R2R3-MYB Protein Research Articles

T and Z, partial seed coat patterning genes in common bean, provide insight into the structure and protein interactions of a plant MBW complex.

Flavonoids are secondary metabolites associated with plant seed coat and flower color. These compounds provide health benefits to humans as anti-inflammatory and antioxidant compounds. The expression of the late biosynthetic genes in the flavonoid pathway is controlled by a ternary MBW protein complex consisting of interfacing MYB, beta-helix-loop-helix (bHLH), and WD40 Repeat (WDR) proteins. P, the master regulator gene of the flavonoid expression in common bean (Phaseolus vulgaris L.), was recently determined to encode a bHLH protein. The T and Z genes control the distribution of color in bean seeds and flowers and have historically been considered regulators of the flavonoid gene expression. T and Z candidates were identified using reverse genetics based on genetic mapping, phylogenetic analysis, and mutant analysis. Domain and AlphaFold2 structure analyses determined that T encodes a seven-bladed β-propeller WDR protein, while Z encodes a R2R3 MYB protein. Deletions and SNPs in T and Z mutants, respectively, altered the 3D structure of these proteins. Modeling of the Z MYB/P bHLH/T WDR MBW complex identified interfacing sequence domains and motifs in all three genes that are conserved in dicots. One Z MYB motif is a possible beta-molecular recognition feature (β-MoRF) that only appears in a structured state when Z MYB is modeled as a component of a MBW complex. Complexes containing mutant T and Z proteins changed the interaction of members of the complex in ways that would alter their role in regulating the expression of genes in the flavonoid pathway.

R2R3-MYB repressor, BrMYB32, regulates anthocyanin biosynthesis in Chinese cabbage.

Anthocyanin-enriched Chinese cabbage has health-enhancing antioxidant properties. Although various regulators of anthocyanin biosynthesis have been identified, the role of individual repressors in this process remains underexplored. This study identifies and characterizes the R2R3-MYB BrMYB32 in Chinese cabbage (Brassica rapa), which acts as a repressor in anthocyanin biosynthesis. BrMYB32 expression is significantly upregulated under anthocyanin inductive conditions, such as sucrose and high light treatment. Transgenic tobacco plants overexpressing BrMYB32 show decreased anthocyanin levels and downregulation of anthocyanin biosynthesis genes in flowers, highlighting BrMYB32's repressive role. Located in the nucleus, BrMYB32 interacts with the TRANSPARENT TESTA 8 (BrTT8), a basic helix-loop-helix protein, but no interaction was detected with the R2R3-MYB protein PRODUCTION OF ANTHOCYANIN PIGMENT 1 (BrPAP1). Functional assays in Chinese cabbage cotyledons and tobacco leaves demonstrate that BrMYB32 represses the transcript level of anthocyanin biosynthesis genes, thereby inhibiting pigment accumulation. Promoter activation assays further reveal that BrMYB32 inhibits the transactivation of CHALCONE SYNTHASE and DIHYDROFLAVONOL REDUCTASE through the C1 and C2 motifs. Notably, BrMYB32 expression is induced by BrPAP1, either alone or in co-expression with BrTT8, and subsequently regulates the expression of these activators. It verifies that BrMYB32 not only interferes with the formation of an active MYB-bHLH-WD40 complex but also downregulates the transcript levels of anthocyanin biosynthesis genes, thereby fine-tuning anthocyanin biosynthesis. Our findings suggest a model in which anthocyanin biosynthesis in Chinese cabbage is precisely regulated by the interplay between activators and repressors.

Mechanistic and structural insight into R2R3-MYB transcription factor in plants: molecular dynamics based binding free energy analysis

