Cloning of Atr MYB Transcription Factor Gene from Acer truncatum

[Objective] Cloning of the AtrMYB transcription factor gene from Acer truncatum was conducted to further explore the red leaf development mechanism and breed cultivars of colored-leaf maple. [Method] The Acer truncatum ‘Luhong No.1＇ cultivar was used as the material for cloning the MYB gene by mean of RTPCR and RACE-PCR. [Results] Sequence analysis showed that the fragment contained a full coding region of 831 bp encoding 276 amino acid residues with a molecular weight of 32.17 kD and a molecular formula C_（1430）H_（14052）N_（2247）O_（406）S_（14）. The gene was named as AtrMYB with a Gen Bank accession number of 1825712. This coded protein had apI of 9.44. The results showed that the AtrMYB exhibited typical features of the R2R3-MYB domain. The AtrMYB was highly homologous with the MYB of other species at nucleotide and amino acid levels. The AtrMYB had no signal peptide, but a nuclear localization signal. The phylogenetic tree showed that the AtrMYB was at the same clade as the MYB from Citrus sinensis. [Conclusion] The AtrMYB was cloned from Acer truncatum ‘Luhong No.1＇ cultivar. These results have provided a foundation for further purification and identification of target protein and function study of the AtrMYB.

Genome-wide identification and expression profiling of MYB transcription factor genes in radish(Raphanus sativus L.)

Radish(Raphanus sativus L.), an important root vegetable crop of the Brassicaceae family, has a high level of anthocyanin accumulation in its pigment root tissues. It was reported that MYB transcription factors(TFs) play vital roles in plant development and anthocyanin metabolism, and the PAP1/2 could promote expression of anthocyanin biosynthesis genes. In this study, a total of 187 radish MYB genes(Rs MYBs) were identified in the radish genome and clustered into 32 subfamilies. Among them, 159 Rs MYBs were localized on nine radish chromosomes. Interestingly, 14 Rs MYBs exhibited differential expression profiles in different taproot developmental stages among four differently colored radish lines. A number of Rs MYBs were highly expressed in the pigmented root tissues at the maturity stage, several genes including Rs MYB41, Rs MYB117, and Rs MYB132 being homologous to PAP1/2, showed high expression levels in the red skin of NAU-YH(red skin-white flesh) taproot, while Rs MYB65 and Rs MYB159 were highly expressed in the purple root skin of NAU-YZH(purple skin-red flesh), indicating that these Rs MYBs might positively regulate the process of anthocyanin accumulation in radish taproot. These results would provide valuable information for further functional characterization of Rs MYBs, and facilitate clarifying the molecular mechanism underlying anthocyanin biosynthesis in radish.

Identifying potential flavonoid biosynthesis regulator in Zanthoxylum bungeanum Maxim. by genome-wide characterization of the MYB transcription factor gene family

Plant MYB transcription factors(TFs) play crucial roles in regulating the biosynthesis of flavonoids but current analysis on their role in Zanthoxylum bungeanum Maxim.(ZBM) is far from comprehensive. In this study, we identified 270 MYB genes in ZBM and divided them into four subfamilies. The R2 R3-MYB(ZbMYB) category contained 251 genes and was classified into 33 subfamilies according to their phylogenetic results and sequence similarity. These subfamilies included 24 subgroups containing both MYBs of ZBM plants and AtMYBs, and nine subgroups containing only ZBM MYBs or AtMYBs. ZbMYBs with similar functions clustered into the same subgroup, indicating functional conservation. The subcellular localization analysis predicted that most ZbMYB genes were found in the nucleus. The transposed duplications appeared to play a major role in the expansion of the MYB gene family in ZBM. Through phylogenetic analysis and transcriptome profiling, it was found that 28 ZbMYB genes may regulate the biosynthesis of flavonoids in ZBM, and these genes expression presented distinct temporal and spatial expression patterns. In different fruit development stages of ZBM, the expression patterns of EVM0042160 and EVM0033809 genes obtained by qRT-PCR analysis are very similar to the flavonoid and anthocyanin content curves in ZBM. Further correlation analysis showed that the content of flavonoids in different fruit development stages and the transcript abundance levels of 28 ZbMYB genes have different degrees of correlation relationship. These results indicated that the ZbMYB genes might be involved in the flavonoid metabolic pathway. This comprehensive and systematic analysis of MYB family genes provided a solid foundation for further functional analysis of MYB TFs in ZBM.

