R2R3-type MYB Transcription Factor Research Articles

GmMYB114 Facilitates the Synthesis of Anthocyanins in Soybean Sprouts under Blue Light.

Soybean sprouts constitute a significant segment of the vegetable market due to their nutritional richness, particularly in various flavonoids, which contribute to numerous health benefits. The augmentation of the flavonoid content in soybean sprouts is pivotal for enhancing their economic value. While research has established the potential of blue light in promoting the synthesis of anthocyanins, a subclass of flavonoids known for their health advantages, the precise regulatory mechanisms remain elusive. In this study, we identified a notable upregulation of an R2R3 type MYB transcription factor, GmMYB114, in response to blue light exposure, exhibiting a significant positive correlation with anthocyanin accumulation in soybean sprouts. The functional role of GmMYB114 was validated in soybean hairy roots, wherein its overexpression substantially augmented anthocyanin synthesis. Further investigations employing yeast one-hybrid (Y1H), dual-luciferase reporter (LUC), and GUS assays revealed that GmMYB114 indirectly influences anthocyanin synthesis as it does not directly bind to the promoters of anthocyanin synthesis genes to activate their expression. These findings contribute to elucidating the mechanism underlying blue light-mediated enhancement of anthocyanin synthesis in soybean sprouts, offering valuable insights for harnessing molecular technologies to obtain anthocyanin-enriched soybean sprouts.

EpMYB2 positively regulates chicoric acid biosynthesis by activating both primary and specialized metabolic genes in purple coneflower.

Chicoric acid is the major active ingredient of the world-popular medicinal plant purple coneflower (Echinacea purpurea (L.) Menoch). It is recognized as the quality index of commercial hot-selling Echinacea products. While the biosynthetic pathway of chicoric acid in purple coneflower has been elucidated recently, its regulatory network remains elusive. Through co-expression and phylogenetic analysis, we found EpMYB2, a typical R2R3-type MYB transcription factor (TF) responsive to methyl jasmonate (MeJA) simulation, is a positive regulator of chicoric acid biosynthesis. In addition to directly regulating chicoric acid biosynthetic genes, EpMYB2 positively regulates genes of the upstream shikimate pathway. We also found that EpMYC2 could activate the expression of EpMYB2 by binding to its G-box site, and the EpMYC2-EpMYB2 module is involved in the MeJA-induced chicoric acid biosynthesis. Overall, we identified an MYB TF that positively regulates the biosynthesis of chicoric acid by activating both primary and specialized metabolic genes. EpMYB2 links the gap between the JA signaling pathway and chicoric acid biosynthesis. This work opens a new direction toward engineering purple coneflower with higher medicinal qualities.

Deciphering the Genetic Basis of Allelopathy in japonica Rice Cultivated in Temperate Regions Using a Genome-Wide Association Study

Allelopathy has been considered as a natural method of weed control. Despite the nature of allelochemical compounds has been studied, little is known about the genetic basis underlying allelopathy. However, it is known that rice exhibits diverse allelopathic potentials across varieties, and breeding for rice plants exhibiting allelopathic potential conferring an advantage against weeds in paddy fields would be highly desirable. Knowledge of the gene factors and the identification of the genomic regions responsible for allelopathy would facilitate breeding programs. Taking advantage of the existing genetic diversity in rice, particularly in temperate japonica rice, we conducted a comprehensive investigation into the genetic determinants that contribute to rice allelopathy. Employing Genome-Wide Association Study, we identified four Quantitative Trait Loci, with the most promising loci situated on chromosome 2 and 5. Subsequent inspection of the genes located within these QTLs revealed genes associated with the biosynthesis of secondary metabolites such as Phenylalanine Ammonia Lyase (PAL), a key enzyme in the synthesis of phenolic compounds, and two genes coding for R2R3-type MYB transcription factors. The identification of these two QTLs associated to allelopathy in rice provides a useful tool for further exploration and targeted breeding strategies.