The MYB transcription factor (TF) family is essential for various plant growth and development processes, including responses to biotic and abiotic stresses. This study investigated the R2R3-MYB protein structure from five plants, including cereal crops. The R2R3–MYB protein structure was docked with the DNA structure, and the best complexes were selected for two runs of molecular dynamics (MD) simulations to investigate the key interacting residues and conformational changes in the R2R3-MYB proteins caused by DNA binding. The MM/PBSA method calculated the binding free energy for each R2R3–MYB protein–DNA complex, showing strong interaction. Hydrophobic and hydrogen bonds significantly stabilized the R2R3–MYB protein–DNA complexes. The principal component analysis showed high restrictions on the movement of protein atoms in the phase space. A similar MD simulation analysis was performed using the crystal structure of the R2R3–MYB protein–DNA complex from Arabidopsis thaliana, and the generated complexes resembled the X-ray crystal structure. This is the first detailed study on the R2R3–MYB protein–DNA complex in cereal crops, providing a cost-effective solution to identify the key interacting residues and analyze the conformational changes in the MYB domain before and after DNA binding. Communicated by Ramaswamy H. Sarma

PgMYB1 Positively Regulates Anthocyanin Accumulation by Activating PgGSTF6 in Pomegranate

The peel color of pomegranates is an important exterior quality that determines market value. Anthocyanins are biosynthesized in the cytosol and then transported to the vacuole for storage. However, the molecular mechanism that determines the color variation between red and white pomegranates remains unclear. In this study, we identified an R2R3-MYB protein (PgMYB1) that interacts with the PgGSTF6 promoter and regulates its transcriptional expression, thus promoting the accumulation of anthocyanins in pomegranate. The expression of PgMYB1 and PgGSTF6 was positively correlated with the anthocyanin content in red and white pomegranates. Further investigation showed that the knockdown of PgMYB1 in red pomegranate 'Taishanhong' (TSH), by the virus-induced gene-silencing system, inhibited anthocyanin accumulation. Together, our results indicate that PgMYB1 controls the transport of anthocyanin via PgGSTF6 and thus promotes anthocyanin accumulation in red pomegranates. Our results have a certain reference value for further clarifying the regulation of anthocyanin synthesis and transport in pomegranate fruits.

Coexpression Network Analysis Based Characterisation of the R2R3-MYB Family Genes in Tolerant Poplar Infected with Melampsora larici-populina

R2R3-MYB protein is the most abundant class of MYB transcription factor family in plants. The transcript profiles of two E4 races of Melampsora larici-populina-tolerant poplars and an intolerant poplar were investigated to characterise the role of the R2R3-MYB family genes in the poplar–E4 interaction. In this study, 217 R2R3-MYBs were identified, and 83 R2R3-MYB genes were assigned to 22 different coexpression modules by weighted gene coexpression network analysis. Most R2R3-MYB genes were unchanged in the early period of E4 infection in both tolerant and intolerant poplars. However, there were obvious increases in differentially expressed R2R3-MYB genes in tolerant poplars at 2 and 4 dpi when defence responses occurred, suggesting that differently expressed R2R3-MYB genes at these time points may play an important role in poplar resistance to E4 infection. In total, 34 R2R3-MYB genes showed differential expression at 2 and 4 dpi between tolerant and intolerant poplars. Among them, 16 differentially expressed R2R3-MYB genes were related to 43 defence-related genes that had significant differences between tolerant and intolerant poplars. There might be coregulatory relationships between R2R3-MYBs and other TFs during poplar–E4 interaction. Some differentially expressed R2R3-MYB genes were related to genes involved in flavonoid biosynthesis and IAA or free SA signal transduction and might help activate defence response during poplar–E4 interaction. MYB194 could be an important node in the convergence of IAA and SA signalling.

The Pea R2R3-MYB Gene Family and Its Role in Anthocyanin Biosynthesis in Flowers.