Mining Myb transcription factors related to wood development in Larix olgensis

Larix olgensis A.Henry is a fast-growing tree used for aff orestation in northeastern China and has great ecological and economic value.For studying developmental genes in the xylem of this species,we investigated the Myb transcription factor family,one of the largest families of transcription factors in plants,which plays an important role in the regulation of lignifi cation in plant secondary walls.By sequencing a L.olgensis cDNA library using the Illumina HiSeq2500 high-throughput sequencing platform,we obtained 58,683 unigene sequences,of which 16,554 unigenes were longer than 1000 bp,accounting for 28.2%of the total database.The alignment of these genes with the GO,COG,KEGG,Swiss-Prot and NR databases resulted in annotated 29,350 unigenes.We obtained a total of 1460 differentially expressed genes,of which 453 were upregulated and 1007 were downregulated at the two developmental stages analyzed.The gene annotations showed a wide range of biological functions and metabolic pathways.The 10 Myb transcription factors that were obtained from the diff erentially expressed genes were analyzed by real-time quantitative PCR(qRT-PCR).The results showed that four Myb transcription factors may be associated with xylem development in L.olgensis.Due to the large genome size of conifers,genomics research on these species has lagged behind that for other plant groups.Our data provide the basis for further studies on xylem development in L.olgensis.

Alternative Splicing in the Anthocyanin Fruit Gene Encoding an R2R3 MYB Transcription Factor Affects Anthocyanin Biosynthesis in Tomato Fruits

Tomato(Solanum lycopersicum)fruits are typically red at ripening,with high levels of carotenoids and a low content in flavonoids.Considerable work has been done to enrich the spectrum of their healthbeneficial phytochemicals,and interspecific crosses with wild species have successfully led to purple anthocyanin-colored fruits.The Aft(Anthocyanin fruit)tomato accession inherited from Solanum chilense the ability to accumulate anthocyanins in fruit peel through the introgression of loci controlling anthocyanin pigmentation,including four R2R3 MYB transcription factor-encoding genes.Here,we carried out a comparative functional analysis of these transcription factors in wild-type and Aft plants,and tested their ability to take part in the transcriptional complexes that regulate the biosynthetic pathway and their effi-ciency in inducing anthocyanin pigmentation.Significant differences emerged for SlAN2like,both in the expression level and protein functionality,with splicing mutations determining a complete loss of function of the wild-type protein.This transcription factor thus appears to play a key role in the anthocyanin fruit pigmentation.Our data provide new clues to the long-awaited genetic basis of the Aft phenotype and contribute to understand why domesticated tomato fruits display a homogeneous red coloration without the typical purple streaks observed in wild tomato species.

Isolation and functional analysis of SrMYB1,a direct transcriptional repressor of SrUGT76G1 in Stevia rebaudiana

SrUGT76G1,the most well-studied diterpene glycosyltransferase in Stevia rebaudiana,is key to the biosynthesis of economically important steviol glycosides(SGs).However,the molecular regulatory mechanism of SrUGT76G1 has rarely been explored.In this study,we identified a MYB transcription factor,SrMYB1,using a yeast one-hybrid screening assay.SrMYB1 belongs to the typical R2R3-type MYB protein and is specifically localized in the nucleus with strong transactivation activity.The transcript of SrMYB1 is predominantly accumulated in flowers,but is also present at a lower level in leaves.Yeast one-hybrid and electrophoretic mobility shift assays verified that SrMYB1 binds directly to the MYB binding sites in the F4-3 fragment(+50–(–141))of the SrUGT76G1 promoter.Furthermore,we found that SrMYB1 could significantly repress the expression of SrUGT76G1 in both epidermal cells of tobacco leaves and stevia callus.Taken together,our results demonstrate that SrMYB1 is an essential upstream regulator of SrUGT76G1 and provide novel insight into the regulatory network for the SGs metabolic pathway in S.rebaudiana.