Identification and characterization of sub-group 4 R2R3-MYB members and their functions in phenylpropanoid and flavonoid regulation in tobacco

Sub-group 4 R2R3-type MYB transcription factors (TFs) are involved in repression of the phenylpropanoid pathway. In this study, we identified and characterized three MYB transcriptional repressors in common tobacco, named NtMYB1, NtMYB2, and NtMYB4, which belong to sub-group 4 members. Tissue expression properties and specificity of stress-induced expression indicated that the NtMYB1, NtMYB2, and NtMYB4 may perform different functions in tobacco. Overexpression of NtMYB1, NtMYB2, and NtMYB4 reduced phenylpropanoids and flaonoid compounds, such as caffeic acid, chlorogenic acid, neochlorogenic acid, and rutin, respectively. Meanwhile, the expression of the most structural genes involved in phenylpropanoids and flavonoids were reduced in the overexpression transgenic lines. In addition, results based on co-expression and expression analyses revealed possible interactions between NtMYB1, NtMYB2, and NtMYB4 and multiple bHLH TFs. Our results showed that the three sub-group 4 R2R3 members exhibited different expression pattern in various tissues and under abiotic stress conditions. Meanwhile, we hypothesized that the three of them involved in the inhibition of phenylpropanoid and flavonoid biosynthesis might be interaction with bHLH TFs.

The transcription factor MYB110 regulates plant height, lodging resistance, and grain yield in rice.

The high-yielding Green Revolution varieties of cereal crops are characterized by a semidwarf architecture and lodging resistance. Plant height is tightly regulated by the availability of phosphate (Pi), yet the underlying mechanism remains obscure. Here, we report that rice (Oryza sativa) R2R3-type Myeloblastosis (MYB) transcription factor MYB110 is a Pi-dependent negative regulator of plant height. MYB110 is a direct target of PHOSPHATE STARVATION RESPONSE 2 (OsPHR2) and regulates OsPHR2-mediated inhibition of rice height. Inactivation of MYB110 increased culm diameter and bending resistance, leading to enhanced lodging resistance despite increased plant height. Strikingly, the grain yield of myb110 mutants was elevated under both high- and low-Pi regimes. Two divergent haplotypes based on single nucleotide polymorphisms in the putative promoter of MYB110 corresponded with its transcript levels and plant height in response to Pi availability. Thus, fine-tuning MYB110 expression may be a potent strategy for further increasing the yield of Green Revolution cereal crop varieties.

ThDIV2, an R-R-type MYB transcription factor of Tamarix hispida, negatively regulates cadmium stress by modulating ROS homeostasis

Cadmium (Cd) is a nonessential and toxic metal that is rapidly taken up by roots and accumulates in various plant tissues, limiting crop growth and productivity. ROS generation and signal transduction play an important role in the detoxification and tolerance of heavy metals (e.g., Cd stress). Nonetheless, the mechanism underlying the regulation of ROS in T. hispida under Cd stress remains largely unknown. Here, we have demonstrated a previously unknown role of the R2R3-type MYB transcription factor ThDIV2 in regulating Cd tolerance by downregulating the expression of ThAO1 and ThAO2. To investigate the role of R2R3-type MYB transcription factor ThDIV2 in regulating Cd tolerance, we monitored the expression level of ThDIV2 in response to Cd stress using qRTPCR. Additionally, we conducted a Qu-2 cell conversion experiment to determine the subcellular localization of ThDIV2. The results demonstrated that ThDIV2, which is localized in the nucleus, was significantly suppressed under Cd stress. To further investigate the function of ThDIV2 under Cd stress, we generated ThDIV2-overexpressing plants and empty an pBI121 vector (used as the control; VC) through transient transformation. Biochemical staining and physiological parameter analysis showed that overexpression of ThDIV2 resulted in higher ROS, H2O2 and proline contents, but lower T-AOC, SOD, POD, and CAT activity, and GSH content compared with VC under Cd treatment. Moreover, expression pattern analysis showed that ThDIV2 repressed the expression of ThAO1, ThAO2, ThAO3, ThSOD1, ThSOD2 and ThSOD3. Further Y1H and ChIP-PCR analyses indicated that ThDIV2 could directly bind to the Myb motif (CAACCA) of the ThAO1 and ThAO2 promoters. In summary, our results suggest that ThDIV2 regulates the expression of ThAO1 and ThAO2, leading to alterations in ROS content and increased sensitivity of T. hispida to Cd stress.