Pea (Pisum sativum L.) is one of the most important legume crops in the world, and it has attracted great attention for its high nutritive values. Recently, the crop breeding program has been focused on the crop metabolic engineering (i.e., color, flavor, nutrition) to improve the quality of crop. As a major group of transcription factors forming the ternary MYB–bHLH–WD repeat protein (MBW) complex to regulate the anthocyanin biosynthesis pathway, members of R2R3-MYB gene family have always been the focus of research targets to improve the valuable metabolic product of crops. Until now, few report about the R2R3-MYB gene family of pea has been released. In this study, we identified 119 R2R3-MYB genes in the assembled pea genome (Version 1a), of which 111 were distributed across 14 chromosomes. Combining with the 126 R2R3-MYB protein sequences of Arabidopsis, we categorized 245 R2R3-MYB proteins into 36 subgroups according to sequence similarity and phylogenetic relationships. There was no member from subgroup 12, 15 and 29 existing in pea genome, whereas three novel subgroups were found in pea and named as N1-N3. Further analyses of conserved domains and Motifs, gene structures, and chromosomal locations showed that the typical R2 and R3 domains were present across all R2R3-MYB proteins, and Motif 1, 2, and 3 were identified in most members. Most of them had no more than two introns. Additionally, 119 pea R2R3-MYB genes did not experience large-scale duplication events. Finally, we concluded that several candidate genes may be responsible for the spatiotemporal accumulation of anthocyanins in pea petals. PsMYB116 was predominantly expressed in the dorsal petals to presumably activate the anthocyanin biosynthesis pathway, while PsMYB37 and PsMYB32 may positively regulates the anthocyanin accumulation in the lateral petals. This study not only provides a good reference to further characterize the diverse functions of R2R3-MYB genes but also helps researchers to understand the color formation of pea flowers.

A rice R2R3‐MYB (OsC1) transcriptional regulator improves oxidative stress tolerance by modulating anthocyanin biosynthesis

The R2R3 type MYB transcription factors participate in controlling flavonoid production in plants, including anthocyanin and proanthocyanin. Black rice with high anthocyanin content is an important candidate for understanding R2R3-MYB-based regulation of the anthocyanin biosynthesis pathway (ABP). This study was undertaken to draw the functional relationship of an R2R3-MYB protein with anthocyanin biosynthesis and oxidative stress tolerance in plants. The expression levels of the late ABP genes in the panicle stage of black rice were in good agreement with the accumulation of anthocyanin, especially cyanidin 3-glucoside. Among all MYB genes present in rice, an R2R3 type (C1) regulates anthocyanin biosynthesis and was studied further. The positive correlation between the expression of ABP genes and OsC1 along with the nuclear localization of OsC1 are in line with its possible involvement as a transcriptional regulator of ABP genes. Interestingly, OsC1 overexpressed in white rice plants triggered anthocyanin production through augmentation of the transcript level of late ABP genes. Moreover, OsC1-transformed plants exhibited a lower amount of reactive oxygen species upon exposure to oxidative stress. The increased anthocyanin content in white rice seedlings resulted in higher photosynthetic efficiency, less membrane damage and consequently lower oxidative stress. The OsC1 transcriptional regulator helps to ameliorate oxidative stresses in plants owing to its anthocyanin modulating ability.

Genetic variation in MYB5_A12 is associated with fibre initiation and elongation in tetraploid cotton

The MYB gene family, which is predominantly composed of the R2R3-MYB family, is large and functionally diverse in plants including cotton (Gossypium hirsutum L.) (Guan et al., 2014; Tian and Zhang, 2021; Walford et al., 2011; Wan et al., 2016; Wang et al., 2019; Wu et al., 2018). In Arabidopsis, the R2R3-MYB protein AtMYB5, which belongs to the GL1 branch in the phylogenetic tree, is a component of the MYB-bHLH-WD40 protein complex that directly regulates the expression of GL2 to control the fate of seed coat outer epidermal cells and plant hairs (Gonzalez et al., 2009). However, the role of the homolog of AtMYB5 in cotton is currently unknown.

Identification and expression profiling of MYB transcription factors related to l-theanine biosynthesis in Camellia sinensis

The MYB proteins belong to a large family of transcription factors in plant genomes and play significant roles in primary and secondary metabolism. Although several CsMYB genes have been identified in Camellia sinensis, few CsMYBs involved in l-theanine biosynthesis have been analyzed. In this study, we screened and identified 20 CsMYBs related to l-theanine biosynthesis. Transcriptomic analysis revealed that the expression profiles of the CsMYBs were positively or negatively related to dynamic changes in the l-theanine content. Validation of selected l-theanine biosynthetic and CsMYB genes was conducted by qRT-PCR. The results illustrated that most of the structural and CsMYB genes were downregulated with a decrease in the l-theanine levels. Protein–protein interaction networks of CsMYB5, CsMYB12 and CsMYB94 proteins demonstrated that they might form complexes with bHLH and WD 40 proteins. Multiple DNA-binding sites of the R2R3-MYB protein were observed in promoter regions of structural genes, indicating CsMYB family proteins might be involved in l-theanine metabolism via the attachment of AC elements. Moreover, CsMYB73 demonstrated binding specificity to the promoter region of CsGDH2 (CsGDH2-pro). These findings provide fundamental understanding of specific members of the CsMYBs related to the l-theanine biosynthesis pathway.

