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Lignin biosynthesis in the sclerenchyma cells is strictly controlled by a complex network of genetic and environmental signals. In the last decades, the transcriptional regulation of lignin synthesis in woody species has been established. However, the role of microRNA-mediated post-transcriptional modulation in secondary cell wall biosynthesis remains poorly understood. Here, we identified a microRNA, miR828, involved in the regulation specific to lignin biosynthesis during stem development in Populus tomentosa Carr. miR828 is preferentially expressed in the secondary vascular tissues during stem development. Two MYB genes (MYB171 and MYB011) were validated as direct targets of miR828 by degradome analysis and green fluorescent protein signal detection. Overexpression of miR828 in poplar downregulated genes for lignin biosynthesis, resulting in reduced lignin content in cell walls. Conversely, suppression of miR828 in plants by the short tandem target mimics elevated the expression of lignin biosynthetic genes and increased lignin deposition. We further revealed that poplar MYB171, as the most abundant miR828 target in the stem, is a positive regulator for lignin biosynthesis. Transient expression assays showed that both MYB171 and MYB011 activated PAL1 and CCR2 transcription, whereas the introduction of miR828 significantly suppressed their expression that was induced by MYB171 or MYB011. Collectively, our results demonstrate that the miR828-MYBs module precisely regulates lignin biosynthesis during the stem development in P. tomentosa through transcriptional and post-transcriptional manners.

Iron (Fe) is an essential element for plant growth and development. Fe deficiency can trigger leaf chlorosis and reduce fruit yield. Therefore, it is necessary to explore transcription factors in response to Fe deficiency stress. A total of 29 MhR2R3-MYB transcription factors were identified based on the transcriptome of Malus halliana under Fe deficiency stress. A comprehensive analysis of physical and chemical properties, gene structures, conserved motif composition, evolutionary relationship and chromosome distribution was performed. Subsequently, based on the transcriptome, 14 genes with the most significant expression under Fe deficiency stress were screened for qRT-PCR verification. Among them,the functional characteristics of MhR2R3-MYB4 (MD05G1089600) were further studied in Arabidopsis thaliana. Expression of 13 out of these 14 genes was upregulated, only one was downregulated. Maximum upregulation of MhR2R3-MYB4 under Fe deficiency was 36.39-fold and 58.21-fold compared with day 0 in leaves and roots, respectively. Overexpression of MhR2R3-MYB4 enhanced tolerance to Fe deficiency in A. thaliana and led to multiple biochemical changes: transgenic lines have higher chl a, chl b and Fe

The MYB transcription factor (TF) family is one of the largest and most important TF families, regulating the growth and response of microalgae to stresses. However, the gene structure and characteristics of Phaeodactylum tricornutum MYB TFs, and their functions under nitrogen deficiency have not been explored yet. To identify all P. tricornutum MYB (PtMYB) genes, the MYB gene family was analyzed at the genome-wide level in this study. Total 26 PtMYB genes were identified from the genome of P. tricornutum. These PtMYB genes were further divided into 5 subfamilies (i.e., 5R-MYB, 4R-MYB, R2R3-MYB, R1R2R3-MYB and MYB-related proteins). Phylogenetical motif and gene structure analyses of MYB genes indicated that the number and proportion of MYB TFs were species-specific, and MYB genes exhibited a lot of duplication events from microalgae to higher plants. Furthermore, the differentially expressed patterns of all 26 PtMYB TFs implied that PtMYB genes might have functional specificity under nitrogen deficiency. Homology analysis of MYB genes revealed that PtMYB3, PtMYB15 and PtMYB21 might play important roles in the regulation of diurnal cycle and response to nitrogen stress in P. tricornutum. PtMYB3, PtMYB15 and PtMYB21 genes might be used as potential candidate genes for further studying the regulatory mechanisms of P. tricornutum under nitrogen deficiency. Totally, this work provides an important foundation for the future research of the potential functions of PtMYB genes and its diurnal regulatory mechanisms under nitrogen deficiency.