OsMYB7 determines leaf angle at the late developmental stage of lamina joints in rice.

Leaf angle shapes plant architecture, allowing for optimal light interception to maximize photosynthesis and yield, and therefore is a crucial agronomic trait. Here, we show that the rice (Oryza sativa L.) R2R3-type MYB transcription factor OsMYB7 determines leaf angle in a developmental stage-specific manner. OsMYB7-overexpressing lines produced wide-angled leaves and osmyb7 knockout mutants exhibited erect leaves. This phenotype was restricted to the lamina joints at the late developmental stage. In agreement with these observations, OsMYB7 was preferentially expressed in the lamina joints of post-mature leaves. Since OsMYB7 homologs are transcriptional repressors of lignin biosynthesis, we examined whether OsMYB7 might inhibit thickening ofsecondary cell walls. Although OsMYB7 repressed lignin biosynthesis, it enhanced thickening of sclerenchyma cell walls by elevating cellulose contents at the lamina joints. Furthermore, we found that OsMYB7 affects endogenous auxin levels in lamina joints, and the adaxial cells of lamina joints in OsMYB7-overexpressing lines and osmyb7 knockout mutants exhibited enhanced and reduced elongation, respectively, compared to the wild type. These results suggest that OsMYB7 promotes leaf inclination partially through decreasing free auxin levels and promoting cell elongation at the adaxial side of lamina joints.

Comprehensive Genome-Wide Analyses of Poplar R2R3-MYB Transcription Factors and Tissue-Specific Expression Patterns under Drought Stress.

R2R3-type MYB transcription factors are implicated in drought stress, which is a primary factor limiting the growth and development of woody plants. The identification of R2R3-MYB genes in the Populus trichocarpa genome has been previously reported. Nevertheless, the diversity and complexity of the conserved domain of the MYB gene caused inconsistencies in these identification results. There is still a lack of drought-responsive expression patterns and functional studies of R2R3-MYB transcription factors in Populus species. In this study, we identified a total of 210 R2R3-MYB genes in the P. trichocarpa genome, of which 207 genes were unevenly distributed across all 19 chromosomes. These poplar R2R3-MYB genes were phylogenetically divided into 23 subgroups. Collinear analysis demonstrated that the poplar R2R3-MYB genes underwent rapid expansion and that whole-genome duplication events were a dominant factor in the process of rapid gene expansion. Subcellular localization assays indicated that poplar R2R3-MYB TFs mainly played a transcriptional regulatory role in the nucleus. Ten R2R3-MYB genes were cloned from P. deltoides × P. euramericana cv. Nanlin895, and their expression patterns were tissue-specific. A majority of the genes showed similar drought-responsive expression patterns in two out of three tissues. This study provides a valid cue for further functional characterization of drought-responsive R2R3-MYB genes in poplar and provides support for the development of new poplar genotypes with elevated drought tolerance.

LiMYB108 is involved in floral monoterpene biosynthesis induced by light intensity in Lilium 'Siberia'.

We find that the MYB family transcription factor, LiMYB108, has a novel function to regulate the floral fragrance affected by light intensity. Floral fragrance determines the commercial value of flowers and is influenced by many environmental factors, especially light intensity. However, the mechanism by which light intensity affects the release of floral fragrance is unclear. Here, we isolated an R2R3-type MYB transcription factor LiMYB108, the expression of which was induced by light intensity and located in the nucleus. Light of 200 and 600μmolm-1s-1 significantly increased the expression of LiMYB108, which was consistent with the improving trend of monoterpene synthesis under light. Virus-induced gene silencing (VIGS) of LiMYB108 in Lilium not only significantly inhibited the synthesis of ocimene and linalool, but also decreased the expression of LoTPS1; however, transient overexpression of LiMYB108 exerted opposite effects. Furthermore, yeast one-hybrid assays, dual-luciferase assays, and electrophoretic mobility shift assays (EMSA) demonstrated that LiMYB108 directly activated the expression of LoTPS1 by binding to the MYB binding site (MBS) (CAGTTG). Our findings demonstrate that light intensity triggered the high expression of LiMYB108, and then LiMYB108 as a transcription factor to activate the expression of LoTPS1, thus promoting the synthesis of the ocimene and linalool, which are important components of floral fragrance. These results provide new insights into the effects of light intensity on floral fragrance synthesis.