Soybean GmMYB76, GmMYB92, and GmMYB177 genes confer stress tolerance in transgenic Arabidopsis plants

MYB-type transcription factors contain the conserved MYB DNA-binding domain of approximately 50 amino acids and are involved in the regulation of many aspects of plant growth, development, metabolism and stress responses. From soybean plants, we identified 156 GmMYB genes using our previously obtained 206 MYB unigenes, and 48 were found to have full-length open-reading frames. Expressions of all these identified genes were examined, and we found that expressions of 43 genes were changed upon treatment with ABA, salt, drought and/or cold stress. Three GmMYB genes, GmMYB76, GmMYB92 and GmMYB177, were chosen for further analysis. Using the yeast assay system, GmMYB76 and GmMYB92 were found to have transactivation activity and can form homodimers. GmMYB177 did not appear to have transactivation activity but can form heterodimers with GmMYB76. Yeast onehybrid assay revealed that all the three GmMYBs could bind to cis-elements TAT AAC GGT TTT TT and CCG GAA AAA AGG AT, but with different affinity, and GmMYB92 could also bind to TCT CAC CTA CC. The transgenic Arabidopsis plants overexpressing GmMYB 76 or GmMYB177 showed better performance than the GmMYB92-transgenic plants in salt and freezing tolerance. However, these transgenic plants exhibited reduced sensitivity to ABA treatment at germination stage in comparison with the wild-type plants. The three GmMYB genes differentially affected a subset of stress-responsive genes in addition to their regulation of a common subset of stress-responsive genes. These resuits indicate that the three GmMYB genes may play differential roles in stress tolerance, possibly through regulation of stress-responsive genes.

Genome-wide Analysis of MYB Gene Family in Potato Provides Insights into Tissue-specific Regulation of Anthocyanin Biosynthesis

The MYB transcription factor is one of the largest gene families in plants,playing an important role in regulating plant growth,development,response to stress,senescence,and especially the anthocyanin biosynthesis.In this study,A total of 217 MYB genes,including 901R-MYBs,124 R2R3-MYBs,and 3 R1R2R3-MYBs have been identified from the potato genome.The 1R-MYB and R2R3-MYB family members could be divided into 20 and 35 subgroups respectively.Analysis of gene structure and protein motifs revealed that members within the same subgroup presented similar exon/intron and motif organization,further supporting the results of phylogenetic analysis.Potato is an ideal plant to reveal the tissue-specific anthocyanins biosynthesis regulated by MYB,as the anthocyanins could be accumulated in different tissues,showing colorful phenotypes.Five pairs of colored and colorless tissues,stigma,petal,stem,leaf,and tuber flesh,were applied to the transcriptomic analysis.A total of 70 MYB genes were found to be differentially expressed between colored and colorless tissues,and these differentially expressed genes were suspected to regulate the biosynthesis of anthocyanin of different tissues.Co-expression analysis identified numerous potential interactive regulators of anthocyanins biosynthesis,involving 39 MYBs,24 bHLHs,2 WD-repeats,and 29 biosynthesis genes.Genome-wide association study(GWAS)of tuber flesh color revealed amajor signal at the end of Chromosome 10,which was co-localized with reported I gene(StMYB88),controlling tuber peel color.Analyses of DEGs(Differentially Expression Genes)revealed that both StMYB88 and StMYB89 were closely related to regulating anthocyanin biosynthesis of tuber flesh.This work offers a comprehensive overview of the MYB family in potato and will lay a foundation for the functional validation of these genes in the tissue-specific regulation of anthocyanin biosynthesis.

