demonstrated that shading treatment induces significant changes in the number of trichomes in younger leaves, alters the width of epidermal cells, and affects the expression levels of CsCPCs and genes related to the phenylpropanoid pathway. These findings have the potential to enhance tea leaf quality, and offer pragmatic implications for tea farming.

TRANSPARENT TESTA GLABRA2 defines trichome cell shape by modulating actin cytoskeleton in Arabidopsis thaliana.

The Arabidopsis (Arabidopsis thaliana) TRANSPARENT TESTA GLABRA2 (TTG2) gene encodes a WRKY transcription factor that regulates a range of development events like trichome, seed coat, and atrichoblast formation. Loss-of-function of TTG2 was previously shown to reduce or eliminate trichome specification and branching. Here, we report the identification of an allele of TTG2, ttg2-6. In contrast to the ttg2 mutants described before, ttg2-6 displayed unique trichome phenotypes. Some ttg2-6 mutant trichomes were hyper-branched, whereas others were hypo-branched, distorted, or clustered. Further, we found that in addition to specifically activating R3 MYB transcription factor TRIPTYCHON (TRY) to modulate trichome specification, TTG2 also integrated cytoskeletal signaling to regulate trichome morphogenesis. The ttg2-6 trichomes displayed aberrant cortical microtubules (cMTs) and actin filaments (F-actin) configurations. Moreover, genetic and biochemical analyses showed that TTG2 could directly bind to the promoter and regulate the expression of BRICK1 (BRK1), which encodes a subunit of the actin nucleation promoting complex suppressor of cyclic AMP repressor (SCAR)/Wiskott-Aldrich syndrome protein family verprolin homologous protein (WAVE). Collectively, taking advantage of ttg2-6, we uncovered a function for TTG2 in facilitating cMTs and F-actin cytoskeleton-dependent trichome development, providing insight into cellular signaling events downstream of the core transcriptional regulation during trichome development in Arabidopsis.

Overexpression of BnMYBL2-1 improves plant drought tolerance via the ABA-dependent pathway

Drought stress is a major environmental challenge that poses considerable threats to crop survival and growth. Previous research has indicated anthocyanins play a crucial role in alleviating oxidative damage, photoprotection, membrane stabilization, and water retention under drought stress. However, the presence of MYBL2 (MYELOBBLASTOSIS LIKE 2), an R3-MYB transcription factor (TF) which known to suppress anthocyanin biosynthesis. In this study, four BnMYBL2 members were cloned from Brassica napus L, and BnMYBL2-1 was overexpressed in Triticum aestivum L (No BnMYBL2 homologous gene was detected in wheat). Subsequently, the transgenic wheat lines were treated with drought, ABA and anthocyanin. Results showed that transgenic lines exhibited greater drought tolerance compared to the wild-type (WT), characterized by improved leaf water content (LWC), elevated levels of soluble sugars and chlorophyll, and increased antioxidant enzyme activity. Notably, transgenic lines also exhibited significant upregulation in abscisic acid (ABA) content, along with the transcriptional levels of key enzymes involved in ABA signalling under drought. Results also demonstrated that BnMYBL2-1 promoted the accumulation of ABA and anthocyanins in wheat. Overall, the study highlights the positive role of BnMYBL2-1 in enhancing crop drought tolerance through ABA signalling and establishes its close association with anthocyanin biosynthesis. These findings offer valuable insights for the development of drought-resistant crop varieties and enhance the understanding of the molecular mechanisms underlying plant responses to drought stress.

CmNAC25 targets CmMYB6 to positively regulate anthocyanin biosynthesis during the post-flowering stage in chrysanthemum

BackgroundAnthocyanin is a class of important secondary metabolites that determines colorful petals in chrysanthemum, a famous cut flower. ‘Arctic Queen’ is a white chrysanthemum cultivar that does not accumulate anthocyanin during the flowering stage. During the post-flowering stage, the petals of ‘Arctic Queen’ accumulate anthocyanin and turn red. However, the molecular mechanism underlying this flower color change remains unclear.ResultsIn this study, by using transcriptome analysis, we identified CmNAC25 as a candidate gene promoting anthocyanin accumulation in the post-flowering stage of ‘Arctic Queen’. CmNAC25 is directly bound to the promoter of CmMYB6, a core member of the MBW protein complex that promotes anthocyanin biosynthesis in chrysanthemum, to activate its expression. CmNAC25 also directly activates the promoter of CmDFR, which encodes the key enzyme in anthocyanin biosynthesis. CmNAC25 was highly expressed during the post-flowering stage, while the expression level of CmMYB#7, a known R3 MYB transcription factor interfering with the formation of the CmMYB6–CmbHLH2 complex, significantly decreased. Genetic transformation of both chrysanthemum and Nicotiana tabacum verified that CmNAC25 was a positive regulator of anthocyanin biosynthesis. Another two cultivars that turned red during the post-flowering stages also demonstrated a similar mechanism.ConclusionsAltogether, our data revealed that CmNAC25 positively regulates anthocyanin biosynthesis in chrysanthemum petals during the post-flowering stages by directly activating CmMYB6 and CmDFR. Our results thus revealed a crucial role of CmNAC25 in regulating flower color change during petal senescence and provided a target gene for molecular design breeding of flower color in chrysanthemum.