Genome-wide DNA methylation analysis of paulownia with phytoplasma infection.

DNA methylation, an important epigenetic modification, regulates a wide range of biological processes. Previous MSAP results showed that the occurrence of PaWB related to changes of DNA methylation level; however, the relationship between DNA methylation and gene expression remains obscure in paulownia. Therefore, in the present study, we applied WGBS and RNA-seq techniques to investigate the DNA methylation and gene expression changes between healthy Paulownia fortunei seedlings and the phytoplasma-infected ones. A map of methylated cytosines at the single base pair resolution of paulownia was constructed. Compared to the healthy seedlings, the DNA methylation level increased after phytoplasma infection, and the change of mCHH was the main methylation pattern. DMR analysis showed that 422,662 DMRs in the genome were identified, in which, 27,871 DMR-associated genes were differentially expressed. Finally, 436 genes with significant differences in their methylation levels and mRNA expression profiles were identified through integrated analysis of the DNA methylomic and transcriptomic. KEGG pathway analysis revealed that these genes are mainly involved in plant hormone signal transduction, carbon metabolism, and starch and sucrose metabolism pathways. Two of DMR-associated genes were verified by BS- PCR. Finally, we selected TRP 1 and R2R3-MYB protein were closely related to the occurrence of PaWB. Our findings provide valuable insight into the mechanism of PaWB at the epigenetic level.

A minor QTL, SG3, encoding an R2R3-MYB protein, negatively controls grain length in rice.

SG3, an R2R3 MYB protein coding gene that tightly linked to a major QTLGS3, negatively regulates grain length while dependent on the status ofGS3in rice. It is often very difficult to isolate a minor QTL that is closely linked to a major QTL in rice. In this study, we focused on the isolation of a minor grain length QTL, small grain 3 (SG3), which is closely linked to the major QTL grain size 3 (GS3). The genetic effect of SG3 on grain length was dependent on GS3 status. Its genetic effect was larger in the presence of nonfunctional sg3 than functional SG3. A large number of near-isogenic F2 plants in which GS3 was fixed with nonfunctional alleles were developed to clone SG3. A gene encoding an R2R3 MYB domain transcriptional regulator was identified as the candidate gene for SG3. SG3 overexpression and knockdown plants showed shortened and elongated grains, respectively, which demonstrated that SG3 acts as a negative regulator of grain length. SG3 was preferentially expressed in panicles after flowering, and SG3 acted as a transcription activator. Comparative sequencing analysis identified a 12-bp insertion in the third exon of NYZ that led to a frameshift and resulted in a premature stop codon. The insertion/deletion was associated with grain length in the presence of functional GS3 in the indica subspecies. SG3 and GS3 were frequently in coupling phase in indica rice, making them good targets for the breeding of cultivars with short or long grains. The isolation of the SG3 MYB gene provides new gene resource and contributes to the regulatory network of grain length in rice.