The MYB gene family is involved in the regulation of plant growth, development and stress responses. In this paper, to identify

The function of the root system is crucial for plant survival, such as anchoring plants, absorbing nutrients and water from the soil, and adapting to stress. MYB transcription factors constitute one of the largest transcription factor families in plant genomes with structural and functional diversifications. Members of this superfamily in plant development and cell differentiation, specialized metabolism, and biotic and abiotic stress processes are widely recognized, but their roles in plant roots are still not well characterized. Recent advances in functional studies remind us that MYB genes may have potentially key roles in roots. In this review, the current knowledge about the functions of MYB genes in roots was summarized, including promoting cell differentiation, regulating cell division through cell cycle, response to biotic and abiotic stresses (e.g., drought, salt stress, nutrient stress, light, gravity, and fungi), and mediate phytohormone signals. MYB genes from the same subfamily tend to regulate similar biological processes in roots in redundant but precise ways. Given their increasing known functions and wide expression profiles in roots, MYB genes are proposed as key components of the gene regulatory networks associated with distinct biological processes in roots. Further functional studies of MYB genes will provide an important basis for root regulatory mechanisms, enabling a more inclusive green revolution and sustainable agriculture to face the constant changes in climate and environmental conditions.

R2R3-MYB transcription factors participate in multiple critical biological processes, particularly as relates to the regulation of secondary metabolites. The dried root of

Plants are affected by various abiotic stresses during their growth and development. In plants, MYB transcription factors are involved in various physiological and biochemical processes, including biotic and abiotic stress responses. In this study, we functionally analyzed MdMYB108L. We examined the transcriptional activity of MdMYB108L under salt stress and determined that the N-terminal domain of MdMYB108L, which was significantly induced under salt stress, has transcriptional activity. MdMYB108L overexpression increased the germination rate, main root length, and the antioxidant activity of catalase and peroxidase in transgenic

Main conclusion: We identified 119 typical CaMYB encoding genes and reveal the major components of the proanthocyanidin regulatory network. CaPARs emerged as promising targets for genetic engineering toward improved agronomic traits in C. arietinum. Chickpea (Cicer arietinum) is among the eight oldest crops and has two main types, i.e., desi and kabuli, whose most obvious difference is the color of their seeds. We show that this color difference is due to differences in proanthocyanidin content of seed coats. Using a targeted approach, we performed in silico analysis, metabolite profiling, molecular, genetic, and biochemical studies to decipher the transcriptional regulatory network involved in proanthocyanidin biosynthesis in the seed coat of C. arietinum. Based on the annotated C. arietinum reference genome sequence, we identified 119 typical CaMYB encoding genes, grouped in 32 distinct clades. Two CaR2R3-MYB transcription factors, named CaPAR1 and CaPAR2, clustering with known proanthocyanidin regulators (PARs) were identified and further analyzed. The expression of CaPAR genes correlated well with the expression of the key structural proanthocyanidin biosynthesis genes CaANR and CaLAR and with proanthocyanidin levels. Protein–protein interaction studies suggest the in vivo interaction of CaPAR1 and CaPAR2 with the bHLH-type transcription factor CaTT8. Co-transfection analyses using Arabidopsis thaliana protoplasts showed that the CaPAR proteins form a MBW complex with CaTT8 and CaTTG1, able to activate the promoters of CaANR and CaLAR in planta. Finally, transgenic expression of CaPARs in the proanthocyanidin-deficient A. thaliana mutant tt2-1 leads to complementation of the transparent testa phenotype. Taken together, our results reveal main components of the proanthocyanidin regulatory network in C. arietinum and suggest that CaPARs are relevant targets of genetic engineering toward improved agronomic traits.