Cloning and functional analysis of RADIALIS-like 1 gene from Antirrhinum majus.

MYB is one of the largest transcription factor families in plants. Among them, the R3-MYB transcription factor RADIALIS (RAD) plays a very important role in the flowers development in Antirrhinum majus. In this study, a R3-MYB gene similar to RAD was found by analyzing the genome of A. majus, which was named AmRADIALIS-like 1 (AmRADL1). The gene function was predicted through bioinformatics. The relative expression levels in different tissues and organs of wild-type A. majus were analyzed by qRT-PCR. AmRADL1 was overexpressed in A. majus, and the transgenic plants were analyzed by morphological observation and histological staining. The results showed that the open reading frame (ORF) of AmRADL1 gene was 306 bp in length, encoding 101 amino acids. It has typical SANT domain, and the C-terminal contains a CREB motif, which was highly homologous to tomato SlFSM1. The results of qRT-PCR showed that AmRADL1 was expressed in roots, stems, leaves and flowers, and the expression level was higher in flowers. Further analysis of its expression in different floral organs showed that AmRADL1 had the highest expression in carpel. The results of histological staining analysis of the transgenic plants showed that compared with the wild type, although the size of the carpel cells of the transgenic plants did not change significantly, the placenta area in the carpel became smaller and the number of cell decreased. In summary, AmRADL1 may be involved in the regulation of carpel development, but the specific mechanism of action in carpel remains to be further studied.

Complementation and protein localization analyses of R3 MYBs in an Arabidopsis caprice mutant.

Root hairs play vital roles in plant growth since they enable the efficient absorption of water and nutrients from the soil. Recent advances in Arabidopsis research have provided a deeper understanding of the molecular genetic mechanisms underlying root hair differentiation. CAPRICE (CPC) and its four homologs, which belong to the CPC gene family and encode R3 MYB transcription factors, play central roles in root hair differentiation. In this study, to better understand the functional specificity and contribution of these five CPC family genes, we conducted phenotypic and expression analyses of the CPC family proteins in a cpc mutant background. As a result, ENHANCER OF TRY AND CPC1 (ETC1) and ETC3 were found to complement the hairless root phenotype of the cpc mutant, as did CPC, whereas TRIPTYCHON (TRY) and ETC2 did not rescue the cpc phenotype. Protein expression analysis revealed that GFP fluorescence was nearly undetectable in pCPC::TRY:GFP/cpc and pCPC::ETC2:GFP/cpc plants, supporting the incapability of root hair formation in these plants. Interestingly, the fluorescence intensity of the CPC:GFP fusion protein was weaker than that of ETC1:GFP and ETC3:GFP fusion proteins. These results were inconsistent with the result of the phenotypic analysis, in which the three genes promoted root hair formation to almost the same degree in the cpc mutant background. We further discuss the discrepancy between the root hair phenotypes and the expression levels of CPC family proteins.

The R3-Type MYB Transcription Factor BrMYBL2.1 Negatively Regulates Anthocyanin Biosynthesis in Chinese Cabbage (Brassica rapa L.) by Repressing MYB–bHLH–WD40 Complex Activity

