Myb-related Genes Research Articles

Overexpression of an MYB-Related Gene FvMYB1 from Fraxinus velutina Increases Tolerance to Salt Stress in Transgenic Tobacco

MYB transcription factors (TFs) in plants play important roles in abiotic stress tolerance. Here, we report that an MYB-related TF gene, named FvMYB1, was isolated from the salt-tolerant tree species Fraxinus velutina Torr. The real-time PCR analysis showed that the FvMYB1 transcript increased remarkably in response to salt treatment. Subsequently, the FvMYB1 gene was transferred into the tobacco genome via the Agrobacterium-mediated approach. PCR and Southern blot verified that the FvMYB1 gene had been expressed in transgenic tobacco. The overexpressing transgenic seedlings displayed better salt tolerance in root length, shoot fresh weight, and shoot/root ratio compared to the wild type. Moreover, along with the superoxide dismutase and peroxidase activities and proline content that were significantly increased in the transgenic plants, the transcriptions of a series of stress-responsive genes were up-regulated under salt treatment. Further, an analysis of the upstream sequence of the FvMYB1 gene revealed that the promoter could enhance GUS gene expression in transgenic tobacco with NaCl treatment. These results indicate that FvMYB1 functions as a transcriptional activator that plays a regulatory role in salt stress tolerance of F. velutina.

MYB-related transcription factors function as regulators of the circadian clock and anthocyanin biosynthesis in Arabidopsis

ABSTRACTIn Arabidopsis, the MYB (myeloblastosis) gene family contains more than 190 members, which play a number of roles in plant growth and development. Based on their protein structure, this gene family was divided into several subclasses, including the MYB-related class. Currently, an MYB-related gene designated as MYB-like Domain (AtMYBD) has been shown to function as a positive regulator of anthocyanin biosynthesis in Arabidopsis. This gene was found to belong to the CCA1-like (circadian clock-associated 1) group, which represents several genes that are master regulators of the circadian clocks of plants. Here, we speculate that AtMYBD is able to regulate anthocyanin biosynthesis in Arabidopsis thaliana in a circadian clock-related manner.

MYBA2 gene involved in anthocyanin and flavonol biosynthesis pathways in grapevine.

MYBA2 transcription factor (Myb-related gene) affects the coloring in grapevine berry and plays an important role in the biosynthesis of anthocyanin. The MYBA2 gene was cloned from Vitis vinifera L. cv. Cabernet Sauvignon and polyclonal antibodies for VvmybA2 were prepared. The VvmybA2 gene expression patterns were observed in seven tissues (the leaf, stem, flower, bud, root, berry, and tendril) and during the berry development stage at transcriptional and translational levels, respectively. The results indicated that the expression of VvmybA2 was approximately 11-fold higher in the berry than that in the other six tissues, and increased rapidly from 60 days after full bloom reaching a maximum on day 80. Furthermore, both the anthocyanin content and UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT) gene expression levels increased rapidly 60 days after full bloom. Moreover, correlation analysis indicated that the transcriptional and translational expression levels of the VvmybA2 gene were significantly positively correlated with not only UFGT and DFR genes but also with the anthocyanin content during berry development. These results suggested that VvmybA2 could not only regulate the transcription of both UFGT and DFR but also is involved in the expression of the UFGT gene associated with color determination in grape berries.