The R2R3-MYB transcription factor is one of the largest transcription factor families in plants. R2R3-MYBs play a variety of functions in plants, such as cell fate determination, organ and tissue differentiations, primary and secondary metabolisms, stress and defense responses and other physiological processes. The Japanese morning glory (Ipomoea nil) has been widely used as a model plant for flowering and morphological studies. In the present study, 126 R2R3-MYB genes were identified in the Japanese morning glory genome. Information, including gene structure, protein motif, chromosomal location and gene expression, were assigned to the InR2R3-MYBs. Phylogenetic tree analysis revealed that the 126 InR2R3-MYBs were classified into 29 subfamilies (C1-C29). Herein, physiological functions of the InR2R3-MYBs are discussed based on the functions of their Arabidopsis orthologues. InR2R3-MYBs in C9, C15, C16 or C28 may regulate cell division, flavonol biosynthesis, anthocyanin biosynthesis or response to abiotic stress, respectively. C16 harbors the known anthocyanin biosynthesis regulator, InMYB1 (INIL00g10723), and putative anthocyanin biosynthesis regulators, InMYB2 (INIL05g09650) and InMYB3 (INIL05g09651). In addition, INIL05g09649, INIL11g40874 and INIL11g40875 in C16 were suggested as novel anthocyanin biosynthesis regulators. We organized the R2R3-MYB transcription factors in the morning glory genome and assigned information to gene and protein structures and presuming their functions. Our study is expected to facilitate future research on R2R3-MYB transcription factors in Japanese morning glory.

Brassica species include important oil crops and vegetables in the world. The R2R3-MYB gene participates in a variety of plant functions, including the activation or inhibition of anthocyanin biosynthesis. Although previous studies have reported its phylogenetic relationships, gene structures, and expression patterns in Arabidopsis, the number and sequence variation of this gene family in Brassica crops and its involvement in the natural quantitative variation in anthocyanin biosynthesis regulation are still largely unknown. In this study, by using whole genome sequences and comprehensive genome-wide comparative analysis among the six cultivated Brassica species, 1990 R2R3-MYB genes were identified in six Brassica species, respectively. These R2R3-MYB genes were phylogenetically clustered into 12 groups. The R2R3-MYB family between A and C subgenomes showed better collinearity than between B and C and between A and B. From comparing transcriptional changes of five Brassica species with the purple and green leaves for the detection of the R2R3-MYB genes associated with anthocyanin biosynthesis, 7 R2R3-MYB genes were co-differentially expressed. The promoter and structure analysis of these genes showed that some variations between non-coding region, but they were highly conserved at the protein level and spatial structure. Co-expression analysis of anthocyanin-related genes and R2R3-MYBs indicated that MYB90 was strongly co-expressed with TT8, and they were co-expressed with structural genes F3H, LDOX, ANS and UF3GT at the same time. These results further clarified the roles of the R2R3-MYBs for leaf coloration in Brasica species, which provided new insights into the functions of the R2R3-MYB gene family in Brasica species.

The spatially and temporally distinct expression of R2R3-MYB positive regulators is among the major mechanisms that create various anthocyanin color patterns in many flowers. However, we do not know how these positive regulators have gained different expression profiles. In the Asiatic hybrid lily 'Lollypop' (derived from the crosses of species belonging to Sinomartagon/Daurolirion section), MYB12 and MYB19S regulate the pigmentation at whole tepals and raised tepal spots, respectively. In the Oriental hybrid lily 'Sorbonne' (derived from the crosses of species belonging to the Archelirion section), MYB12 regulates both whole tepal and raised spot pigmentation. The genes have similar amino acid sequences with similar protein functions but exhibit different expression profiles in lily flowers. As promoters are among the most significant factors affecting gene expression profiles, their promoter sequences were determined in this study. The three genes had very different promoter sequences, and putative cis-regulatory elements were not conserved in numbers or order. To further confirm the promoter functions, tobacco plants were transformed with native promoter-driven MYB12 or MYB19S genes of 'Lollypop.' Expression levels of MYB12 were higher in corolla tubes than in lobes, while those of MYB19S were higher in corolla lobes than in tubes. Thus, the diverse promoter functions were likely to be the leading causes of their different expression profiles and generation of unique color patterns. Finally, the history of R2R3-MYB gene establishment during lily evolution was estimated using sequence data.

