R2R3-type MYB Transcription Factor Research Articles

The R2R3-type MYB transcription factor MdMYB90-like is responsible for the enhanced skin color of an apple bud sport mutant

The anthocyanin content in apple skin determines its red coloration, as seen in a Fuji apple mutant. Comparative RNA-seq analysis was performed to determine differentially expressed genes at different fruit development stages between the wild-type and the skin color mutant. A novel R2R3-MYB transcription factor, MdMYB90-like, was uncovered as the key regulatory gene for enhanced coloration in the mutant. The expression of MdMYB90-like was 21.3 times higher in the mutant. MdMYB90-like regulates anthocyanin biosynthesis directly through the activation of anthocyanin biosynthesis genes and indirectly through the activation of other transcription factors that activate anthocyanin biosynthesis. MdMYB90-like bound to the promoters of both structural genes (MdCHS and MdUFGT) and other transcription factor genes (MdMYB1 and MdbHLH3) in the yeast one-hybrid system, electrophoretic mobility shift assay, and dual-luciferase assay. Transgenic analysis showed that MdMYB90-like was localized in the nucleus, and its overexpression induced the expression of other anthocyanin-related genes, including MdCHS, MdCHI, MdANS, MdUFGT, MdbHLH3, and MdMYB1. The mutant had reduced levels of DNA methylation in two regions (−1183 to −988 and −2018 to −1778) of the MdMYB90-like gene promoter, which might explain the enhanced expression of the gene and the increased anthocyanin content in the mutant apple skin.

Role of Xanthoceras sorbifolium MYB44 in tolerance to combined drought and heat stress via modulation of stomatal closure and ROS homeostasis

Yellowhorn (Xanthoceras sorbifolium) is an important edible woody oil tree species that is endemic to China. Drought and heat stresses are factors severely limiting the high-quality development of the yellowhorn industry. Transcription factors (TFs) play critical roles in regulating the response of woody plant species to water deficit or high temperature. However, the MYB TFs that respond to combined drought and heat stress in yellowhorn remain unclear.Here, we first investigated the physiological changes in 5 yellowhorn varieties in response to combined stress treatments. We observed significant changes in antioxidant enzyme activities and photosynthesis. The Maigaiti variety yielded the best results and was selected for subsequent experiments. An R2R3-type MYB TF, designated XsMYB44, was isolated from the leaves of yellowhorn. XsMYB44 expression was strongly induced by combined stress. Suppression of XsMYB44 expression via virus-induced gene silencing weakened yellowhorn tolerance to both individual and combined drought and heat stress, and the increased susceptibility was coupled with decreased plant height, fresh weight and relative water content and inhibited stomatal closure. Moreover, compared with the individual stresses, the combined stress caused increased reactive oxygen species levels and decreased antioxidant enzyme activities and proline content in XsMYB44-silenced plants. Furthermore, the expression levels of several defense-related genes were reduced in the XsMYB44-silenced plants. Overall, we studied the physiological characteristics of 5 yellowhorn varieties, and the results demonstrated that XsMYB44 acts as a positive regulator in the yellowhorn response to combined stress by triggering stomatal closure to maintain water levels and by modulating ROS homeostasis.

An R2R3-type myeloblastosis transcription factor MYB103 is involved in phosphorus remobilization

The members of myeloblastosis transcription factor (MYB TF) family are involved in the regulation of biotic and abiotic stresses in plants. However, the role of MYB TF in phosphorus remobilization remains largely unexplored. In the present study, we show that an R2R3 type MYB transcription factor, MYB103, is involved in phosphorus (P) remobilization. MYB103 was remarkably induced by P deficiency in cabbage (Brassica oleracea var. capitata L.). As cabbage lacks the proper mutant for elucidating the mechanism of MYB103 in P deficiency, another member of the crucifer family, Arabidopsis thaliana was chosen for further study. The transcript of its homologue AtMYB103 was also elevated in response to P deficiency in A. thaliana, while disruption of AtMYB103 (myb103) exhibited increased sensitivity to P deficiency, accompanied with decreased tissue biomass and soluble P concentration. Furthermore, AtMYB103 was involved in the P reutilization from cell wall, as less P was released from the cell wall in myb103 than in wildtype, coinciding with the reduction of ethylene production. Taken together, our results uncover an important role of MYB103 in the P remobilization, presumably through ethylene signaling.