‘Zhengmei’: A new early-ripening table grape

Introduction: As a result of natural hybridization and human selection over centuries, the grape berry skin colour has become greatly diversified. The colour of grape skins is determined by the accumulation of anthocyanins (HARBONE et al. 2000). Anthocyanin content is a trait of major interest in Vitis vinifera L. They have very important role in wine and grape industry, confer colour and contribute to other sensory characteristics (BOSS et al. 1996). Molecular analyses of structural genes involved in the anthocyanin biosynthetic pathway showed that expression of the UDP glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene is a critical point in the control of berry colour (KOBAYASHI et al. 2001). It was shown that Myb-related gene, VvMybA1, regulates anthocyanin biosynthesis in grapes via UFGT gene expression (KOBAYASHI et al. 2004, 2005) and the colouring of grape skin depends on the genotype of VvMybA1 (LIJAVETZKY et al. 2006, AZUMA et al. 2007). Recently, it has been shown that the presence of Gret1, a Ty3–gypsy-type retrotransposon, in the promoter region of VvMybA1 gene, is associated with white-berry cultivars when present in a homozygous state (KOBAYASHI et al. 2004). Coloured cultivars hold at least one allele at the VvMybA1 locus not containing this large insertion: VvMybA1b or VvMybA1c (KOBAYASHI et al. 2004, AZUMA et al. 2007, YAKUSHIJI et al. 2006). It was revealed that the VvMybA1b and VvMybA1c alleles are functional. The VvMybA1b allele has a single long terminal repeat (LTR) of Gret1 in the 5 flanking region near the coding region of VvMybA1 gene (AZUMA et al. 2007). VvMybA1c, the wild type allele, is the original sequence of VvMybA1 gene before the insertion of Gret1 (YAKUSHIJI et al. 2006). In most examined white-skinned grapes, the colour locus is homozygous for VvMybA1a, whereas the genotype of colour skinned berries are heterozygous (VvMybA1a/VvMybA1b or VvMybA1a/ VvMybA1c) or are homozygous (VvMybA1c) (AZUMA et al. 2008). Due to the lack of data for Armenian grapevines, the present study has the objective to analyze the role of VvmybA1 gene in berry color variation. In order to ascertain the existent allelic composition for this gene and its association with berry color variation, thirty autochthonous Armenian grapevine cultivars were analyzed.

Overexpression of sheepgrass R1-MYB transcription factor LcMYB1 confers salt tolerance in transgenic Arabidopsis

Sheepgrass [Leymus chinensis (Trin.) Tzvel.] is a dominant, rhizomatous grass that has extensive plasticity in adapting to various harsh environments. Based on data from 454 high-throughput sequencing (GS FLX) exposure to salt stress, an unknown functional MYB-related gene LcMYB1 was identified from sheepgrass. Tissue specific expression profiles showed that the LcMYB1 gene was expressed ubiquitously in different tissues, with higher expression levels observed in the rhizome and panicle. The expression of LcMYB1 was induced obviously by high salt, drought and abscisic acid and was induced slightly by cold. A fusion protein of LcMYB1 with green fluorescent protein (GFP) was localized to the nucleus, and yeast one-hybrid analysis indicated that LcMYB1 was an activator of transcriptional activity. LcMYB1-overexpressing plants were more tolerant to salt stress than WT plants. The amounts of proline and soluble sugars were higher in transgenic Arabidopsis than in WT plants under salt stress conditions. The overexpression of LcMYB1 enhanced the expression levels of P5CS1 and inhibited other salt stress response gene markers. These findings demonstrate that LcMYB1 influences the intricate salt stress response signaling networks by promoting different pathways than the classical DREB1A- and MYB2-mediated signaling pathway. Additionally, LcMYB1 is a promising gene resource for improving salinity tolerance in crops.

Genome-Wide Identification and Evolutionary and Expression Analyses of MYB-Related Genes in Land Plants

MYB proteins constitute one of the largest transcription factor families in plants. Recent evidence revealed that MYB-related genes play crucial roles in plants. However, compared with the R2R3-MYB type, little is known about the complex evolutionary history of MYB-related proteins in plants. Here, we present a genome-wide analysis of MYB-related proteins from 16 species of flowering plants, moss, Selaginella, and algae. We identified many MYB-related proteins in angiosperms, but few in algae. Phylogenetic analysis classified MYB-related proteins into five distinct subgroups, a result supported by highly conserved intron patterns, consensus motifs, and protein domain architecture. Phylogenetic and functional analyses revealed that the Circadian Clock Associated 1-like/R-R and Telomeric DNA-binding protein-like subgroups are >1 billion yrs old, whereas the I-box-binding factor-like and CAPRICE-like subgroups appear to be newly derived in angiosperms. We further demonstrated that the MYB-like domain has evolved under strong purifying selection, indicating the conservation of MYB-related proteins. Expression analysis revealed that the MYB-related gene family has a wide expression profile in maize and soybean development and plays important roles in development and stress responses. We hypothesize that MYB-related proteins initially diversified through three major expansions and domain shuffling, but remained relatively conserved throughout the subsequent plant evolution.