The v-myb avian myeloblastosis viral oncogene homolog (MYB) family of transcription factors is extensively distributed across the plant kingdom. However, the functional significance of red maple (Acer rubrum) MYB transcription factors remains unclear. Our research identified 393 MYB transcription factors in the Acer rubrum genome, and these ArMYB members were unevenly distributed across 34 chromosomes. Among them, R2R3 was the primary MYB sub-class, which was further divided into 21 sub-groups with their Arabidopsis homologs. The evolution of the ArMYB family was also investigated, with the results revealing several R2R3-MYB sub-groups with expanded membership in woody species. Here, we report on the isolation and characterization of ArMYB89 in red maple. Quantitative real-time PCR analysis revealed that ArMYB89 expression was significantly up-regulated in red leaves in contrast to green leaves. Sub-cellular localization experiments indicated that ArMYB89 was localized in the nucleus. Further experiments revealed that ArMYB89 could interact with ArSGT1 in vitro and in vivo. Overexpression of ArMYB89 in tobacco enhances the anthocyanin content of transgenic plants. In conclusion, our results contribute to the elucidation of a theoretical basis for the ArMYB gene family, and provide a foundation for further characterization of the biological roles of MYB genes in the regulation of Acer rubrum leaf color.

Background The rattan is a valuable plant resource with multiple applications in tropical forests. Calamus simplicifolius and Daemonorops jenkinsiana are the two most representative rattan species, supplying over 95% of the raw materials for the rattan industry. Hence, the wood properties of both rattans have always attracted researchers’ attention. Results We re-annotated the genomes, obtained 81 RNA-Seq datasets, and developed an improved pipeline to increase the reliability of co-expression networks of both rattans. Based on the data and pipeline, co-expression relationships were detected in 11 NACs, 49 MYBs, and 86 lignin biosynthesis genes in C. simplicifolius and four NACs, 59 MYBs, and 76 lignin biosynthesis genes in D. jenkinsiana, respectively. Among these co-expression pairs, several genes had a close relationship to the development of wood properties. Additionally, we detected the enzyme gene on the lignin biosynthesis pathway was regulated by either NAC or MYB, while LACCASES was regulated by both NAC and MYB. For D. jenkinsiana, the lignin biosynthesis regulatory network was characterized by positive regulation, and MYB possible negatively regulate non-expressed lignin biosynthesis genes in stem tissues. For C. simplicifolius, NAC may positively regulate highly expressed genes and negatively regulate non-expressed lignin biosynthesis genes in stem tissues. Furthermore, we established core regulatory networks of NAC and MYB for both rattans. Conclusions This work improved the accuracy of rattan gene annotation by integrating an efficient co-expression network analysis pipeline, enhancing gene coverage and accuracy of the constructed network, and facilitating an understanding of co-expression relationships among NAC, MYB, and lignin biosynthesis genes in rattan and other plants.

Breeding for flower cold resistance is a priority for flower breeding research in northern China. The identification of cold resistance genes will not only provide genetic resources for cold resistance breeding, but also form a basis for the study of plant cold resistance mechanisms. Based on the flower transcriptome of Iris laevigata, 20 R2R3-MYBs were identified and comprehensive analysis, including conservative domain, phylogenetic analyses and functional distribution, were performed for R2R3-MYBs. Expression patterns of the abiotic stress genes under cold stress were detected, the upregulated gene was genetically transformed into tobacco, and the related physiological indicators of the transgenic tobacco were measured. A novel cold resistance gene, IlMYB306, was obtained. qRT-PCR indicated that IlMYB306 was dramatically induced by cold stress and was significantly upregulated in roots. The free proline content, MDA, SOD and POD activity of the transgenic tobacco improved after cold stress, and the chlorophyll content decreased slowly. In addition, overexpression of IlMYB306 improved cold resistance of the seeds. SEM results showed leaves of transgenic tobacco had obvious folds, more grooves and bulges on the lower leaf surface. Overall, we report a novel cold resistance R2R3-MYB gene, IlMYB306, in the flower of I. laevigata, which could improve tobacco cold stress tolerance by thickening the waxy layer, increasing antioxidant activity and the content of proline.