Chinese cabbage (Brassica rapa L.) leaves are purple in color due to anthocyanin accumulation and have nutritional and aesthetic value, as well as antioxidant properties. Here, we identified the R3 MYB transcription factor BrMYBL2.1 as a key negative regulator of anthocyanin biosynthesis. A Chinese cabbage cultivar with green leaves harbored a functional BrMYBL2.1 protein, designated BrMYBL2.1-G, with transcriptional repressor activity of anthocyanin biosynthetic genes. By contrast, BrMYBL2.1 from a Chinese cabbage cultivar with purple leaves carried a poly(A) insertion in the third exon of the gene, resulting in the insertion of multiple lysine residues in the predicted protein, designated BrMYBL2.1-P. Although both BrMYBL2.1 variants localized to the nucleus, only BrMYBL2.1-G interacted with its cognate partner BrTT8. Transient infiltration assays in tobacco leaves revealed that BrMYBL2.1-G, but not BrMYBL2.1-P, actively represses pigment accumulation by inhibiting the transcription of anthocyanin biosynthetic genes. Transient promoter activation assay in Arabidopsis protoplasts verified that BrMYBL2.1-G, but not BrMYBL2.1-P, can repress transcriptional activation of BrCHS and BrDFR, which was activated by co-expression with BrPAP1 and BrTT8. We determined that BrMYBL2.1-P may be more prone to degradation than BrMYBL2.1-G via ubiquitination. Taken together, these results demonstrate that BrMYBL2.1-G blocks the activity of the MBW complex and thus represses anthocyanin biosynthesis, whereas the variant BrMYBL2.1-P from purple Chinese cabbage cannot, thus leading to higher anthocyanin accumulation.

TRIPTYCHON-LIKE regulates aspects of both fruit flavor and color in citrus

Deciphering the genetic basis of organoleptic traits is critical for improving the quality of fruits, which greatly shapes their appeal to consumers. Here, we characterize the citrus R3-MYB transcription factor TRIPTYCHON-LIKE (CitTRL), which is closely associated with the levels of citric acid, proanthocyanidins (PAs), and anthocyanins. Overexpression of CitTRL lowered acidity levels and PA contents in citrus calli as well as anthocyanin and PA contents in Arabidopsis leaves and seeds. CitTRL interacts with the two basic helix-loop-helix (bHLH) proteins CitbHLH1 and ANTHOCYANIN 1 (CitAN1) to regulate fruit quality. We show that CitTRL competes with the R2R3-MYB CitRuby1 for binding to CitbHLH1 or CitAN1, thereby repressing their activation of anthocyanin structural genes. CitTRL also competes with a second R2R3-MYB, CitPH4, for binding to CitAN1, thus altering the expression of the vacuolar proton-pump gene PH5 and Leucoanthocyanidin reductase, responsible for vacuolar acidification and proanthocyanidins biosynthesis, respectively. Moreover, CitPH4 activates CitTRL transcription, thus forming an activator-repressor loop to prevent the overaccumulation of citric acid and PAs. Overall, this study demonstrates that CitTRL acts as a repressor of the accumulation of citric acid, PAs, and anthocyanins by a cross-regulation mechanism. Our results provide an opportunity to simultaneously manipulate these key traits as a means to produce citrus fruits that are both visually and organoleptically appealing.

R3-MYB transcription factor LcMYBx from Litchi chinensis negatively regulates anthocyanin biosynthesis by ectopic expression in tobacco.

Anthocyanin accumulation is one of the remarkable physiological changes during fruit ripening. In plants, anthocyanin synthesis is regulated by MYB activators, but the MYB repressors has been recognized recently. Here, we isolated a repressor of anthocyanin synthesis, LcMYBx, from Litchi chinensis Sonn. LcMYBx encoded a typical R3-MYB protein and contained a conserved [D/E]Lx2[R/K]x3Lx6Lx3R motif for interacting with bHLH proteins. Overexpression of LcMYBx in tobacco suppressed anthocyanin accumulation resulting in faded petals from pale-pink to almost white. Gene expression analysis showed the strong down-regulation of endogenous anthocyanin structural and regulatory genes by LcMYBx overexpression. Yeast two-hybrid and bimolecular fluorescence complementation assays indicated that LcMYBx could interact with the transcription factors LcbHLH1 and LcbHLH3. Transient promoter activation assays showed that LcMYBx could inhibit the activation capacity of LcMYB1-LcbHLH3 complex for LcDFR gene. These results suggest that LcMYBx competed with LcMYB1 to LcbHLHs, thus preventing the activation of LcDFR by LcMYB1-LcbHLHs complex and negatively controlling anthocyanin biosynthesis.