CmMYB44 might interact with CmAPS2-2 to regulate starch metabolism in oriental melon fruit

Sugar content is one of the determining factors for melon fruit maturity. Studies have shown that starch gradually degrades during fruit ripening, resulting in sugar accumulation. But the specific relationship between starch metabolism and sucrose accumulation was still unknown. Here, the starch and sugar contents, the activities of key enzymes and the expression patterns of genes related to starch-sucrose metabolism were determined in the fruit of high sugar and starch variety ‘HS’ and low sugar and starch variety ‘LW’. It was found that starch accumulated during fruit development process, and then degraded at 30 days after anthesis (DAA), which was synchronized with sucrose accumulation in ‘HS’ fruit, while starch and sucrose contents were always at a lower level during ‘LW’ fruit maturation. Furthermore, starch metabolism-related enzymes (Adenine dinucleotide phosphate -glucose pyrophosphorylase (AGPase), α-amylase (AMY), β-amylase (BMY)) and the key enzymes for sucrose accumulation (sucrose phosphate synthase (SPS) and sucrose synthase (SS)) were significantly increased at ripening stage of ‘HS’ fruit, and their activities were consistent with the expressions of CmAPS2-2, CmAMY2, CmBAM1, CmBAM9 and CmSPS1. However, the contents of starch and sucrose and the activities of AGPase and SPS in ‘LW’ fruit didn't change significantly. We discovered an R2R3-type MYB transcription factor, CmMYB44, screened from yeast one hybrid library, could directly bind to the promoter of CmAPS2-2 to inhibit its transcription. These results revealed that the targeted down-regulation of CmAPS2-2 by CmMYB44 might be involved in the starch accumulation process, which affect the flavor quality of oriental melon fruit.

Cloning and Expression Analysis of MaMYB308 Gene in Mulberry

In order to analyze the function of MYB transcription factors in mulberry abiotic stress and development, the full-length cDNA sequence of MYB transcription factor MaMYB308 was cloned from the mulberry cultivar ‘Guiyou 62’. The amino acid sequence of MYB transcription factor MaMYB308 was analyzed by bioinformatics, and its self activation was verified by yeast single hybridization. The expression pattern of the gene under different tissue and abiotic stresses was detected by real-time fluorescence quantitative PCR. The results showed that the full length of  MaMYB308  gene is 1 266 bp, including an open reading frame of 1 026 bp, which can encode 341 amino acids, and its molecular weight is 39 kD; it has two SANT domains, belonging to typical R2R3 type MYB transcription factor, and has self activating activity. Blast results showed that MaMYB308 had high homology with a variety of R2R3-MYB transcription factors from higher plants, and had the highest homology with MYB308 from Morus notabilis , and the consistency of amino acid level was 96.48%. The results of real-time fluorescence quantitative PCR showed that the expression of MaMYB308 in roots, stems and leaves of mulberry was significantly higher than that in stems and leaves, and it was responsive to abiotic stresses such as high temperature, low temperature, high salt and drought. Among them, the expression of MaMYB308  was the most significant under low temperature stress, about 40 times of the control. The results of this study could be helpful for further study on the biological function of mulberry MaMYB308  gene and the stress resistance mechanism of mulberry.

Structural insights into partner selection for MYB and bHLH transcription factor complexes.