Phloem parenchyma transfer cells in Arabidopsis – an experimental system to identify transcriptional regulators of wall ingrowth formation

In species performing apoplasmic loading, phloem cells adjacent to sieve elements often develop into transfer cells (TCs) with wall ingrowths. The highly invaginated wall ingrowths serve to amplify plasma membrane surface area to achieve increased rates of apoplasmic transport, and may also serve as physical barriers to deter pathogen invasion. Wall ingrowth formation in TCs therefore plays an important role in phloem biology, however, the transcriptional switches regulating the deposition of this unique example of highly localized wall building remain unknown. Phloem parenchyma (PP) TCs in Arabidopsis veins provide an experimental system to identify such switches. The extent of ingrowth deposition responds to various abiotic and applied stresses, enabling bioinformatics to identify candidate regulatory genes. Furthermore, simple fluorescence staining of PP TCs in leaves enables phenotypic analysis of relevant mutants. Combining these approaches resulted in the identification of GIGANTEA as a regulatory component in the pathway controlling wall ingrowth development in PP TCs. Further utilization of this approach has identified two NAC (NAM, ATAF1/2 and CUC2)-domain and two MYB-related genes as putative transcriptional switches regulating wall ingrowth deposition in these cells.

Flavonoid biosynthesis-related genes in grape skin are differentially regulated by temperature and light conditions

Temperature and light are important environmental factors that affect flavonoid biosynthesis in grape berry skin. However, the interrelationships between temperature and light effects on flavonoid biosynthesis have not been fully elucidated at the molecular level. Here, we investigated the effects of temperature and light conditions on the biosynthesis of flavonoids (anthocyanins and flavonols) and the expression levels of related genes in an in vitro environmental experiment using detached grape berries. Sufficient anthocyanin accumulation in the grape skin was observed under a low temperature (15 °C) plus light treatment, whereas high temperature (35 °C) or dark treatment severely suppressed anthocyanin accumulation. This indicates that the accumulation of anthocyanins is dependent on both low temperature and light. qRT-PCR analysis showed that the responses of three MYB-related genes (VlMYBA1-3, VlMYBA1-2, and VlMYBA2) to temperature and light differed greatly even though the products of all three genes had the ability to regulate anthocyanin biosynthesis pathway genes. Furthermore, the expression levels of other MYB-related genes and many flavonoid biosynthesis pathway genes were regulated independently by temperature and light. We also found that temperature and light conditions affected the anthocyanin composition in the skin through the regulation of flavonoid biosynthesis pathway genes. Our results suggest that low temperature and light have a synergistic effect on the expression of genes in the flavonoid biosynthesis pathway. These findings provide new information about the relationships between environmental factors and flavonoid accumulation in grape berry skin.

Haplotype composition at the color locus is a major genetic determinant of skin color variation in Vitis ×labruscana grapes

Skin color is one of the most important fruit traits in grape, and has become greatly diversified due to hybridization and human selection. Many studies concerning the genetic control of grape color in European species (Vitis vinifera L.), especially the role of MYB-related genes, have been reported. On the other hand, there have been few studies of the MYB-related genes in grapes belonging to V.×labruscana L.H. Bailey, a subgroup of grapes that originated from the hybridization of V. labrusca with V. vinifera. In the present study, we found a novel functional haplotype, HapE2 (consisting of the genes VlMYBA2 and VlMYBA1-3), in diploid V.×labruscana. Moreover, we developed a method to determine the haplotype compositions of tetraploid grapes by means of quantitative real-time PCR, and investigated the relationship between haplotype composition and skin color. The color locus in V.×labruscana grapes usually consists of functional haplotypes (HapE1 and/or HapE2), and non-functional haplotype HapA. The number of functional haplotypes in the genome was found to be correlated with the level of anthocyanin in the skin. Anthocyanin contents of grapes that contained HapE2 were significantly higher than those containing HapE1. These results suggest that the number and kind of functional haplotypes at the color locus are the major genetic factors that determine skin color variation. These findings provide new knowledge about the unique genetic control of color in V.×labruscana grapes, and should contribute to development of new cultivars that have the desired color and anthocyanin content.