Comparative transcriptomic analysis of Rosa sterilis inflorescence branches with different trichome types reveals an R3-MYB transcription factor that negatively regulates trichome formation

Rosa sterilis S. D. Shi is an important economic tree in China that produces fruits with high nutritional and medicinal value. Many of R. sterilis' organs are covered with different types of trichomes or prickles that directly affect fruit appearance and plant management. This study used RNA sequencing technology to analyze the transcriptomes of two parts of the inflorescence branch, namely inflorescence stems with flagellated trichomes and pedicels with both flagellated and glandular trichomes. Comparative transcriptomic analysis showed that many transcription factors (TFs) are potentially involved in the formation and development of trichomes. The accumulation of RsETC1, a TF of the R3-MYB family, was significantly higher in inflorescence stems than in pedicels; quantitative reverse transcription PCR (qRT-PCR) verified that its expression was significantly higher in inflorescence stems than in pedicels during the first three development stages, indicating its inhibitory action on the initiation of glandular trichomes in R. sterilis. The mRNA level of RsETC1 accumulated to significantly higher levels in trichomeless tissues than in tissues with trichromes, suggesting that this gene may inhibit the formation of trichomes in R. sterilis. Over-expression of RsETC1 in Arabidopsis resulted in glabrous phenotypes, and the expression of trichome-related endogenous genes, except for TTG1, was markedly reduced. In addition, the contents of the phytohormones jasmonic acid (JA), gibberellin A3 (GA3), and cytokinins (CKs) in pedicels were significantly higher than those in inflorescence stems, and the expression patterns of the genes related to hormone biosynthesis and signal transduction presented consistent responses, suggesting that the transduction of these hormones might be crucial for trichome initiation and development. These data provide a new perspective for revealing the molecular mechanism of trichome formation in R. sterilis.

Identification and expression analysis of R2R3-MYB gene family in Andrographis paniculata

The plant growth, development, and secondary metabolism are regulated by R2 R3-MYB transcription factors. This study identified the R2 R3-MYB genes in the genome of Andrographis paniculata and analyzed the chromosomal localization, gene structure, and conserved domains, phylogenetic relationship, and promoter cis-acting elements of these R2 R3-MYB genes. Moreover, the gene expression profiles of R2 R3-MYB genes under abiotic stress and hormone treatments were generated by RNA-seq and validated by qRT-PCR. The results showed that A. paniculata contained 73 R2 R3-MYB genes on 21 chromosomes. These members belonged to 34 subfamilies, 19 of which could be classified into the known subfamilies in Arabidopsis thaliana. The 73 R2 R3-MYB members included 36 acidic proteins and 37 basic proteins, with the lengths of 148-887 aa. The domains, motifs, and gene structures of R2 R3-MYBs in A. paniculata were conserved. The promoter regions of these genes contains a variety of cis-acting elements related to the responses to environmental factors and plant hormones including light, ABA, MeJA, and drought. Based on the similarity of functions of R2 R3-MYBs in the same subfamily and the transcription profiles, ApMYB13/21/35/67/73(S22) may regulate drought stress through ABA pathway; ApMYB20(S11) and ApMYB55(S2) may play a role in the response of A. paniculata to high temperature and UV-C stress; ApMYB5(S7) and ApMYB33(S20) may affect the accumulation of andrographolide by regulating the expression of key enzymes in the MEP pathway. This study provides theoretical reference for further research on the functions of R2 R3-MYB genes in A. paniculata and breeding of A. paniculata varieties with high andrographolide content.

Identification and expression profiling of R2R3-MYB transcription factors in Erigeron breviscapus

R2 R3-MYB transcription factors are ubiquitous in plants, playing a role in the regulation of plant growth, development, and secondary metabolism. In this paper, the R2 R3-MYB transcription factors were identified by bioinformatics analysis of the genomic data of Erigeron breviscapus, and their gene sequences, structures, physical and chemical properties were analyzed. The functions of R2 R3-MYB transcription factors were predicted by cluster analysis. Meanwhile, the expression patterns of R2 R3-MYB transcription factors in response to hormone treatments were analyzed. A total of 108 R2 R3-MYB transcription factors, named EbMYB1-EbMYB108, were identified from the genome of E. breviscapus. Most of the R2 R3-MYB genes carried 2-4 exons. The phylogenetic tree of MYBs in E. breviscapus and Arabidopsis thaliala was constructed, which classified 234 MYBs into 30 subfamilies. The MYBs in the five MYB subfamilies of A.thaliala were clustered into independent clades, and those in E. breviscapus were clustered into four clades. The transcriptome data showed that MYB genes were differentially expressed in different tissues of E. breviscapus and in response to the treatments with exogenous hormones such as ABA, SA, and GA for different time. The transcription of 13 R2 R3-MYB genes did not change significantly, and the expression patterns of some genes were up-regulated or down-regulated with the extension of hormone treatment time. This study provides a theoretical basis for revealing the mechanisms of R2 R3-MYB transcription factors in regulating the growth and development, stress(hormone) response, and active ingredient accumulation in E. breviscapus.