MYB and basic helix-loop-helix (bHLH) transcription factors form complexes to regulate diverse metabolic and developmental processes in plants. However, the molecular mechanisms responsible for MYB-bHLH interaction and partner selection remain unclear. Here, we report the crystal structures of three MYB-bHLH complexes (WER-EGL3, CPC-EGL3 and MYB29-MYC3), uncovering two MYB-bHLH interaction modes. WER and CPC are R2R3- and R3-type MYBs, respectively, but interact with EGL3 through their N-terminal R3 domain in a similar mode. A single amino acid of CPC, Met49, is crucial for competition with WER to interact with EGL3. MYB29, a R2R3-type MYB transcription factor, interacts with MYC3 by its C-terminal MYC-interaction motif. The WER-EGL3 and MYB29-MYC3 binding modes are widely applied among MYB-bHLH complexes in Arabidopsis and evolve independently in plants.

CRISPR/Cas9-Mediated Targeted Mutagenesis of FtMYB45 Promotes Flavonoid Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum).

The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is an efficient genome editing tool used in multiple plant species. However, it has not been applied to Tartary buckwheat (Fagopyrum tataricum), which is an important edible and medicinal crop rich in rutin and other flavonoids. FtMYB45 is an R2R3-type MYB transcription factor that negatively regulates flavonoid biosynthesis in Tartary buckwheat. Here, the CRISPR/Cas9 system polycistronic tRNA-sgRNA (PTG)/Cas9 was employed to knock out the FtMYB45 gene in Tartary buckwheat. Two single-guide RNAs (sgRNAs) were designed to target the second exon of the FtMYB45 gene. Twelve transgenic hairy roots were obtained using Agrobacterium rhizogenes-mediated transformation. Sequencing data revealed that six lines containing six types of mutations at the predicted double-stranded break site were generated using sgRNA1. The mutation frequency reached 50%. A liquid chromatography coupled with triple quadrupole mass spectrometry (LC-QqQ-MS) based metabolomic analysis revealed that the content of rutin, catechin, and other flavonoids was increased in hairy root mutants compared with that of lines transformed with the empty vector. Thus, CRISPR/Cas9-mediated targeted mutagenesis of FtMYB45 effectively increased the flavonoids content of Tartary buckwheat. This finding demonstrated that the CRISPR/Cas9 system is an efficient tool for precise genome editing in Tartary buckwheat and lays the foundation for gene function research and quality improvement in Tartary buckwheat.

GhODO1, an R2R3-type MYB transcription factor, positively regulates cotton resistance to Verticillium dahliae via the lignin biosynthesis and jasmonic acid signaling pathway

In plants, MYB transcription factors play diverse roles in growth, development, and response to abiotic and biotic stresses. However, the signaling processes of these transcription factors in defense against pathogen attacks remain largely unknown. This study isolated a novel R2R3-type MYB transcription factor GhODO1 from cotton (Gossypium hirsutum) and functionally characterized its positive role in tolerance to Verticillium dahliae. GhODO1 was induced by V. dahliae and jasmonic acid (JA) and transient expression of fused GhODO1-GFP in onion epidermal cells showed that GhODO1 protein was localized in the cell nucleus. Knockdown of GhODO1 significantly reduced the resistance of cotton to V. dahliae, whereas GhODO1 ectopic overexpression in Arabidopsis conferred enhanced resistance to V. dahliae. Lignin deposition was significantly decreased in GhODO1-silenced cotton plants after V. dahliae inoculation and mock treatment. The expression levels of genes and activities of enzymes involved in lignin biosynthesis were reduced in GhODO1-silenced cotton plants compared to the TRV:00. Yeast one-hybrid assays revealed that GhODO1 protein interacts with the promoters of lignin biosynthesis-related genes Gh4CL1 and GhCAD3, directly activates their expression, and enhances total lignin accumulation. Moreover, GhODO1 silencing compromised JA-mediated defense signaling and JA accumulation. These results show that GhODO1 is involved in cotton resistance to V. dahliae by involving the lignin biosynthesis and the JA signaling pathway.

The R2R3-MYB transcription factor FaMYB63 participates in regulation of eugenol production in strawberry.