TaMYB86B encodes a R2R3-type MYB transcription factor and enhances salt tolerance in wheat

The MYB transcription factor family is important for plant responses to abiotic stresses. In this study, we identified three wheat TaMYB86 genes encoding R2R3-type MYB transcription factors. Analyses of the phylogenetic relationships and gene structures of TaMYB86A, TaMYB86B, and TaMYB86D revealed considerable similarities in gene structures and the encoded amino acid sequences. Additionally, TaMYB86B was highly expressed in the roots, stems, and leaves, suggesting it is critical for regulating salt stress responses in wheat. Moreover, TaMYB86B expression was induced by NaCl, abscisic acid (ABA), methyl jasmonate (MeJA), gibberellin (GA), auxin and low temperature treatments. The TaMYB86B protein localized in the nucleus and exhibited transcriptional activation activity. Under salt stress, TaMYB86B-overexpressing plants had a higher biomass and potassium ion (K+) content, but lower MDA, H2O2, O2−., and sodium ion (Na+) contents, when compared with the wild-type plants. Quantitative real-time PCR results indicated that the overexpression of TaMYB86B improved the expression of many stress-related genes. These findings suggest that TaMYB86B influences the salt tolerance of wheat by regulating the ion homeostasis to maintain an appropriate osmotic balance and decrease ROS levels.

Identification and Functional Characterization of PtoMYB055 Involved in the Regulation of the Lignin Biosynthesis Pathway in Populus tomentosa

Wood, which is mainly composed of lignified secondary cell wall, is the most abundant biomass in woody plants. Previous studies have revealed that R2R3-type MYB transcription factors are important regulators of the formation of the secondary cell wall in vascular plants. In this study, we isolated the R2R3-type MYB transcription factor gene PtoMYB055, which is mainly expressed in xylem and phloem tissue, from Populus tomentosa and demonstrate that PtoMYB055 is a key regulator of lignin biosynthesis. PtoMYB055 as a transcriptional activator is localized to the nucleus. Overexpression of PtoMYB055 upregulates expression of lignin biosynthetic genes in transgenic poplar plants, resulting in ectopic deposition of lignin in phloem tissue and an increase in thickness of the secondary cell wall. In sum, PtoMYB055 is a transcriptional activator that is involved in regulating lignin biosynthesis during the formation of the secondary cell wall in poplar.

Anthocyanin, a novel and user-friendly reporter for convenient, non-destructive, low cost, directly visual selection of transgenic hairy roots in the study of rhizobia-legume symbiosis

BackgroundAgrobacterium rhizogenes-mediated hairy root transformation provides a powerful tool for investigating the functions of plant genes involved in rhizobia-legume symbiosis. However, in the traditional identification methods of transgenic hairy roots based on reporter genes, an expensive chemical substrate or equipment is required.ResultsHere, we report a novel, low cost, and robust reporter for convenient, non-destructive, and directly visual selection of transgenic hairy roots by naked eye, which can be used in the study of rhizobia-legume symbiosis. The reporter gene AtMyb75 in Arabidopsis, encoding an R2R3 type MYB transcription factor, was ectopically expressed in hairy roots-mediated by A. rhizogenes, which induced purple/red colored anthocyanin accumulation in crop species like soybean (Glycine max (L.) Merr.) and two model legume species, Lotus japonicas and Medicago truncatula. Transgenic hairy roots of legumes containing anthocyanin can establish effective symbiosis with rhizobia. We also demonstrated the reliability of AtMyb75 as a reporter gene by CRISPR/Cas9-targeted mutagenesis of the soybean resistance to nodulation Rfg1 gene in the soybean PI377578 (Nod-) inoculated with Sinorhizobium fredii USDA193. Without exception, mature nitrogen-fixation nodules, were formed on purple transgenic hairy roots containing anthocyanin.ConclusionsAnthocyanin is a reliable, user-friendly, convenient, non-destructive, low cost, directly visual reporter for studying symbiotic nitrogen-fixing nodule development and could be widely applied in broad leguminous plants.