Function of the TRY C-terminal region artificially fused with its homologous transcription factors inducing root hair differentiation in Arabidopsis.

TRIPTYCHON (TRY) is one of the R3-MYB transcription factors. Its extended C-terminal 19 amino-acid region (CTRY) is considered to affect the ability of root hair differentiation in Arabidopsis. Here, to further understand the function of CTRY, it, together with GFP, was artificially fused with TRY homologs, CPC and ETC1, which do not contain such extended regions and induce root hair differentiation. Arabidopsis transgenic plants carrying the fusion proteins, CPC-CTRY-GFP and ETC1-CTRY-GFP, induced root hair differentiation as observed in those carrying the original proteins without CTRY. The expression levels of the fusion proteins in the transgenic plants were essentially the same as those of the original proteins, although their subcellular localization to nuclei of root epidermal cells was slightly changed by CTRY. Therefore, CTRY does not affect the ability of CPC and ETC1 to induce root hair differentiation when artificially fused, and its function may be restricted in TRY.

CmMYB#7, an R3 MYB transcription factor, acts as a negative regulator of anthocyanin biosynthesis in chrysanthemum.

'Jimba', a well-known white flowered chrysanthemum cultivar, occasionally and spontaneously produces red colored petals under natural cultivation, but there is little information about the molecular regulatory mechanism underlying this process. We analysed the expression patterns of 91 MYB transcription factors in 'Jimba' and 'Turning red Jimba' and identified an R3 MYB, CmMYB#7, whose expression was significantly decreased in 'Turning red Jimba' compared with 'Jimba', and confirmed it is a passive repressor of anthocyanin biosynthesis. CmMYB#7 competed with CmMYB6, which together with CmbHLH2 is an essential component of the anthocyanin activation complex, for interaction with CmbHLH2 through the bHLH binding site in the R3 MYB domain. This reduced binding of the CmMYB6-CmbHLH2 complex and inhibited its ability to activate CmDFR and CmUFGT promoters. Moreover, using transient expression assays we demonstrated that changes in the expression of CmMYB#7 accounted for alterations in anthocyanin content. Taken together, our findings illustrate that CmMYB#7 is a negative regulator of anthocyanin biosynthesis in chrysanthemum.

Repression of anthocyanin biosynthesis by R3-MYB transcription factors in lily (Lilium spp.).

Lily R3-MYB transcription factors are involved in negative regulation to limit anthocyanin accumulation in lily flowers and leaves and create notable color patterns on ectopically expressed petunia flowers. In eudicots, both positive and negative regulators act to precisely regulate the level of anthocyanin accumulation. The R3-MYB transcription factor is among the main factors repressing anthocyanin biosynthesis. Although, in monocots, the positive regulators have been well characterized, the negative regulators have not been examined. Two R3-MYBs, LhR3MYB1 and LhR3MYB2, which were identified in lily transcriptomes, were characterized in this study to understand the regulatory mechanisms of anthocyanin biosynthesis. LhR3MYB1 and LhR3MYB2 had a C2 suppressor motif downstream of a single MYB repeat; the similar amino acid motif appears only in AtMYBL2 among the eudicot R3-MYB proteins. Stable and transient overexpression of LhR3MYB1 and LhR3MYB2 in tobacco plants showed suppression of anthocyanin biosynthesis by both; however, suppression by LhR3MYB2 was stronger than that by LhR3MYB1. In the lily plant, the LhR3MYB2 transcript was detected in leaves with light stimulus-induced anthocyanin accumulation and in pink tepals. Although LhR3MYB1 was expressed in some, but not all tepals, its expression was not linked to anthocyanin accumulation. In addition, LhR3MYB1 expression levels in the leaves remained unchanged by the light stimulus, and LhR3MYB1 transcripts predominantly accumulated in the ovaries, which did not accumulate anthocyanins. Thus, although LhR3MYB1 and LhR3MYB2 have an ability to repress anthocyanin accumulation, LhR3MYB2 is more strongly involved in the negative regulation to limit the accumulation than that by LhR3MYB1. In addition, the overexpression of LhR3MYB2 generated notable color patterns in petunia flowers; thus, the usefulness of the LhR3MYB genes for creating unique color patterns by genetic engineering is discussed.