The biosynthetic pathway of volatile phenylpropanoids, including 4-allyl-2-methoxyphenol (eugenol), has been investigated in petunia (Petunia hybrida). However, the regulatory network for eugenol accumulation in strawberry (Fragaria × ananassa Duch.) fruit remains unclear. Here, an R2R3-type MYB transcription factor (TF; FaMYB63) was isolated from strawberry by yeast one-hybrid (Y1H) screening using the promoter of the FaEGS1 (eugenol synthase 1 [EGS 1]) gene, which encodes the enzyme responsible for the last step in eugenol biosynthesis. FaMYB63 is phylogenetically distinct from other R2R3-MYB TFs, including FaEOBІІ (EMISSION OF BENZENOID II [EOBII]), which also participates in regulating eugenol biosynthesis in strawberry receptacles. Reverse transcription quantitative PCR (RT-qPCR) assays showed that the expression of FaMYB63 was tissue-specific and consistent with eugenol content through strawberry fruit development, was repressed by abscisic acid, and was activated by auxins (indole-3-acetic acid). Overexpression and RNA interference-mediated silencing of FaMYB63 resulted in marked changes in the transcript levels of the biosynthetic genes FaEGS1, FaEGS2, and FaCAD1 (cinnamyl alcohol dehydrogenase 1 [CAD1]) and, thereby, the accumulation of eugenol. Electrophoretic mobility shift, Y1H, GUS activity, and dual-luciferase activity assays demonstrated that the transcript levels of FaEOBІІ and FaMYB10 were regulated by FaMYB63, but not the other way around. Together, these results demonstrate that FaMYB63 directly activates FaEGS1, FaEGS2, FaCAD1, FaEOBІІ, and FaMYB10 to induce eugenol biosynthesis during strawberry fruit development. These findings deepen the understanding of the regulatory network that influences eugenol metabolism in an edible fruit crop.

FvMYB79 Positively Regulates Strawberry Fruit Softening via Transcriptional Activation of FvPME38.

Strawberry is a soft fruit with short postharvest life, due to a rapid loss of firmness. Pectin methylesterase (PME)-mediated cell wall remodeling is important to determine fruit firmness and softening. Previously, we have verified the essential role of FvPME38 in regulation of PME-mediated strawberry fruit softening. However, the regulatory network involved in PME-mediated fruit softening is still largely unknown. Here, we identified an R2R3-type MYB transcription factor FvMYB79, which activates the expression level of FvPME38, thereby accelerating fruit softening. During fruit development, FvMYB79 co-expressed with FvPME38, and this co-expression pattern was opposite to the change of fruit firmness in the fruit of ‘Ruegen’ which significantly decreased during fruit developmental stages and suddenly became very low after the color turning stage. Via transient transformation, FvMYB79 could significantly increase the transcriptional level of FvPME38, leading to a decrease of firmness and acceleration of fruit ripening. In addition, silencing of FvMYB79 showed an insensitivity to ABA-induced fruit ripening, suggesting a possible involvement of FvMYB79 in the ABA-dependent fruit softening process. Our findings suggest FvMYB79 acts as a novel regulator during strawberry ripening via transcriptional activation of FvPME38, which provides a novel mechanism for improvement of strawberry fruit firmness.

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.

A single amino acid mutant in the EAR motif of IbMYB44.2 reduced the inhibition of anthocyanin accumulation in the purple-fleshed sweetpotato