Identification and Analysis of <i>R2R3-MYB</i> Genes in Sweet Potato Genome

The plant MYB is a transcription factor family large in number and with important functions. In this study, the MYB family genes were screened and identified via bioinformatic methods from the raw sequence of sweet potato ( Ipomoea batatas ) genome and the gene structure and function of R2R3-MYB were analyzed. The results showed that there were 88 R2R3-MYB genes with intact R2 and R3 conservative domains, which contained 8 and 9 highly conserved basic amino acids. The results of MEME analysis showed that there were 10 conserved motifs within the I. batatas R2R3-MYB protein sequences. For the I. batatas R2R3-MYB protein sequences, over 80% contained motif 1, motif 2, motif 3, motif 4, motif 5 and motif 7. The R2R3-MYB genes were distributed unevenly across the 15 chromosomes. The number of R2R3-MYB genes in chromosome No.5 was 15, which was the largest; the numbers of R2R3-MYB genes in chromosome No. 4 and 13 were both only 2, which was the smallest. Analysis of the sequence alignment showed that there were 6 pairs of interchromosomal duplication and there were 20 pairs of intrachromosomal duplication, 19 of which existed in clusters. The function prediction and categorization via sequence analysis showed that 44 R2R3-MYB genes of the I. batatas could be categorized to the 13 subgroups of the A. thaliana R2R3-MYB genes, which were involved in the responses to biotic stress and abiotic stress, anthocyanin biosynthesis, anther development, etc. Further analysis showed that 36 R2R3-MYB genes might play important roles in dealing with biotic stress and abiotic stress, 9 of which showed significant up/down-regulation under Fusarium oxysporum f. sp. batatas stress and 27 of which showed significant up/down-regulation under low temperature stress. The domains of I. batatas R2R3-MYB transcription factors were highly conservative, which contained highly conserved motifs within R2 and R3 domains. The phylogenetic tree and transcriptomics data analysis showed that some R2R3-MYB genes might play roles in growth and development, metabolism regulation, biotic stress and abiotic stress, which lent support to I. batatas breeding.

The R2R3-MYB transcription factor BnaMYB111L from rapeseed modulates reactive oxygen species accumulation and hypersensitive-like cell death

As one of the largest families of transcription factors in plants, the R2R3-MYB proteins play important roles in diverse biological processes including growth and development, primary and secondary metabolism such as flavonoid and anthocyanin biosynthesis as well as abiotic and biotic stress responses. However, functions of R2R3-MYB genes in rapeseed (Brassica napus L.) remain elusive. Here, we characterized BnaMYB111L, which is homologous to Arabidopsis MYB111 and encodes an R2R3-MYB protein in rapeseed. BnaMYB111L is responsive to abscisic acid (ABA), heat, cold, hydrogen peroxide and fungal pathogen Sclerotinia scelerotiorum treatments through quantitative RT-PCR assay. BnaMYB111L encodes a transcriptional activator and is localized exclusively to nuclei. Interestingly, overexpression of BnaMYB111L in tobacco (Nicotiana benthamiana) and rapeseed protoplasts promoted reactive oxygen species (ROS) production and hypersensitive response-like cell death, accumulation of malondialdehyde (MDA) as well as degradation of chlorophyll. Furthermore, BnaMYB111L expression evoked the alterations of transcript levels of genes encoding ROS-producing enzyme, vacuolar processing enzymes and genes implicated in defense responses. A further dual luciferase reporter assay indicated that BnaMYB111L activated the expression of RbohB, MC4 and ACRE132, which are involved in ROS generation, cell death as well as defense responses. Taken together, this study characterized the function of rapeseed MYB111L and identified its putative target genes involved in ROS production and cell death.

Evolution of the R2R3-MYB gene family in six Rosaceae species and expression in woodland strawberry

R2R3-MYB gene family play important roles in plants development, metabolism, and responses to various biotic and abiotic stresses. In this study, 838 R2R3-MYB genes were identified from six Rosaceae species, including 105 in woodland strawberry (Fragaria vesca), 173 in European pear (Pyrus communis), 219 in apple (Malus domestica), 121 in peach (Prunus persica), 121 in Chinese rose (Rosa chinensis), and 99 in black raspberry (Rubus occidentalis). All R2R3-MYB genes in the six Rosaceae species were clustered into 51 species-specific duplicated clades with 109 genes and 50 lineage-specific duplicated clades with 242 genes according to phylogenetic analysis. R2R3-MYB genes were distributed on all chromosomes in each of the six species, with a small amount of tandem duplication events. The proportion of tandem repeat genes ranged from 0 to 25.1%. The R2R3-MYB protein was conserved in a clade and likely to share similar functions. The distribution of Ks showed the duplication times of R2R3-MYB genes in six Rosaceae species. Furthermore, most of the R2R3-MYB genes had Ka/Ks values less than 1, which indicated they were driven by purifying selection during the evolutionary processes. The GO term enrichment analysis revealed that R2R3-MYB genes in strawberry and black raspberry were more divergent than in other Rosaceae species. Analysis of transcriptomes of 42 different tissues and development stages of woodland strawberry showed that high expression levels of R2R3-MYB suggested that the R2R3-MYB genes in strawberry played a key role in growth and development of both vegetative tissues and fruits. The strawberry R2R3-MYB genes in sub-group of S1, S2, S11, S20, and S22 had high expression levels both in young leaves (YL) and old leaves (OL) strawberry tissues under drought treatments.