Background MYBs are among the largest transcription factor families in plants. Consequently, members of this family are involved in a plethora of processes including development and specialized metabolism. The MYB families of many plant species were investigated in the last two decades since the first investigation looked at Arabidopsis thaliana. This body of knowledge and characterized sequences provide the basis for the identification, classification, and functional annotation of candidate sequences in new genome and transcriptome assemblies. Results A pipeline for the automatic identification and functional annotation of MYBs in a given sequence data set was implemented in Python. MYB candidates are identified, screened for the presence of a MYB domain and other motifs, and finally placed in a phylogenetic context with well characterized sequences. In addition to technical benchmarking based on existing annotation, the transcriptome assembly of Croton tiglium and the annotated genome sequence of Castanea crenata were screened for MYBs. Results of both analyses are presented in this study to illustrate the potential of this application. The analysis of one species takes only a few minutes depending on the number of predicted sequences and the size of the MYB gene family. This pipeline, the required bait sequences, and reference sequences for a classification are freely available on github: https://github.com/bpucker/MYB_annotator. Conclusions This automatic annotation of the MYB gene family in novel assemblies makes genome-wide investigations consistent and paves the way for comparative studies in the future. Candidate genes for in-depth analyses are presented based on their orthology to previously characterized sequences which allows the functional annotation of the newly identified MYBs with high confidence. The identification of orthologs can also be harnessed to detect duplication and deletion events.

The plant growth, development, and secondary metabolism are regulated by R2 R3-MYB transcription factors. This study identified the R2 R3-MYB genes in the genome of Andrographis paniculata and analyzed the chromosomal localization, gene structure, and conserved domains, phylogenetic relationship, and promoter cis-acting elements of these R2 R3-MYB genes. Moreover, the gene expression profiles of R2 R3-MYB genes under abiotic stress and hormone treatments were generated by RNA-seq and validated by qRT-PCR. The results showed that A. paniculata contained 73 R2 R3-MYB genes on 21 chromosomes. These members belonged to 34 subfamilies, 19 of which could be classified into the known subfamilies in Arabidopsis thaliana. The 73 R2 R3-MYB members included 36 acidic proteins and 37 basic proteins, with the lengths of 148-887 aa. The domains, motifs, and gene structures of R2 R3-MYBs in A. paniculata were conserved. The promoter regions of these genes contains a variety of cis-acting elements related to the responses to environmental factors and plant hormones including light, ABA, MeJA, and drought. Based on the similarity of functions of R2 R3-MYBs in the same subfamily and the transcription profiles, ApMYB13/21/35/67/73(S22) may regulate drought stress through ABA pathway; ApMYB20(S11) and ApMYB55(S2) may play a role in the response of A. paniculata to high temperature and UV-C stress; ApMYB5(S7) and ApMYB33(S20) may affect the accumulation of andrographolide by regulating the expression of key enzymes in the MEP pathway. This study provides theoretical reference for further research on the functions of R2 R3-MYB genes in A. paniculata and breeding of A. paniculata varieties with high andrographolide content.

Conical epidermal cells occur on the tepals (perianth organs, typically petals and/or sepals) of the majority of animal-pollinated angiosperms, where they play both visual and tactile roles in pollinator attraction, providing grip to foraging insects, and enhancing colour, temperature, and hydrophobicity. To explore the evolutionary history of conical epidermal cells in angiosperms, we surveyed the tepal epidermis in representative species of the ANA-grade families, the early-diverging successive sister lineages to all other extant angiosperms, and analysed the function of a candidate regulator of cell outgrowth from Cabomba caroliniana (Nymphaeales). We identified conical cells in at least two genera from different families (Austrobaileya and Cabomba). A single SBG9 MYB gene was isolated from C. caroliniana and found to induce strong differentiation of cellular outgrowth, including conical cells, when ectopically expressed in Nicotiana tabacum. Ontogenetic analysis and quantitative reverse transcription–PCR established that CcSBG9A1 is spatially and temporally expressed in a profile which correlates with a role in conical cell development. We conclude that conical or subconical cells on perianth organs are ancient within the angiosperms and most probably develop using a common genetic programme initiated by a SBG9 MYB transcription factor.