Single-repeat R3 MYB transcription factors from Platanus acerifolia negatively regulate trichome formation in Arabidopsis.

Four R3 MYB genes were cloned and identified from Platanus acerifolia and analysed according to endogenous gene expression profiles, protein-protein interaction patterns, phenotypic effects and related gene expression profiles in transgenic Arabidopsis, suggesting that London plane R3 MYB genes inhibit trichome formation in Arabidopsis. The CPC-like MYB transcription factors including CAPRICE (CPC), TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC 1, 2 and 3 (ETC1, ETC2 and ETC3), TRICHOMELESS1 (TCL1) and TRICHOMELESS2(TCL2) play important roles in controlling trichome patterning in Arabidopsis. In this study, four sequences homologous with the Arabidopsis CPC family were identified from London plane and named PaTRY, PaCPC-like1, PaCPC-like2 and PaCPC-like3. Over-expression of PaTRY, PaCPC-like1, PaCPC-like2 and PaCPC-like3 in Arabidopsis resulted in glabrous phenotypes. In addition, expression of endogenous GL2, GL1, MYB23, TTG2 and a set of R3 MYB-encoding genes was markedly reduced. Furthermore, the protein products of PaTRY, PaCPC-like1, PaCPC-like2 and PaCPC-like3 were shown to interact with PaGL3 in yeast two-hybrid assays. Together, these results likely suggest that the mechanisms of trichome regulation in London plane have similarities with those in Arabidopsis.

Genetic evidence suggests that GIS functions downstream of TCL1 to regulate trichome formation in Arabidopsis

BackgroundTrichome formation in Arabidopsis is regulated by a MBW complex formed by MYB, bHLH and WD40 transcriptional factors, which can activate GLABRA2 (GL2) and the R3 MYB transcription factor genes. GL2 promotes trichome formation, whereas R3 MYBs are able to block the formation of the MBW complex. It has been reported that the C2H2 transcription factor GIS (GLABROUS INFLORESCENCE STEMS) functions upstream of the MBW activator complex to regulate trichome formation, and that the expression of TCL1 is not regulated by the MBW complex. However, gis and the R3 MYB gene mutant tcl1 (trichomeless 1) have opposite inflorescence trichome phenotypes, but their relationship in regulating trichome formation remained unknown.ResultsBy generating and characterization of the gis tcl1 double mutant, we found that trichome formation in the gis tcl1double and the tcl1 single mutants were largely indistinguishable, but the trichome formation in the 35S:TCL1/gis transgenic plant was similar to that in the gis mutant. By using quantitative RT-PCR analysis, we showed that expression level of GIS was increased in the triple mutant tcl1 try cpc, but the expression level of TCL1 was not affected in the gis mutant. On the other hand, trichome morphology in both gis tcl1 and 35S:TCL1/gis plants was similar to that in the gis mutant.ConclusionsIn summary, our results indicate that GIS may work downstream of TCL1 to regulate trichome formation, and GIS has a dominant role in controlling trichome morphology.

Identification and Functional Characterization of R3 MYB Transcription Factor Genes in Soybean

In Arabidopsis, trichome formation is regulated by a MYB-bHLH-WD40 (MBW) transcriptional activator complex, which can activate the expression of GLABRA2 (GL2) and R3 MYB genes. GL2 is required for trichome formation, whereas R3 MYBs inhibit trichome formation by blocking the formation of the MBW complex, thus inhibiting the expression of GL2. By using the amino acid sequence of the Arabidopsis R3 MYB transcription factor TRICHOMELESS1 (TCL1) to BLAST the soybean (Glycine max) protein database, we found that there are a total of six R3 MYB genes in soybean, namely Glycine max TRICHOMELESS1 through 6 (GmTCL1-GmTCL6). By generating transgenic plants, we found that trichome formation in soybean plants overexpressing each of the GmTCLs remained largely unchanged, and the expression of putative GL1 and GL2 genes in the transgenic plants was unaffected. However, all the GmTCLs interacted with GLABRA3 (GL3) in transfected Arabidopsis protoplasts, expression each of the GmTCLs in Arabidopsis inhibited trichome formation, and the expression levels of GL1 and GL2 were greatly reduced in the Arabidopsis transgenic plants. Moreover, phenotypic complementary analysis showed that GmTCL1 is functionally equivalent to TCL1. Taken together, these results suggest that GmTCLs are functional R3 MYBs, however, they do not regulate trichome formation in soybean.