Structural insights into target DNA recognition by R2R3-MYB transcription factors.

As the largest group of MYB family transcription factors, R2R3-MYB proteins play essential roles during plant growth and development. However, the structural basis underlying how R2R3-MYBs recognize the target DNA remains elusive. Here, we report the crystal structure of Arabidopsis WEREWOLF (WER), an R2R3-MYB protein, in complex with its target DNA. Structural analysis showed that the third α-helices in both the R2 and R3 repeats of WER fit in the major groove of the DNA, specifically recognizing the DNA motif 5′-AACNGC-3′. In combination with mutagenesis, in vitro binding and in vivo luciferase assays, we showed that K55, N106, K109 and N110 are critical for the function of WER. Although L59 of WER is not involved in DNA binding in the structure, ITC analysis suggested that L59 plays an important role in sensing DNA methylation at the fifth position of cytosine (5mC). Like 5mC, methylation at the sixth position of adenine (6mA) in the AAC element also inhibits the interaction between WER and its target DNA. Our study not only unravels the molecular basis of how WER recognizes its target DNA, but also suggests that 5mC and 6mA modifications may block the interaction between R2R3-MYB transcription factors and their target genes.

The MYB Activator WHITE PETAL1 Associates with MtTT8 and MtWD40-1 to Regulate Carotenoid-Derived Flower Pigmentation in Medicago truncatula.

Carotenoids are a group of natural tetraterpenoid pigments with indispensable roles in the plant life cycle and the human diet. Although the carotenoid biosynthetic pathway has been well characterized, the regulatory mechanisms that control carotenoid metabolism, especially in floral organs, remain poorly understood. In this study, we identified an anthocyanin-related R2R3-MYB protein, WHITE PETAL1 (WP1), that plays a critical role in regulating floral carotenoid pigmentation in Medicago truncatula Carotenoid analyses showed that the yellow petals of the wild-type M. truncatula contained high concentrations of carotenoids that largely consisted of esterified lutein and that disruption of WP1 function via Tnt1 insertion led to substantially reduced lutein accumulation. WP1 mainly functions as a transcriptional activator and directly regulates the expression of carotenoid biosynthetic genes including MtLYCe and MtLYCb through its C-terminal acidic activation motif. Further molecular and genetic analyses revealed that WP1 physically interacts with MtTT8 and MtWD40-1 proteins and that this interaction facilitates WP1's function in the transcriptional activation of both carotenoid and anthocyanin biosynthetic genes. Our findings demonstrate the molecular mechanism of WP1-mediated regulation of floral carotenoid pigmentation and suggest that the conserved MYB-basic-helix-loop-helix-WD40 regulatory module functions in carotenoid biosynthesis in M. truncatula, with specificity imposed by the MYB partner.

PgMYB2, a MeJA-Responsive Transcription Factor, Positively Regulates the Dammarenediol Synthase Gene Expression in Panax Ginseng.

The MYB transcription factor family members have been reported to play different roles in plant growth regulation, defense response, and secondary metabolism. However, MYB gene expression has not been reported in Panax ginseng. In this study, we isolated a gene from ginseng adventitious root, PgMYB2, which encodes an R2R3-MYB protein. Subcellular localization revealed that PgMYB2 protein was exclusively detected in the nucleus of Allium cepa epidermis. The highest expression level of PgMYB2 was found in ginseng root and it was significantly induced by plant hormones methyl jasmonate (MeJA). Furthermore, the binding interaction between PgMYB2 protein and the promoter of dammarenediol synthase (DDS) was found in the yeast strain Y1H Gold. Moreover, the electrophoretic mobility shift assay (EMSA) identified the binding site of the interaction and the results of transiently overexpressing PgMYB2 in plants also illustrated that it may positively regulate the expression of PgDDS. Based on the key role of PgDDS gene in ginsenoside synthesis, it is reasonable to believe that this report will be helpful for the future studies on the MYB family in P. ginseng and ultimately improving the ginsenoside production through genetic and metabolic engineering.