Loss of anthocyanin pigments is a common transition during cereal domestication, diversification, and improvement. However, the genetic basis for this convergent transition in cereal remains largely unknown. Here, we identified a chromosomal syntenic block across different species that contained R2R3-MYB genes (c1/pl1) responsible for the convergent decoloring of anthocyanins in cereals. Quantitative trait locus (QTL) mapping identified a major QTL for aerial root color corresponding to pl1 and a major QTL for spikelet color corresponding to c1 on maize chromosomes 6 and 9, respectively. One insertion in the regulatory region that led to transcriptional down-regulation was present in maize pl1, and several insertions in the coding region resulting in loss of function occurred in maize c1. A transposable element insertion in the third exon of c1, leading to three new non-functional transcripts, was responsible for decoloring in foxtail millet. The c1/pl1 genes enhanced the transcription of the core enzyme-encoding genes, including pr1, fht1, a1, a2, bz1, and aat1 in the anthocyanin pathway, while they repressed the expression of fnsii1 in flavones, sm2 in maysin, and bx3, bx4, bx5, and bx10 in DIMBOA. Our results indicated that the convergent decoloring of these plants shared the same genetic basis across different cereal species.

Background The R2R3-MYB transcription factor is one of the largest gene families in plants and involved in the regulation of plant development, hormone signal transduction, biotic and abiotic stresses. Tobacco is one of the most important model plants. Therefore, it will be of great significance to investigate the R2R3-MYB gene family and their expression patterns under abiotic stress and senescence in tobacco. Results A total of 174 R2R3-MYB genes were identified from tobacco (Nicotiana tabacum L.) genome and were divided into 24 subgroups based on phylogenetic analysis. Gene structure (exon/intron) and protein motifs were especially conserved among the NtR2R3-MYB genes, especially members within the same subgroup. The NtR2R3-MYB genes were distributed on 24 tobacco chromosomes. Analysis of gene duplication events obtained 3 pairs of tandem duplication genes and 62 pairs of segmental duplication genes, suggesting that segmental duplications is the major pattern for R2R3-MYB gene family expansion in tobacco. Cis-regulatory elements of the NtR2R3-MYB promoters were involved in cellular development, phytohormones, environmental stress and photoresponsive. Expression profile analysis showed that NtR2R3-MYB genes were widely expressed in different maturity tobacco leaves, and however, the expression patterns of different members appeared to be diverse. The qRT-PCR analysis of 15 NtR2R3-MYBs confirmed their differential expression under different abiotic stresses (cold, salt and drought), and notably, NtMYB46 was significantly up-regulated under three treatments. Conclusions In summary, a genome-wide identification, evolutionary and expression analysis of R2R3-MYB gene family in tobacco were conducted. Our results provided a solid foundation for further biological functional study of NtR2R3-MYB genes in tobacco.

Background Sensing and responding to stresses determine the tolerance of plants to adverse environments. The triploid Chinese white poplar is widely cultivated in North China because of its adaptation to a wide range of habitats including highly saline ones. However, its triploid genome complicates any detailed investigation of the molecular mechanisms underlying its adaptations. Results We report a haplotype-resolved genome of this triploid poplar and characterize, using reverse genetics and biochemical approaches, a MYB gene, SALT RESPONSIVE MYB TRANSCRIPTION FACTOR (SRMT), which combines NUCLEAR FACTOR Y SUBUNIT C 9 (PtoNF-YC9) and RESPONSIVE TO DESICCATION 26 (PtoRD26), to regulate an ABA-dependent salt-stress response signaling. We reveal that the salt-inducible PtoRD26 is dependent on ABA signaling. We demonstrate that ABA or salt drives PtoNF-YC9 shuttling into the nucleus where it interacts with SRMT, resulting in the rapid expression of PtoRD26 which in turn directly regulates SRMT. This positive feedback loop of SRMT-PtoRD26 can rapidly amplify salt-stress signaling. Interference with either component of this regulatory module reduces the salt tolerance of this triploid poplar. Conclusion Our findings reveal a novel ABA-dependent salt-responsive mechanism, which is mediated by the PtoNF-YC9-SRMT-PtoRD26 module that confers salt tolerance to this triploid poplar. These genes may therefore also serve as potential and important modification targets in breeding programs.