Purple-fleshed sweetpotato (Ipomoea batatas（L.）Lam.) is rich in anthocyanins. R2R3-type MYB transcription factors（TFs）with EAR motifs inhibiting anthocyanin biosynthesis have been reported, and there is still a lack of information on how mutations in the EAR motifs of MYBs affect anthocyanin accumulation. In this study, we obtained three IbMYB44 TFs by bioinformatics. Among these TFs, IbMYB44.1, IbMYB44.3 with a complete EAR motif and IbMYB44.2 with a single amino acid mutant in the EAR motif caused an amino acid substitution from leucine to valine. RT-qPCR analysis showed that IbMYB44s was expressed at lower levels in the purple-fleshed sweetpotato than in nonpurple-fleshed sweetpotato (P < 0.01). Transient expression assays showed that the inhibitory effect of IbMYB44.1/3 was stronger than IbMYB44.2 in tobacco leaves and red-skinned pears. RT-qPCR analysis further proved that IbMYB44.1/3 significantly inhibited the expression of anthocyanin biosynthesis-related genes compared with IbMYB44.2 in tobacco leaves and red-skinned pears. A dual luciferase reporter assay showed that IbMYB44s cannot directly activate the IbANS promoter, and the result was also verified by yeast one-hybrid (Y1H) experiments. Moreover, we identified the interaction of IbMYB340 with IbMYB44.1, IbMYB44.2 and IbMYB44.3 via yeast two-hybrid (Y2H) assays. Thus, IbMYB44.1/3 could interact with IbMYB340 to negatively regulate anthocyanin biosynthesis. This study enriched the regulatory network of anthocyanins and also provided a theoretical basis for a single amino acid mutant from leucine to valine in the EAR motif of IbMYB44.2 affecting anthocyanin biosynthesis in the purple-fleshed sweetpotato.

Expression analysis of MYB genes in different apple cultivars with distinct coloration pattern during fruit maturation

Abstract Anthocyanins are water-soluble pigments responsible for red, pink, purple or blue coloration in the flesh and/or skin of apple fruit. Since consumers prefer anthocyanin rich fruits due to their health benefits, anthocyanin content is an important trait for marketability of apples. Synthesis and accumulation of anthocyanins are controlled by environmental and genetic factors such as transcription factors (TFs). Two-repeat (R2R3) MYB TFs are involved in the regulation of anthocyanin biosynthesis in plants. In this study, the expression of the MdMYBA and MdMYB3 genes encoding R2R3 type MYB TFs were analyzed in apple cultivars with fruit skin color from green to dark red at different growth stages. Fruit samples were collected from “Scarlet Spur”, “Galaxy Gala”, Golden Delicious” “Granny Smith” apple cultivars with dark red, light red, yellow and green fruits, respectively, at four different time periods after full-bloom. Total RNA was isolated from the peel of the collected fruits and the expression of the MdMYBA and MdMYB3 genes was analyzed by real-time RT-PCR. The expression of the MdMYBA gene started to increase at 92 DAFB and thereafter in red-skin apples; however, no expression was observed at any time points in yellow or green-skin apple cultivars. On the other hand, the MdMYB3 gene was expressed in red-skin and yellow-skin apple cultivars starting at 55 DAFB, but no expression was observed in green apple cultivar at any time of fruit maturation. The expression levels of the MdMYBA and MdMYB3 genes varied in apple fruits with different skin coloration indicating that they play a role regulation of the anthocyanin biosynthesis in apple cultivars during fruit maturation.

FvMYB24, a strawberry R2R3-MYB transcription factor, improved salt stress tolerance in transgenic Arabidopsis

Salinity is one of the major environmental stresses that limit crop growth and productivity. In this study, the FvMYB24 gene that encodes an R2R3-type MYB transcription factor was cloned and characterized. An expression analysis showed that FvMYB24 had a tissue- and stage-specific profile and was induced by salt treatment. Arabidopsis plants that overexpressed transgenic FvMYB24 exhibited a higher germination rate, fresh weight, chlorophyll content, and longer root length than the wild type (WT) under salt stress. The transgenic plants had higher activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) and the accumulation of proline, while these plants accumulated lower amounts of malondialdehyde (MDA) compared with the WT. Furthermore, our results also revealed that the overexpression of FvMYB24 up-regulated the expression of several stress-related genes (AtSOS1, AtSOS2, AtSOS3, AtSOD, AtPOD, AtCAT1, AtNHX1, and AtLEA3) in response to salt stress, thus, enhancing the tolerance of transgenic Arabidopsis. An analysis of the cis-acting elements in the SOS1, SOS2, and SOS3 promoters revealed MYB-binding sites. However, FvMYB24 could only bind to the SOS1 promoter to mediate salt tolerance but not to the SOS2 and SOS3 promoters. These findings suggest that FvMYB24 could potentially be used as a positive regulator in transgenic plant breeding to improve the tolerance of strawberry plants to salt.