The R2R3-MYB transcription factor AtMYB49 modulates salt tolerance in Arabidopsis by modulating the cuticle formation and antioxidant defence.

Salt stress activates defence responses in plants, including changes in leaf surface structure. Here, we showed that the transcriptional activation of cutin deposition and antioxidant defence by the R2R3-type MYB transcription factor AtMYB49 contributed to salt tolerance in Arabidopsis thaliana. Characterization of loss-of-function myb49 mutants, and chimeric AtMYB49-SRDX-overexpressing SRDX49 transcriptional repressor and AtMYB49-overexpressing (OX49) overexpressor plants demonstrated a positive role of AtMYB49 in salt tolerance. Transcriptome analysis revealed that many genes belonging to the category "cutin, suberin and wax biosyntheses" were markedly up-regulated and down-regulated in OX49 and SRDX49 plants, respectively, under normal and/or salt stress conditions. Some of these differentially expressed genes, including MYB41, ASFT, FACT and CYP86B1, were also shown to be the direct targets of AtMYB49 and activated by AtMYB49. Biochemical analysis indicated that AtMYB49 modulated cutin deposition in the leaves. Importantly, cuticular transpiration, chlorophyll leaching and toluidine blue-staining assays revealed a link between increased AtMYB49-mediated cutin deposition in leaves and enhanced salt tolerance. Additionally, increased AtMYB49 expression elevated Ca2+ level in leaves and improved antioxidant capacity by up-regulating genes encoding peroxidases and late embryogenesis abundant proteins. These results suggest that genetic manipulation of AtMYB49 may provide a novel way to improve salt tolerance in plants.

SlMYB102, an R2R3-type MYB gene, confers salt tolerance in transgenic tomato

Salinity threatens the productivity of tomato (Solanum lycopersicum L.). R2R3-type MYB transcription factors are important regulators in response to environmental stress. Here, we analyzed the function of the tomato R2R3-type MYB gene SlMYB102. A transcriptional activation assay showed that SlMYB102 had transactivation activity in yeast. Promoter analysis showed that multiple stress-related elements were found in the promoter of SlMYB102. Furthermore, SlMYB102 was induced by osmotic stress, particularly by salt stress. The overexpression of SlMYB102 in tomato affected multiple parameters under salinity stress. Under long-term salt stress, the degree of growth inhibition was significantly reduced in the two overexpression (OE) lines. In addition, the two OE lines maintained a better K+/Na+ ratio, lower reactive oxygen species (ROS) generation (O2•− production rate and H2O2 content) and lower electrolytic leakage rates than the wild type (WT). The activity of ROS scavenging enzymes including superoxide dismutase, peroxidase, catalase and ascorbate peroxidase, and the accumulation of antioxidants (ascorbic acid and glutathione) and proline was higher in the two OE lines compared with WT. The qRT-PCR analysis confirmed that the transcript abundance of many salt stress-related genes (SlSOS1, SlSOS2, SlNHX3, SlNHX4, SlHAK5, SlCPK1 and SlCPK3) was upregulated in two OE lines under salt stress. Collectively, these results suggest that SlMYB102 participates in tomato tolerance through the regulation of a series of molecular and physiological processes.

The grapevine R2R3-type MYB transcription factor VdMYB1 positively regulates defense responses by activating the stilbene synthase gene 2 (VdSTS2)

BackgroundResveratrol is a naturally occurring plant stilbene that exhibits a wide range of valuable biological and pharmacological properties. Although the beneficial effects of trans-resveratrol to human health and plant protection against fungal pathogens and abiotic stresses are well-established, yet little is known about the molecular mechanisms regulating stilbene biosynthesis in plant defense progress.ResultsHere, we cloned and identified the Chinese wild grape (Vitis davidii) R2R3-MYB transcription factor VdMYB1, which activates defense responses against invading pathogen. VdMYB1 transcripts were significantly upregulated after inoculation with the grapevine powdery mildew fungus Erysiphe necator (Schw.) Burr. Transient expression analysis using onion epidermal cells and Arabidopsis thaliana protoplasts showed that VdMYB1 was localized in the nucleus. Yeast one-hybrid assays revealed that VdMYB1 acts as a transcriptional activator. Grapevine leaves transiently overexpressing VdMYB1 showed a lower number of fungal conidiophores compared with wild-type leaves. Overexpression of VdMYB1 in grapevine leaves did not alter the expression of genes in salicylic acid- and jasmonate-dependent pathways, but affected the expression of stilbene synthase (STS) genes, key regulators of flavonoid metabolism. Results of electrophoretic mobility shift assays and in vivo transcriptional activation assays showed that VdMYB1 binds to the MYB binding site (MYBBS) in the STS2 gene promoter, thus activating STS2 transcription. In heterologous expression assays using tobacco leaves, VdMYB1 activated STS2 gene expression and increased the accumulation of resveratrol.ConclusionsOur study showed that VdMYB1 activates STS2 gene expression to positively regulate defense responses, and increases the content of resveratrol in leaves.