Short-term salt and PEG stresses regulate expression of MicroRNA, miR159 in sugarcane leaves

MicroRNAs (miRNAs) are involved in the regulation of stress-responsive gene expression and stress adaptation. Transcript expression of mature miR159 was studied in sugarcane leaves stressed for long-term (15 days) NaCl (150 mM) or iso-osmotic PEG 8000 (20% w/v) and for short period (up to 24 h) with NaCl (200 mM) or PEG (20% w/v). The results revealed no significant changes in transcript levels of the miRNA in response to the long-term stress however, short-term salt or PEG stress led to significant up-regulation over the control. Progressive increase in transcripts of the miRNA was observed under short-term PEG stress with exposure period. Using computational tools, various members of the MYB transcription factor family were predicted as the potential targets of miR159. The transcript expression of the MYB-related gene indicated up-regulation at 1 h of salt stress with concomitant slight downregulation of the miRNA. In addition, under short-term PEG stress, the transcript levels of MYB and miR159 were the opposite of each other, suggesting MYB as a potential target of miR159. To our knowledge, this is the first preliminary evidence on the involvement of miR159 in abiotic stress responses in sugarcane.

Resistant and Susceptible Responses in Tomato to Cyst Nematode are Differentially Regulated by Salicylic Acid

To understand the machinery underlying a tomato cultivar harboring the Hero A gene against cyst nematode using microarrays, we first analyzed tomato gene expression in response to potato cyst nematode (PCN; Globodera rostochiensis) during the early incompatible and compatible interactions at 3 and 7 days post-inoculation (dpi). Transcript levels of the phenylalanine ammonia lyase (PAL) and Myb-related genes were up-regulated at 3 dpi in the incompatible interaction. Transcription of the genes encoding pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) was also up-regulated at 3 dpi in the incompatible interaction. On the other hand, the four genes (PAL, Myb, PDC and ADH) were down-regulated in the compatible interaction at 3 dpi. When the expression levels of several pathogenesis-related (PR) protein genes in tomato roots were compared between the incompatible and compatible interactions, the salicylic acid (SA)-dependent PR genes were found to be induced in the incompatible interaction at 3 dpi. The PR-1(P4) transcript increased to an exceptionally high level at 3 dpi in the cyst nematode-infected resistant plants compared with the uninoculated controls. The free SA levels were elevated to similar levels in both incompatible and compatible interactions. We then confirmed that PR-1(P4) was not significantly induced in the NahG tomato harboring the Hero A gene, compared with the resistant cultivar. We thus found that PR-1(P4) was a hallmark for the cultivar resistance conferred by Hero A against PCN and that nematode parasitism resulted in the inhibition of the SA signaling pathway in the susceptible cultivars.

Genomic and genetic analysis of Myb-related genes that regulate anthocyanin biosynthesis in grape berry skin

As a result of natural hybridization and human selection over millennia, the skin colors of grapes have become greatly diversified. The color is determined by the quantity and composition of anthocyanins. Color-skinned cultivars accumulate anthocyanins in their skins, whereas white-skinned cultivars do not. Myb-related transcription-factor genes such as VvmybA1 regulate anthocyanin biosynthesis. VvMYBA2r, VlmybA1-1, VlmybA1-2, and VlmybA2, which are homologs of VvmybA1, also regulate anthocyanin biosynthesis. In this study, we isolated a novel Myb-related sequence, VlmybA1-3, from cultivars of Vitis labruscana (Vitis vinifera x Vitis labrusca) by means of inverse PCR, and confirmed by means of transient gene expression assay that the gene regulates anthocyanin biosynthesis in grape berry skin. Seedlings of V. labruscana with two functional haplotypes at a region of berry color loci accumulated more anthocyanins than seedlings with a single functional haplotype. In addition, we investigated the haplotypes at the region in 35 cultivars (both V. vinifera and V. labruscana), and found certain typical characteristics. These findings will contribute to the selection of seedlings with high anthocyanin quantities in breeding programs for wine and table grapes, and will help elucidate the origin and evolution of Vitis species.