R2 R3-MYB transcription factors are ubiquitous in plants, playing a role in the regulation of plant growth, development, and secondary metabolism. In this paper, the R2 R3-MYB transcription factors were identified by bioinformatics analysis of the genomic data of Erigeron breviscapus, and their gene sequences, structures, physical and chemical properties were analyzed. The functions of R2 R3-MYB transcription factors were predicted by cluster analysis. Meanwhile, the expression patterns of R2 R3-MYB transcription factors in response to hormone treatments were analyzed. A total of 108 R2 R3-MYB transcription factors, named EbMYB1-EbMYB108, were identified from the genome of E. breviscapus. Most of the R2 R3-MYB genes carried 2-4 exons. The phylogenetic tree of MYBs in E. breviscapus and Arabidopsis thaliala was constructed, which classified 234 MYBs into 30 subfamilies. The MYBs in the five MYB subfamilies of A.thaliala were clustered into independent clades, and those in E. breviscapus were clustered into four clades. The transcriptome data showed that MYB genes were differentially expressed in different tissues of E. breviscapus and in response to the treatments with exogenous hormones such as ABA, SA, and GA for different time. The transcription of 13 R2 R3-MYB genes did not change significantly, and the expression patterns of some genes were up-regulated or down-regulated with the extension of hormone treatment time. This study provides a theoretical basis for revealing the mechanisms of R2 R3-MYB transcription factors in regulating the growth and development, stress(hormone) response, and active ingredient accumulation in E. breviscapus.

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Background R2R3-MYB is a class of transcription factor crucial in regulating secondary cell wall development during wood formation. The regulation of wood formation in gymnosperm has been understudied due to its large genome size. Using Single-Molecule Real-Time sequencing, we obtained full-length transcriptomic libraries from the developmental stem of Cunninghamia lanceolata, a perennial conifer known as Chinese fir. The R2R3-MYB of C. lanceolata (hereafter named as ClMYB) associated with secondary wall development were identified based on phylogenetic analysis, expression studies and functional study on transgenic line. Results The evolutionary relationship of 52 ClMYBs with those from Arabidopsis thaliana, Eucalyptus grandis, Populus trichocarpa, Oryza sativa, two gymnosperm species, Pinus taeda, and Picea glauca were established by neighbour-joining phylogenetic analysis. A large number of ClMYBs resided in the woody-expanded subgroups that predominated with the members from woody dicots. In contrast, the woody-preferential subgroup strictly carrying the members of woody dicots contained only one candidate. The results suggest that the woody-expanded subgroup emerges before the gymnosperm/angiosperm split, while most of the woody-preferential subgroups are likely lineage-specific to woody dicots. Nine candidates shared the same subgroups with the A. thaliana orthologs, with known function in regulating secondary wall development. Gene expression analysis inferred that ClMYB1/2/3/4/5/26/27/49/51 might participate in secondary wall development, among which ClMYB1/2/5/26/27/49 were significantly upregulated in the highly lignified compression wood region, reinforcing their regulatory role associated with secondary wall development. ClMYB1 was experimentally proven a transcriptional activator that localised in the nucleus. The overexpression of ClMYB1 in Nicotiana benthamiana resulted in an increased lignin deposition in the stems. The members of subgroup S4, ClMYB3/4/5 shared the ERF-associated amphiphilic repression motif with AtMYB4, which is known to repress the metabolism of phenylpropanoid derived compounds. They also carried a core motif specific to gymnosperm lineage, suggesting divergence of the regulatory process compared to the angiosperms. Conclusions This work will enrich the collection of full-length gymnosperm-specific R2R3-MYBs related to stem development and contribute to understanding their evolutionary relationship with angiosperm species.

MYB transcription factors of plants play important roles in flavonoid synthesis, aroma regulation, floral organ morphogenesis, and responses to biotic and abiotic stresses. Cymbidium ensifolium is a perennial herbaceous plant belonging to Orchidaceae, with special flower colors and high ornamental value. In this study, a total of 136 CeMYB transcription factors were identified from the genome of C. ensifolium, including 27 1R-MYBs, 102 R2R3-MYBs, 2 3R-MYBs, 2 4R-MYBs, and 3 atypical MYBs. Through phylogenetic analysis in combination with MYB in Arabidopsis thaliana, 20 clusters were obtained, indicating that these CeMYBs may have a variety of biological functions. The 136 CeMYBs were distributed on 18 chromosomes, and the conserved domain analysis showed that they harbored typical amino acid sequence repeats. The motif prediction revealed that multiple conserved elements were mostly located in the N-terminal of CeMYBs, suggesting their functions to be relatively conserved. CeMYBs harbored introns ranging from 0 to 13 and contained a large number of stress- and hormone-responsive cis-acting elements in the promoter regions. The subcellular localization prediction demonstrated that most of CeMYBs were positioned in the nucleus. The analysis of the CeMYBs expression based on transcriptome data showed that CeMYB52, and CeMYB104 of the S6 subfamily may be the key genes leading to flower color variation. The results lay a foundation for the study of MYB transcription factors of C. ensifolium and provide valuable information for further investigations of the potential function of MYB genes in the process of anthocyanin biosynthesis.