Transgenic expression of MYB15 confers enhanced sensitivity to abscisic acid and improved drought tolerance in Arabidopsis thaliana

Abiotic stresses cause serious crop losses. Knowledge on genes functioning in plant responses to adverse growth conditions is essential for developing stress tolerant crops. Here we report that transgenic expression of MYB15, encoding a R2R3 MYB transcription factor in Arabidopsis thaliana, conferred hypersensitivity to exogenous abscisic acid （ABA） and improved tolerance to drought and salt stresses. The promoter of MYB15 was active in not only vegetative and reproductive organs but also the guard cells of stomata. Its transcript level was substantially upregulated by ABA, drought or salt treatments. Compared with wild type （WT） control, MYB15 overexpression lines were hypersensitive to ABA in germination assays, more susceptible to ABA-elicited inhibition of root elongation, and more sensitive to ABA-induced stomatal closure. In line with the above findings, the transcript levels of ABA biosynthesis （ABA1, ABA2）, signaling （AB13） and responsive genes （AtADH1, RD22, RD29B, AtEM6） were generally higher in MYB15 overexpression seedlings than in WT controls after treatment with ABA. MYB15 overexpression lines displayed improved survival and reduced water loss rates than WT control under water deficiency conditions. These overexpression lines also displayed higher tolerance to NaCI stress. Collectively, our data suggest that overexpression of MYB15 improves drought and salt tolerance in Arabidopsis possibly by enhancing the expression levels of the genes involved in ABA biosynthesis and signaling, and those encoding the stress-protective proteins.

Cotton GhMYB7 is predominantly expressed in developing fibers and regulates secondary cell wall biosynthesis in transgenic Arabidopsis

The secondary cell wall in mature cotton fibers contains over 90%cellulose with low quantities of xylan and lignin.However,little is known regarding the regulation of secondary cell wall biosynthesis in cotton fibers.In this study,we characterized an R2R3-MYB transcription factor,Gh MYB7,in cotton.Gh MYB7 is expressed at a high level in developing fibers and encodes a MYB protein that is targeted to the cell nucleus and has transcriptional activation activity.Ectopic expression of Gh MYB7 in Arabidopsis resulted in small,curled,dark green leaves and also led to shorter inflorescence stems.A cross-sectional assay of basal stems revealed that cell wall thickness of vessels and interfascicular fibers was higher in transgenic lines overexpressing Gh MYB7 than in the wild type.Constitutive expression of Gh MYB7 in Arabidopsis activated the expression of a suite of secondary cell wall biosynthesis-related genes(including some secondary cell wall-associated transcription factors),leading to the ectopic deposition of cellulose and lignin.The ectopic deposition of secondary cell walls may have been initiated before the cessation of cell expansion.Moreover,Gh MYB7 was capable of binding to the promoter regions of At SND1 and At Ces A4,suggesting that Gh MYB7 may function upstream of NAC transcription factors.Collectively,these findings suggest that Gh MYB7 is a potential transcriptional activator,which may participate in regulating secondary cell wall biosynthesis of cotton fibers.

Genomic Survey and Expression Profiling of the MYB Gene Family in Watermelon

Myeloblastosis(MYB)proteins constitute one of the largest transcription factor(TF)families in plants.They are functionally diverse in regulating plant development,metabolism,and multiple stress responses.However,the function of watermelon MYB proteins remains elusive to date.Here,a genome-wide identification of watermelon MYB TFs was performed by bioinformatics analysis.A total of 162 MYB genes were identified from watermelon(Cla MYB).A comprehensive overview of the Cla MYB genes was undertaken,including the gene structures,chromosomal distribution,gene duplication,conserved protein motif,and phylogenetic relationship.According to the analyses,the watermelon MYB genes were categorized into three groups(R1R2R3-MYB,R2R3-MYB,and MYB-related).Amino acid alignments for all MYB motifs of Cla MYBs demonstrated high conservation.Investigation of their chromosomal localization revealed that these Cla MYB genes distributed across the 11watermelon chromosomes.Gene duplication analyses showed that tandem duplication events contributed predominantly to the expansion of the MYB gene family in the watermelon genome.Phylogenetic comparison of the Cla MYB proteins with Arabidopsis MYB proteins revealed that watermelon MYB proteins underwent a more diverse evolution after divergence from Arabidopsis.Some watermelon MYBs were found to cluster into the functional clades of Arabidopsis MYB proteins.Expression analysis under different stress conditions identified a group of watermelon MYB proteins implicated in the plant stress responses.The comprehensive investigation of watermelon MYB genes in this study provides a useful reference for future cloning and functional analysis of watermelon MYB proteins.