The developmental selector AS1 is an evolutionarily conserved regulator of the plant immune response.

The MYB-related gene ASYMMETRIC LEAVES 1 (AS1) and its orthologs have an evolutionarily conserved role in specification of leaf cell identity. AS1 is expressed in leaf founder cells, where it functions as a heterodimer with the structurally unrelated AS2 proteins to repress activity of KNOTTED 1-like homeobox (KNOX) genes. AS1 therefore confines KNOX activity to the shoot apical meristem, where it promotes stem cell function through the regulation of phytohormone activities. Here, we show that loss-of-function mutations in AS1 unexpectedly convey heightened protection against necrotrophic fungi. AS1 operates as a negative regulator of inducible resistance against these pathogens by selectively binding to the promoters of genes controlled by the immune activator, jasmonic acid (JA), damping the defense response. In contrast, AS1 is a positive regulator of salicylic acid (SA)-independent extracellular defenses against bacterial pathogens. Neither the absence of AS2 nor ERECTA function, which enhances the morphological phenotype of as1, nor the conditional or constitutive expression of KNOX genes impacted disease resistance. Thus, the function of AS1 in responses to phytopathogens is independent of its AS2-associated role in development. Loss of function in the AS1 orthologs PHAN in Antirrhinum majus and NSPHAN in Nicotiana sylvestris produced pathogen-response phenotypes similar to as1 plants, and therefore the defense function of AS1 is evolutionarily conserved in plant species with a divergence time of approximately 125 million years.

Constitutive expression of CIR1 (RVE2) affects several circadian‐regulated processes and seed germination in Arabidopsis

Circadian clocks are endogenous auto-regulatory mechanisms that allow organisms, from bacteria to humans, to advantageously time a wide range of activities within 24 h environmental cycles. Here we report the identification and characterization of an MYB-related gene, designated Circadian 1 (CIR1), that is involved in circadian regulation in Arabidopsis. Expression of CIR1 is transiently induced by light and oscillates with a circadian rhythm. The rhythmic expression of CIR1 is controlled by the central oscillator. Constitutive expression of CIR1 resulted in a shorter period length for the rhythms of four central oscillator components, and much lower amplitude for the rhythms of central oscillator components CCA1 and LHY. Furthermore, CIR1 over-expression severely affected the circadian rhythms of its own RNA and those of the slave oscillator EPR1 and effector genes Lhcb and CAT3. Plants that constitutively expressed CIR1 displayed delayed flowering, longer hypocotyls and reduced seed germination in the dark. These results suggest that CIR1 is possibly part of a regulatory feedback loop that controls a subset of the circadian outputs and modulates the central oscillator.

VvmybA1 genotype determines grape skin color

Introduction: The skin color of grapes is deter-mined by the quantity and composition of anthocy-anins. Black and red cultivars accumulate anthocyanins in their skins, whereas white cultivars do not synthesize them. In grape, Myb-related genes such as VlmybA1-1 of Vitis labruscana regulate anthocyanin biosynthesis by controlling expression of the gene for UDP-glu-cose: flavonoid 3-O-glucosyltransferase (K

A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum.

The Rosea1, Rosea2, and Venosa genes encode MYB-related transcription factors active in the flowers of Antirrhinum majus. Analysis of mutant phenotypes shows that these genes control the intensity and pattern of magenta anthocyanin pigmentation in flowers. Despite the structural similarity of these regulatory proteins, they influence the expression of target genes encoding the enzymes of anthocyanin biosynthesis with different specificities. Consequently, they are not equivalent biochemically in their activities. Different species of the genus Antirrhinum, native to Spain and Portugal, show striking differences in their patterns and intensities of floral pigmentation. Differences in anthocyanin pigmentation between at least six species are attributable to variations in the activity of the Rosea and Venosa loci. Set in the context of our understanding of the regulation of anthocyanin production in other genera, the activity of MYB-related genes is probably a primary cause of natural variation in anthocyanin pigmentation in plants.