The fruit surface has an enormous impact on the external appearance and postharvest shelf-life of fruit. Here, we report two functionally redundant genes, PpMYB25 and PpMYB26, involved in regulation of fruit skin texture in peach. PpMYB25 can activate transcription of PpMYB26 and they both induce trichome development and cuticular wax accumulation, resulting in peach fruit with a fuzzy and dull appearance. By contrast, nonfunctional mutation of PpMYB25 caused by an insertional retrotransposon in the last exon in nectarine fails to activate transcription of PpMYB26, resulting in nectarine fruit with a smooth and shiny appearance due to loss of trichome initiation and decreased cuticular wax accumulation. Secondary cell wall biosynthesis in peach fruit pubescence is controlled by a transcriptional regulatory network, including the master regulator PpNAC43 and its downstream MYB transcription factors such as PpMYB42, PpMYB46 and PpMYB83. Our results show that PpMYB25 and PpMYB26 coordinately regulate fruit pubescence and cuticular wax accumulation and their simultaneous perturbation results in the origin of nectarine, which is botanically classified as a subspecies of peach.

MYB transcription factors are crucial in regulating stress tolerance and expression of major genes involved in flavonoid biosynthesis. The functions of MYBs is well explored in a number of plants, yet no study is reported in Apocynum venetum. We identified a total of 163 MYB candidates, that comprised of 101 (61.96%) R2R3, 6 3R, 1 4R and 55 1R. Syntenic analysis of A. venetum R2R3 (AvMYBs) showed highest orthologous pairs with Vitis vinifera MYBs followed by Arabidopsis thaliana among the four species evaluated. Thirty segmental duplications and 6 tandem duplications were obtained among AvMYB gene pairs signifying their role in the MYB gene family expansion. Nucleotide substitution analysis (Ka/Ks) showed the AvMYBs to be under the influence of strong purifying selection. Expression analysis of selected AvMYBs under low temperature and cadmium stresses resulted in the identification of AvMYB48, AvMYB97, AvMYB8, AvMYB4 as potential stress responsive genes and AvMYB10 and AvMYB11 in addition, proanthocyanidin biosynthesis regulatory genes which is consistent with their annotated homologues in Arabidopsis. Tissue specific expression profile analysis of the AvMYBs further supported the qPCR analysis result. MYBs with higher transcript levels in root, stem and leaf like AvMYB4 for example, was downregulated under the stresses and such with low transcript level such as AvMYB48 which had low transcript in the leaf was upregulated under both stresses. Transcriptome and phylogenetic analyses suggested AvMYB42 as a potential regulator of anthocyanin biosynthesis. Thus, this study provided valuable information on AvR2R3-MYB gene family with respect to stress tolerance and flavonoid biosynthesis.

Anthocyanins and proanthocyanidins (PAs) are vital secondary metabolites in Tartary buckwheat because of their antioxidant capacities and radical scavenging functions. It has been demonstrated that R2R3-MYB transcription factors (TFs) are essential regulators of anthocyanin and PA biosynthesis in many plants. However, their regulatory mechanisms in Tartary buckwheat remain to be clarified. Here, we confirmed the role of FtMYB3 in anthocyanin and PA biosynthesis. FtMYB3, which belongs to the subgroup 4 R2R3 family was predominantly expressed in roots. The transcriptional expression of FtMYB3 increased significantly under hormone treatment with SA and MeJA and abiotic stresses including drought, salt, and cold at the seedling stage. Functional analyses showed that FtMYB3 negatively regulated anthocyanin and PA biosynthesis, primarily via downregulating the expression of the