Root Epidermal Cell Patterning Is Modulated by a Critical Residue in the WEREWOLF Transcription Factor.

The Arabidopsis (Arabidopsis thaliana) root epidermis exhibits a position-dependent pattern of root-hair and nonhair cell types. A highly orchestrated network of gene regulatory interactions, including the R2R3-type MYB transcription factor WEREWOLF (WER), is responsible for generating this cell pattern during root development. In this study, we identified a novel wer mutant from a genetic enhancer screen, designated wer-4, that exhibits an abnormal pattern of root-hair and nonhair cells. We established that wer-4 bears a single-residue substitution (D105N) in the DNA-binding R3 MYB repeat of WER, which differentially affects the transcription of WER target genes, including GLABRA2, CAPRICE, TRIPTYCHON, and ENHANCER OF TRY AND CPC1 This modulation of the gene regulatory network leads to altered levels and distributions of cell fate regulators in the differentiating epidermal cells that ultimately generate the abnormal cell-type pattern. We also created several WER variants with substitutions at the Asp-105 position, and these exhibited a variety of gene expression and cell-type pattern alterations, further supporting the critical role of this residue. These findings provide insight into WER protein function and its importance in generating the proper balance of downstream transcriptional factors in the gene regulatory network that establishes root epidermal cell fate.

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.

The R2R3-MYB Transcription Factor MYB49 Regulates Cadmium Accumulation.

Abscisic acid (ABA) reduces accumulation of potentially toxic cadmium (Cd) in plants. How the ABA signal is transmitted to modulate Cd uptake remains largely unclear. Here, we report that the basic region/Leu zipper transcription factor ABSCISIC ACID-INSENSITIVE5 (ABI5), a central ABA signaling molecule, is involved in ABA-repressed Cd accumulation in plants by physically interacting with a previously uncharacterized R2R3-type MYB transcription factor, MYB49. Overexpression of the Cd-induced MYB49 gene in Arabidopsis (Arabidopsis thaliana) resulted in a significant increase in Cd accumulation, whereas myb49 knockout plants and plants expressing chimeric repressors of MYB49:ERF-associated amphiphilic repression motif repression domain (SRDX49) exhibited reduced accumulation of Cd. Further investigations revealed that MYB49 positively regulates the expression of the basic helix-loop-helix transcription factors bHLH38 and bHLH101 by directly binding to their promoters, leading to activation of IRON-REGULATED TRANSPORTER1, which encodes a metal transporter involved in Cd uptake. MYB49 also binds to the promoter regions of the heavy metal-associated isoprenylated plant proteins (HIPP22) and HIPP44, resulting in up-regulation of their expression and subsequent Cd accumulation. On the other hand, as a feedback mechanism to control Cd uptake and accumulation in plant cells, Cd-induced ABA up-regulates the expression of ABI5, whose protein product interacts with MYB49 and prevents its binding to the promoters of downstream genes, thereby reducing Cd accumulation. Our results provide new insights into the molecular feedback mechanisms underlying ABA signaling-controlled Cd uptake and accumulation in plants.