The myb-related gene stonewall induces both hyperplasia and cell death in Drosophila: rescue of fly lethality by coexpression of apoptosis inducers

We carried out gain-of-function mutagenesis screening and identified a mutant in which GAL4 induction led to both hyperplasia and apoptosis. The gene involved was identified as stonewall (stwl), a myb-related gene involved in germ cell proliferation and differentiation during oogenesis. As observed with dmyb, the ectopic expression of stwl(UY823) inhibited endoreplication in salivary glands. We also found that stwl(UY823) overexpression, like overexpression of the wild-type gene, activated G1/S transition and apoptosis. The apoptosis triggered by stwl(UY823) expression is correlated to induction of the proapoptotic gene reaper. Finally, the death of flies induced by ectopic stwl(UY823) expression is efficiently prevented in vivo by triggering cell death in stwl(UY823)-expressing cells. Our results suggest that stwl(UY823) kills flies by causing inappropriate cell cycle entry, and that triggering the death of these overproliferating cells or slowing their proliferation restores viability.

Functional diversification ofMYB23andGL1genes in trichome morphogenesis and initiation

The functional diversification of duplicated genes is one of the driving forces in evolution. To understand the molecular mechanisms of gene diversification, we studied the functional relationship of the two Arabidopsis paralogous MYB-related genes GL1 and MYB23. We show that MYB23 controls trichome branching and trichome initiation at leaf edges. The latter is controlled redundantly together with GL1. We show that the two proteins are functionally equivalent during trichome initiation but not during trichome branching. RT-PCR and reporter construct analysis revealed spatial, temporal and genetic differences in transcriptional regulation of the GL1 and MYB23 genes. Presented data indicate that the diversification of GL1 and MYB23 gene functions occurred at the level of cis-regulatory sequences with respect to trichome initiation, and that, in parallel, the diversification with respect to regulation of trichome branching also involved changes in respective proteins.

Association of VvmybA1 Gene Expression with Anthocyanin Production in Grape (Vitis vinifera) Skin-color Mutants

By using white-skinned cultivars (‘Italia’ and ‘Muscat of Alexandria’) and putative red-skinned sports (‘Ruby Okuyama’ and ‘Flame Muscat’) from the white cultivars, we analyzed the expression and function of a myb-related gene, VvmybA1, involved in the regulation of anthocyanin biosynthesis in grapes. A simple sequence repeat (SSR) analysis showed that ‘Ruby Okuyama’ and ‘Flame Muscat’ are true derivatives by bud mutation from ‘Italia’ and ‘Muscat of Alexandria’, respectively. The VvmybA1 transcript was detected in berry skins of ‘Ruby Okuyama’ after coloring had begun; it was not detectable in those of ‘Italia’. Three VvmybA cDNAs (VvmybA1, VvmybA2, and VvmybA3) from ‘Ruby Okuyama’ under the control of the 35S promoter were introduced into somatic embryos of ‘Kyoho’ by particle bombardment. Upon the introduction of VvmybA1, red cells were induced in the embryos, whereas the introduction of VvmybA2 or VvmybA3 failed to do so. The VvmybA1 cDNA was also shown to have the ability to induce the anthocyanin-producing cells in the skin tissues of ‘Muscat of Alexandria’. The relationship between the skin-color mutations and the detailed structure of the VvmybA1 gene is also described.

The distribution of c-myb immunoreactivities in the adult mouse retina

The myb gene family is composed of three different myb-related genes, A-, B- and c- myb. Among these, the presence of c- myb mRNA in developing and adult retina was previously reported. However, further study on the expression of c-myb in the retina is warranted because the previous study was only performed on developing retinal tissues by in situ hybridization technique. Therefore, in this study, we tried to perform immunohistochemical study, with a focus on the c-myb protein expression in adult retina. Although the cell types were unconfirmed, c-myb protein was likely to have been found in the ganglion, amacrine, horizontal and photoreceptor cells, judging from their locations and morphologies. The experimental data suggested that c-myb immunoreactivities were expressed by the cells in the neural retina, even in the adult stage, especially within some types of cells in specified retinal layers. This suggests that c-myb might play a role in the physiology of the retinal cells, not only in differentiation during retinal development.