Evolutionary Analysis of MBW Function by Phenotypic Rescue in Arabidopsis thaliana.

The MBW complex consisting of the three proteins R2R3MYB, bHLH and WDR regulates five traits in Arabidopsis thaliana including trichome and root hair patterning, seed coat color, anthocyanidin production and seed coat mucilage release. The WDR gene TTG1 regulates each trait in specific combinations with different bHLH and R2R3MYB proteins. In this study we analyze to what extent the biochemical properties of the MBW proteins contribute to trait specificity by expressing them in appropriate A. thaliana mutants. We show that the rescue behavior of A. thaliana bHLH and R2R3MYB protein is sufficient to explain the function as derived previously from mutant analysis. When extending this rescue approach using MBW proteins from other species we find that proteins involved in anthocyanidin regulation typically show a rescue of the anthocyanidin phenotype but not of the other traits. Finally, we correlate the rescue abilities of MBW protein from different species with the A. thaliana proteins.

A R2R3-type MYB transcription factor gene from soybean, GmMYB12, is involved in flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis

The R2R3-type MYB transcription factors have been shown to increase flavonoids accumulation by regulating the expression of key enzyme genes related to flavonoid biosynthesis pathway. However, the roles and underlying mechanisms of the soybean GmMYB12 gene in regulation of flavonoids accumulation and tolerance to abiotic stresses are rarely known. In the present study, the GmMYB12 gene was isolated and its function was characterized. Sequence and yeast one-hybrid analyses showed that GmMYB12 contained two MYB domains and belonged to R2R3-MYB protein with transactivation activity. Subcellular localization analysis in onion epidermal cells indicated that GmMYB12 was localized to the nucleus. Overexpression of GmMYB12 increased the production of downstream flavonoids and the expression of related genes in the flavonoid biosynthesis pathway. It also improved resistance to salt and drought stresses during seed germination, root development, and growing stage. Further component and enzymatic analyses showed significant increases of proline content, pyrroline-5-carboxylate synthase (P5CS), superoxide dismutase (SOD), and peroxidase (POD) activities, as well as significant reduction of H2O2 and malonaldehyde (MDA) content under salt and drought stresses in transgenic plants. Meanwhile, the expression level of AtP5CS, AtSOD and AtPOD genes was up-regulated against salt and drought stresses. Together, our finding indicated that changing the expression level of GmMYB12 in plants alters the accumulation of flavonoids and regulates plantlet tolerance to abiotic stress by regulating osmotic balance, protecting membrane integrity and maintaining ROS homeostasis. The GmMYB12 gene has the potential to be used to increase the content of valuable flavonoids and improve the tolerance to abiotic stresses in plants.

Heterogeneous expression of the cotton R2R3-MYB transcription factor GbMYB60 increases salt sensitivity in transgenic Arabidopsis

MYB proteins form one of the largest transcription factor families in plants. Growing evidence has suggested that MYB has important roles in the regulation of plant growth and development, as well as responses to abiotic and biotic stresses. In the present study, the GbMYB60 gene isolated from Gossypium barbadense was heterogeneously expressed in Arabidopsis. GbMYB60 encodes a R2R3-MYB protein with transcriptional activation activity. Histochemical staining revealed that GbMYB60 was preferential expressed in the leaf vascular and meristem tissues of young seedlings and in the axillary bud tissue of mature plants. Expression pattern analysis indicated that GbMYB60 is induced by ABA, mannitol, and salt, but not H2O2. Transgenic Arabidopsis with constitutively higher expression of GbMYB60 exhibited reduced germination rates under salt and mannitol treatments as well as reduced tolerance to salt at the adult-plant stage. Transgenic Arabidopsis accumulates more reactive oxygen species compared to wild-type plants under stress conditions, corresponding with more severe cell death in transgenic plants. Additionally, ABA-related genes, including ABI5 and ABF3, were down-regulated in GbMYB60-overexpressing plants. This research has revealed that cotton GbMYB60 negatively regulates plant tolerance to salt stress, possibly through the inhibition of ABA signaling pathways and enhancing reactive oxygen species over-production.