Members of R2R3-type MYB transcription factors from subgroups 20 and 22 are involved in abiotic stress response in tea plants

Abiotic stresses, including extreme temperature, drought and salinity, seriously threaten the growth and development of tea plants. The tea plant fights against various threats to innate defensive systems by regulating the expression profiles of related anti-genes. R2R3-type MYB family transcription factors (TFs) contribute to plant defense against abiotic stresses. Five CsMYB TFs were identified from the tea plant according to the transcriptome database. Conserved motif analysis revealed that subgroups 20 and 22 were conserved in the N-terminus with two motifs. All five CsMYB TF sequence homologies had a flexible C-terminal region. Subcellular localization analysis indicated that the protein of CsMYB19 was specifically localized in the nucleus. The expression patterns of CsMYB genes under abiotic stress treatments were significantly higher than that of the control, and the members from subgroup 20 had higher response than those in subgroup 22. During hormonal treatment, CsMYB8 was selected as highly responsive to methyl jasmonate (MeJA) treatment. The expression level of CsMYB20 was highest under ABA treatment and lowest under SA treatment. Our study serves as a foundation for understanding the response mechanisms of MYB genes under abiotic stress in tea plants.

A novel MYB transcription factor regulates ascorbic acid synthesis and affects cold tolerance.

Dehydroascorbate reductase (DHAR) plays an important role in stress responses, but the transcriptional regulation of DHAR in response to abiotic stress is still poorly understood. In this study, we isolated a novel R2R3-type MYB transcription factor from Pyrus betulaefolia by yeast one-hybrid screening, designated as PbrMYB5. PbrMYB5 was localized in the nucleus and could bind specifically to the promoter of PbrDHAR2. PbrMYB5 was greatly induced by cold and salt but slightly by dehydration. Overexpression of PbrMYB5 in tobacco conferred enhanced tolerance to chilling stresses, whereas down-regulation of PbrMYB5 in P.betulaefolia by virus-induced gene silencing resulted in elevated chilling sensitivity. Transgenic tobacco exhibited higher expression levels of NtDHAR2 and accumulated larger amount of ascorbic acid (AsA) than the wild-type plants. Virus-induced gene silencing of PbrMYB5 in P.betulaefolia down-regulated PbrDHAR2 abundance and decreased AsA level, accompanied by an increased sensitivity to the chilling stress. Taken together, these results demonstrated that PbrMYB5 was an activator of AsA biosynthesis and may play a positive role in chilling tolerance, at least in part, due to the modulation of AsA synthesis by regulating the PbrDHAR2 expression.

The MIEL1 E3 Ubiquitin Ligase Negatively Regulates Cuticular Wax Biosynthesis in Arabidopsis Stems

Cuticular wax is an important hydrophobic layer that covers the plant aerial surface. Cuticular wax biosynthesis is shaped by multiple layers of regulation. In particular, a pair of R2R3-type MYB transcription factors, MYB96 and MYB30, are known to be the main participants in cuticular wax accumulation. Here, we report that the MYB30-INTERACTING E3 LIGASE 1 (MIEL1) E3 ubiquitin ligase controls the protein stability of the two MYB transcription factors and thereby wax biosynthesis in Arabidopsis. MIEL1-deficient miel1 mutants exhibit increased wax accumulation in stems, with up-regulation of wax biosynthetic genes targeted by MYB96 and MYB30. Genetic analysis reveals that wax accumulation of the miel1 mutant is compromised by myb96 or myb30 mutation, but MYB96 is mainly epistatic to MIEL1, playing a predominant role in cuticular wax deposition. These observations indicate that the MIEL1-MYB96 module is important for balanced cuticular wax biosynthesis in developing inflorescence stems.

A Wheat R2R3-type MYB Transcription Factor TaODORANT1 Positively Regulates Drought and Salt Stress Responses in Transgenic Tobacco Plants.

MYB transcription factors play important roles in plant responses to biotic and abiotic stress. In this study, TaODORANT1, a R2R3-MYB gene, was cloned from wheat (Triticum aestivum L.). TaODORANT1 was localized in the nucleus and functioned as a transcriptional activator. TaODORANT1 was up-regulated in wheat under PEG6000, NaCl, ABA, and H2O2 treatments. TaODORANT1-overexpressing transgenic tobacco plants exhibited higher relative water content and lower water loss rate under drought stress, as well as lower Na+ accumulation in leaves under salt stress. The transgenic plants showed higher CAT activity but lower ion leakage, H2O2 and malondialdehyde contents under drought and salt stresses. Besides, the transgenic plants also exhibited higher SOD activity under drought stress. Our results also revealed that TaODORANT1 overexpression up-regulated the expression of several ROS- and stress-related genes in response to both drought and salt stresses, thus enhancing transgenic tobacco plants tolerance. Our studies demonstrate that TaODORANT1 positively regulates plant tolerance to drought and salt stresses.