Overexpression of the Wounding-Responsive Gene AtMYB15 Activates the Shikimate Pathway in Arabidopsis

The MYB transcription factor genes play important roles in many developmental processes and various defense responses of plants. The shikimate pathway is a major biosynthetic pathway for the production of three aromatic amino acids and other aromatic compounds that are involved in multiple responses of plants, including protection against UV and defense. Herein, we describe the characterization of the R2R3-MYB gene AtMYB15as an activator of the shikimate pathway in Arabidopsis. The AtMYB15 protein is nuclear localized and a transcriptional activation domain is found in its C-terminal portion. Northern blots showed that AtMYB15 is an early wounding-inducible gene. Resutls of microarray analysis, confirmed using quantitative real-time polymerase chain reaction, showed that overexpression of AtMYB15 in transgenic plants resulted in elevated expression of almost all the genes involved in the shikimate pathway. Bioinformatics analysis showed that one or more AtMYB15-binding AC elements were detected in the promoters of these upregulated genes. Furthermore, these genes in the shikimate pathway were also found to be induced by wounding. These data suggest an important role of AtMYB15as a possible direct regulator of the Arabidopsis shikimate pathway in response to wounding.

MYB106 is a negative regulator and a substrate for CRL3^(BPM) E3 ligase in regulating flowering time in Arabidopsis thaliana

Flowering time is crucial for successful reproduction in plants, the onset and progression of which are strictly controlled. However, flowering time is a complex and environmentally responsive history trait and the underlying mechanisms still need to be fully characterized. Post-translational regulation of the activities of transcription factors(TFs) is a dynamic and essential mechanism for plant growth and development. CRL3 BPME3 ligase is a CULLIN3-based E3 ligase involved in orchestrating protein stability via the ubiquitin proteasome pathway. Our study shows that the mutation of MYB106 induced early flowering phenotype while over-expression of MYB106 delayed Arabidopsis flowering. Transcriptome analysis of myb106 mutants reveals 257 differentially expressed genes between wild type and myb106-1 mutants, including Flowering Locus T(FT) which is related to flowering time. Moreover, in vitro electrophoretic mobility shift assays(EMSA), in vivo chromatin immunoprecipitation quantitative polymerase chain reaction(ChIP-q PCR) assays and dual luciferase assays demonstrate that MYB106 directly binds to the promoter of FT to suppress its expression. Furthermore, we confirm that MYB106 interacts with BPM proteins which are further identified by CRL3 BPME3 ligases as the substrate. Taken together, we have identified MYB106 as a negative regulator in the control of flowering time and a new substrate for CRL3 BPM E3 ligases in Arabidopsis.

The pineapple reference genome:Telomere-to-telomere assembly, manually curated annotation, and comparative analysis

Pineapple is the third most crucial tropical fruit worldwide and available in five varieties.Genomes of different pineapple varieties have been released to date;however,none of them are complete,with all exhibiting substantial gaps and representing only two of the five pineapple varieties.This significantly hinders the advancement of pineapple breeding efforts.In this study,we sequenced the genomes of three varieties:a wild pineapple variety,a fiber pineapple variety,and a globally cultivated edible pineapple variety.We constructed the first gap-free reference genome(Ref)for pineapple.By consolidating multiple sources of evidence and manually revising each gene structure annotation,we identified 26,656 proteincoding genes.The BUSCO evaluation indicated a completeness of 99.2%,demonstrating the high quality of the gene structure annotations in this genome.Utilizing these resources,we identified 7,209 structural variations across the three varieties.Approximately 30.8%of pineapple genes were located within±5 kb of structural variations,including 30 genes associated with anthocyanin synthesis.Further analysis and functional experiments demonstrated that the high expression of AcMYB528 aligns with the accumulation of anthocyanins in the leaves,both of which may be affected by a 1.9-kb insertion fragment.In addition,we developed the Ananas Genome Database,which offers data browsing,retrieval,analysis,and download functions.The construction of this database addresses the lack of pineapple genome resource databases.In summary,we acquired a seamless pineapple reference genome with highquality gene structure annotations,providing a solid foundation for pineapple genomics and a valuable reference for pineapple breeding.