Maintenance of phosphate homeostasis and root development are coordinately regulated by MYB1, an R2R3-type MYB transcription factor in rice

The adaptive responses of plants to phosphate (Pi) starvation stress are fine-tuned by an elaborate regulatory network. In this study, we identified and characterized a novel Pi starvation-responsive gene, MYB1, encoding an R2R3-type transcription factor in rice. MYB1 was transcriptionally induced in leaf sheaths and old leaf blades. It was localized to the nucleus and expressed mainly in vascular tissues. Mutation of MYB1 led to an increase in Pi uptake and accumulation, accompanied by altered expression of a subset of Pi transporters and several genes involved in Pi starvation signaling. Furthermore, MYB1 affected the elongation of the primary root in a Pi-dependent manner and lateral roots in a Pi-independent manner. Moreover, gibberellic acid (GA)-triggered lateral root elongation was largely suppressed in wild-type plants under Pi starvation conditions, whereas this suppression was partially rescued in myb1 mutant lines, correlating with the up-regulation of a GA biosynthetic gene upon MYB1 mutation. Taken together, the findings of this study highlight the role of MYB1 as a regulator involved in both Pi starvation signaling and GA biosynthesis. Such a co-regulator might have broad implications for the study of cross-talk between nutrient stress and other signaling pathways.

The MIEL1 E3 Ubiquitin Ligase Negatively Regulates Cuticular Wax Biosynthesis in Arabidopsis Stems.

Cuticular wax is an important hydrophobic layer that covers the plant aerial surface. Cuticular wax biosynthesis is shaped by multiple layers of regulation. In particular, a pair of R2R3-type MYB transcription factors, MYB96 and MYB30, are known to be the main participants in cuticular wax accumulation. Here, we report that the MYB30-INTERACTING E3 LIGASE 1 (MIEL1) E3 ubiquitin ligase controls the protein stability of the two MYB transcription factors and thereby wax biosynthesis in Arabidopsis. MIEL1-deficient miel1 mutants exhibit increased wax accumulation in stems, with up-regulation of wax biosynthetic genes targeted by MYB96 and MYB30. Genetic analysis reveals that wax accumulation of the miel1 mutant is compromised by myb96 or myb30 mutation, but MYB96 is mainly epistatic to MIEL1, playing a predominant role in cuticular wax deposition. These observations indicate that the MIEL1-MYB96 module is important for balanced cuticular wax biosynthesis in developing inflorescence stems.

An R2R3-type MYB transcription factor, GmMYB29, regulates isoflavone biosynthesis in soybean.

Isoflavones comprise a group of secondary metabolites produced almost exclusively by plants in the legume family, including soybean [Glycine max (L.) Merr.]. They play vital roles in plant defense and have many beneficial effects on human health. Isoflavone content is a complex quantitative trait controlled by multiple genes, and the genetic mechanisms underlying isoflavone biosynthesis remain largely unknown. Via a genome-wide association study (GWAS), we identified 28 single nucleotide polymorphisms (SNPs) that are significantly associated with isoflavone concentrations in soybean. One of these 28 SNPs was located in the 5’-untranslated region (5’-UTR) of an R2R3-type MYB transcription factor, GmMYB29, and this gene was thus selected as a candidate gene for further analyses. A subcellular localization study confirmed that GmMYB29 was located in the nucleus. Transient reporter gene assays demonstrated that GmMYB29 activated the IFS2 (isoflavone synthase 2) and CHS8 (chalcone synthase 8) gene promoters. Overexpression and RNAi-mediated silencing of GmMYB29 in soybean hairy roots resulted in increased and decreased isoflavone content, respectively. Moreover, a candidate-gene association analysis revealed that 11 natural GmMYB29 polymorphisms were significantly associated with isoflavone contents, and regulation of GmMYB29 expression could partially contribute to the observed phenotypic variation. Taken together, these results provide important genetic insights into the molecular mechanisms underlying isoflavone biosynthesis in soybean.

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.